issn: 2150-4091 volume 2, number 5, may 2010 natural cience s vol2 no.5-02-05... · 2011-09-07 ·...

Vol.2, No.5, 419-525 (2010) Natural Science

Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/NS/

TABLE OF CONTENTS

Volume 2, Number 5, May 2010

Research on the plasma dynamics in a magnetically self-insulated ion diode with explosive emission

potential electrode A. Pushkarev, Y. Isakova, R. Sazonov…………………………………………………………………………………………419

The capabilities of the calculated approach for the astroclimatic assessment in radioastronomy N. V. Ruzhentsev, A. S. Mihailov………………………………………………………………………………………………427

Kinetic spectrophotometric determination of certain cephalosporins using iodate/iodide mixture S. R. El-Shaboury, F. A. Mohamed, G. A. Saleh, A. H. Rageh…………………………………………………………………432

Application of microspectral luminescent analysis to study the intracellular metabolism in single cells and cell systems

N. A. Karnaukhova, L. A. Sergievich, V. N. Karnaukhov……………………………………………………………………………444

Wettability alteration by magnesium ion binding in heavy oil/brine/chemical/sand systems—analysis of hydration forces

Q. Liu, M.-Z. Dong, K. Asghari, Y. Tu…………………………………………………………………………………………450

Dicranostigma leptopodum (maxim) fedde induced apoptosis on SMMC-7721 human hepatoma cells and inhibited tumor growth in mice

W.-H. Zhang, M.-H. Lv, J. Hai, Q.-P. Wang, Q. Wang……………………………………………………………………………457

Mesolamellar composite of TiN and CTAB using fluoride ion bridge: synthesis, mechanism & characterization

T. V. Anuradha……………………………………………………………………………………………………………………464

Extraction, identification and adsorption-kinetic studies of a natural color component from G. sepium K. N. Vinod, Puttaswamy, K. N. Gowda, R. Sudhakar…………………………………………………………………………469

Evidence for the existence of localized plastic flow auto-waves generated in deforming metals

L. B. Zuev, Svetlana A. Barannikova…………………………………………………………………………………………476

Higher dimensional bianchi type-V universe in creation-field cosmology

K. S. Adhav, S. D. Katore, A. S. Bansod, P. S. Gadodia…………………………………………………………………………484

Scalar-isovector δ-meson mean-field and mixed phase structure in compact stars

G. B. Alaverdyan…………………………………………………………………………………………………………………489

Stability analysis of primary emulsion using a new emulsifying agent gum odina

A. Samanta, D. Ojha, B. Mukherjee………………………………………………………………………………………………494

Investigation of airborne fungi at different altitudes in Shenzhen University

L. Li, C. Lei, Z.-G. Liu…………………………………………………………………………………………………………506

Effect of latex conversion on glass transition temperature

S.-X. Li, Y.-D. Guan, L.-M. Liu…………………………………………………………………………………………………515

Genomic data provides simple evidence for a single origin of life

K. Sorimachi……………………………………………………………………………………………………………………519

Natural Science

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Vol.2, No.5, 419-426 (2010)doi: 10.4236/ns.2010.25051


Natural Science

Research on the plasma dynamics in a magnetically self-insulated ion diode with explosive emission potential electrode

Alexander Pushkarev*, Yulia Isakova, Roman Sazonov

Tomsk Polytechnic University, Tomsk, Russia; *Corresponding Author: [email protected]

Received 10 January 2010; revised 23 February 2010; accepted 10 March 2010.

ABSTRACT

The results of an experimental investigation of the plasma dynamics in a magnetically insulated ion diode in bipolar-pulse mode are pre-sented. The experiments were done at the pulsed TEMP-4M accelerator by formation of a first negative pulse (100 ns, 150-200 kV) and a second positive pulse (80 ns, 250-300 kV). The voltage-current diode characteristics were used to analyze the plasma behavior in the anode- cathode gap. It is shown that, during the first pulse, a discrete emissive surface is formed on the graphite potential electrode and a plasma forms by explosive-emission, which before the second pulse comes, fills the whole working surface of the electrode and spreads to the anode-cathode gap. An analytical expression is obtained for the total current in the cellular structure approximation. It is shown that the current build-up for a cathode surface with discrete emitting centers is described satisfactorily by a modified Child-Langmuir formula with a form factor decreasing from F = 6 to 1. It is found that, once plasma formation at the graphite potential electrode is complete and until the second positive pulse, the plasma speed is constant and equals 1.3 ± 0.2 cm/μs.

Keywords: Ion Beams; Plasma Generation; Graphite Cathodes; Plasma Speed; Magnetic Insulation; Explosions

1. INTRODUCTION

Modernizing of engineering products is difficult without application of new progressive technological processes, which allow increasing the life and reliability of com-ponents and connections under very severe operating conditions. Powerful ion beam irradiation provides heat-

ing and cooling of boundary layers of a treated item at a rate of more than 108 K/s. This allows compounds and structures to be realized in surface layers which cannot be made by traditional industrial methods. As a result, the characteristics of materials change: solidity, strength, wear resistance; the operational characteristics of items made from these materials improve. For wide industrial implementation of the modification methods of bound-ary layers by high-power ion beams, a reliable and eco-nomical powerful ion beam source with long operational life is necessary. The effective generation of powerful ion beams became

possible when two important problems were solved: formation of dense plasma on the anode surface and suppression of the electronic component of diode current. The ion and electron generation and acceleration proc-esses occur simultaneously when the voltage is applied to the diode. As follows from the Child-Langmuir for-mula [1], the proton current density is 2.3% of the elec-tron current density. As for heavier ions, they are lower. In 1973, Sudan and Lovelace [2] first suggested the con-struction of an ion diode with external magnetic isolation for the suppression of the electronic component of diode current. The efficiency of such a construction was con-firmed experimentally in 1976 by Dreike, Eichenberger, Humphries, and Sudan [3]. In their article, the results of experiments on proton beam generation in an ion diode of coaxial construction with external magnetic isolation were presented. The total diode current was described by the Child-Langmuir ratio for proton current (subject to diode geometry) by changing the accelerating voltage from 120 to 200 kV, with pulse duration of 50 ns and a magnetic induction of 1.03 T in the anode-cathode gap. When the magnetic induction goes down to 0.52 T, the total current exceeds the calculated value of proton cur-rent by a factor of 2. In 1977, Humphries [4] first showed that it is possible to arrange magnetic self-insulation using a special construction of ion diode. A transverse magnetic field is formed in the anode-cathode gap by the diode self current when the current flows in the elec-trodes. In this case an additional magnetic field source is

A. Pushkarev et al. / Natural Science 2 (2010) 419-426

Copyright © 2010 SciRes. http://www.scirp.org/journal/NS/Openly accessible at

420

not required. It significantly simplifies the construction of a powerful ion beam generator.

As for the problem of dense plasma formation on the anode surface, in 1980 Logachev, Remnev and Usov first suggested using the phenomenon of explosive elec-tron emission [5]. They used a double pulse generator of nanosecond duration (without any pause between pulses). The first pulse had negative polarity and the second pulse had positive polarity. During the first pulse, a plasma forms by explosive emission on the potential electrode. It contains ions with 1-4 degrees of ionization, the plasma temperature is 4-5 eV and the density is more than 1020 cm-3. During the second pulse, ions are pulled out from the explosive emission plasma and accelerated. A simple and robust construction of double pulse gen-erator with an adjustable pause between pulses was first suggested by Logachev, Remnev and Usov in 1983 [6]. We modernized this construction of the generator in 2009 [7], as a result the process of plasma formation on the potential electrode surface has improved. This gen-erator construction underlies the accelerator we use; it is described in detail further in this text. For a more de-tailed review of pulsed ion beam generation see [8,9].

In spite of much progress in powerful ion beam gen-eration, many processes in an ion diode with magnetic self insulation and with an explosive emission anode have not been researched enough. In particular, there is no experimental information about the duration of solid emissive surface formation on anode and plasma expen-sion velocity. This can be explained in the following way: during the first 20-25 years, the main application of powerful ion beams was in controlled thermonuclear fusion investigations. The production of ion beams with maximum current density and a pulse power of more than 1012 W were mostly attempted. The relatively mod-ern and broad application of high current ion beam ac-celerators is in surface treatment or material modifica-tion (i.e. smoothing and annealing of metal surfaces, alloying, removal of coatings, thin film deposition, etc.). One of the first attempts to use a high current ion accel-erator for materials modification was made by Ham-mer’s group at Cornell in collaboration with Hodgson’s group at IBM in 1980 [10]. They used a pulsed 180 keV, 80 ns proton beam at several different ion current densi-ties (40, 75 and 380 A/cm2) to anneal ion-implant-dam- aged semiconductors. The first investigations into the possibility of using powerful ion beams for metals alloy materials modification were conducted by Didenko, Ku- snetsov and Remnev in 1981 [11].

The conditions for the development and formation of explosive emission plasma during pulsed ion beam gen-eration in the first stage (negative polarity pulse) are close to the conditions in an electron diode with an ex-plosive emission cathode. Experimental data and theoretical models, describing a change of explosive emis-

sion plasma velocity during electron beam generation, have a conflicting character. A review of the investiga-tions into explosive emission plasma dynamics during pulsed electron beam generation has been given in our earlier article [12]. In 2006-2008 we conducted a com-prehensive investigation of pulse electron beam genera-tion in a diode with an explosive-emitting cathode of a different configuration. For the first time it was shown that, from the moment when the process of plasma for-mation on the cathode is complete until the end of the pulse (80-100 ns), the plasma velocity is constant and equal to 2 ± 0.5 cm/μs for a graphite or carbon fiber cathode, 3 ± 0.5 cm/μs for a tungsten cathode, and 4 ± 0.5 cm/μs for a cathode made from copper [12].

The first attempt at a systematic investigation of ex-plosive emission plasma dynamics during pulsed ion beam generation in a diode with self magnetic insulation was made by Xin, Zhu and Lei in 2008 [13]. To deter-mine the explosive emission plasma expansion velocity, the voltage-current characteristics of the diode and the Child-Langmuir ratio were used. That only applies when a diode operates in a mode of space charge limitation. They found that, during the first 29 ns after the voltage is applied to the diode, the plasma velocity is equal to zero. Then the plasma velocity increases up to a maximum value of 4.5 cm/μs (t = 45 ns), subsequently it decreases to 1.5 cm/μs at t = 70 ns. However, the authors did not adduce proofs of the diode operation during the first 100 ns. Our investigations have shown that the duration of solid plasma layer formation on the potential electrode surface in a diode with a similar construction, exceeds 200 ns. During this period of time, the total diode current is limited by the emissive ability of the potential elec-trode. Thus, it is not correct to use the Child-Langmuir ratio for the determination of the explosive emission plasma velocity.

Also, the explosive emission plasma behavior from the moment when separate emission centers form until the solid plasma layer forms on the potential electrode surface is not thoroughly investigated. In previous works [14,15], numerical modeling was performed (tube of current method) of the change of average electron cur-rent density in a planar diode with the discrete emission surface of the cathode during the evolution from forma-tion on separate emission centers until the solid plasma layer forms. The modeling conditions are a constant ve-locity for the expansion of the plasma forming by explo-sive emission and a rectangular pulse with constant am-plitude. In our work [16], the first experimental investi-gation of the voltage-current characteristics of a flat di-ode with a graphite explosion emission cathode was de-scribed. We show that the rise of electron current on the discrete emission cathode surface is well described by the Child-Langmuir ratio when the form-factor decreases from 6 to 1. Results for plasma layer evolution without


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421421

Openly accessible at

an external magnetic field were obtained. The results of explosive emission plasma dynamics from formation on separate emission centers until a solid plasma layer forms on the potential electrode surface of the ion diode (in a transverse magnetic field) have not been conducted until the present time.

The purpose of this work is an investigation with high temporal resolution of a magnetically insulated ion diode in bipolar-pulse mode during the formation of the plasma on the potential electrode surface.

2. EXPERIMENTAL INSTALLATION

Investigations were conducted at accelerator TEMP-4M (modification of accelerator TEMP-4 [17,18]) with the following parameters: the first impulse is negative (≈ 100 ns, 100-150 kV) and the second one is positive (80 ns, 250-300 kV). Beam composition: ions of carbon and protons, ion current density 10-150 A/cm2 (for different types of diodes), pulse frequency 5-10 pulses per minute. The accelerator contains a high-voltage pulse generator, double forming line (Blumlein line), basic and preliminary gas dischargers, vacuum stripe diode, composed of potential and grounded electrodes. The potential graphite electrode of the diode is connected through a preliminary gas discharger to the internal electrode of the double forming line. The middle electrode of the double form-ing line is connected to the high-voltage pulse generator. In order to optimize the process of plasma formation by explosive emission on the potential electrode, the nano-second generator of the TEMP-4 accelerator was mod-ernized. The positive voltage, which forms during the delay between the first negative pulse and the second positive pulse, was eliminated. This increases the effi-ciency of plasma formation. Figure 1 shows a block diagram of the diode connection of accelerator TEMP- 4M, the circuit for measuring voltage and current in the stripe focusing diode with self-isolation.

For carbon, the electric field threshold, at which ex-plosive emission of electrons begins, is lower than that of copper and other metals. Moreover, in the space charge limitation mode, the ion current density is inversely proportional to the square root of the ion mass. The car-bon ions in the materials of construction have the least mass. Therefore, its use is much more practical for measuring plasma expansion velocity.

We have investigated a focusing diode (4 cm × 25 cm) and a planar diode (4 cm × 20 cm) each with a potential electrode made from graphite. A grounded electrode of the same dimensions is made from stainless steel and has slits 0.5 cm × 5 cm, optical transparency of 60%.

To measure the total current consumed by the diode connection a Rogowski coil with a reverse coil was used. The voltage at the potential electrode was measured by a resistive voltage divider. The recording of the electric

signals coming from sensors was performed on a Te- ktronix 3052 B oscilloscope (500 MHz, 5·109 measurements per second). The inaccuracy of electric signal synchronization did not exceed 0.5 ns. The calibration of the diagnostic equipment showed that it correctly reflected the accelerator operation in short circuit mode (U = 50-60 kV), when operating with a resistive load up to 10 Ω (200-300 kV) and when operating with the diode.

Shown in Figure 2 is a typical oscilloscope trace of the voltage on the potential electrode and of the load impedance. These results were achieved using a 9.5 Ohm resistor during calibration. The voltage measurement accuracy achieved by the resistive divider and of the total electron beam current by the Faraday cup, as well as their frequency performance allows to measure the diode impedance with an accuracy better than ±10%. The stripe diode with magnetic self-isolation performed effectively at a vacuum of 10-3 Torr with a limit of more than 106 pulses. The frequency of generation of these powerful ion beam pulses was restricted only by the rate of heating in the diode.

Figure 1. Diode connection of accelerator TEMP- 4M: 1-potential electrode of diode, 2-grounded electrode, 3-Faraday cup, 4-Rogowski coil, 5-voltage divider.

Figure 2. Oscilloscope trace of voltage (1) and calculated values of impedance (2).



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3. BASIC CALCULATION EQUATIONS

An analysis of plasma behavior in the anode-cathode gap was performed based on the current-voltage characteris-tics of the diode. The electron and ion current densities flowing through the diode in the mode restricted by space charge are determined by the following expres-sions [1]: Electron current:

20

3/26

20

3/2

e

0e 102.33

9

24ε

d

U

d

U

m

eJ (1)

Ion current:

20

2/30

9

24

d

U

m

zJ

i

iion

(2)

where U is the voltage applied to the diode; d0 is the anode-cathode gap, me is electron mass; mi is ionic mass; zi is ionic charge.

Taking into account the reduction of anode-cathode gap due to expansion of plasma from the emissive sur-face of the potential electrode, the electron current den-sity is equal to:

20

3/26

e)(

102.33)(vtd

UtJ

(3)

where v is plasma expansion velocity. If the diode operates in the mode of space charge

limitation, then from correlation (3), we obtain the cath-ode plasma expansion speed as:

6 3/20

0e

2.33 101( )

S Uv t d

t I

(4)

where S0 is the surface area of potential electrode (cath-ode during negative pulsed) of diode. This correlation is used further down in the calculation of plasma expansion speed.

The speed at which the cathode plasma spreads can be correctly calculated from the current-voltage characteris-tics of the diode only under operating conditions corre-sponding to the mode of space charge limitation. The mode operation of the diode can easily be determined by comparing the experimental and calculated values of diode impedance. Their coincidence corresponds to the diode current being limited by space charge. In the initial period (discrete emission surface mode) and in the satu-ration mode, the diode current is limited by the electron emission from cathode. This is why the experimental values of impedance will be larger than the calculated ones. It is evident from Eq.1 and Eq.2, that the ion cur-rent amounts to only a small part of the total current through the diode, therefore the impedance of the diode can be calculated from the following:

1/260

20

e0calc

102.33

)(

US

tvd

JS

UR

(5)

4. DISCRETE EMISSIVE SURFACE MODE

Operation of the magnetic self-isolation diode at the first (negative) pulse and during the pause between pulses is analogous to the operation mode of a planar diode with explosive emission cathode at electron beam generation. In Figure 3, a typical oscilloscope trace of the voltage on the potential graphite electrode and calculated values of the diode impedance are displayed.

Two modes of operation can be singled out [12,19]. From the application of voltage until the formation of a solid plasma surface at the potential electrode (discrete emissive surface mode, 0 < t < 250 ns in Figure 3), the diode current is limited by the emissive ability of the cathode. After covering the potential electrode surface with plasma, the total diode current is limited only by the vacuum electron charge in the anode-cathode gap (250 ns < t < 600 ns in Figure 3).

Reduction of impedance of the diode in discrete emis-sive surface mode is connected with two processes. They are the increase of emissive surface on the graphite elec-trode and the reduction of anode-cathode gap through movement of the explosive emission plasma towards the grounded electrode (Eq.5). The origin of the reduction in diode impedance in space charge limitation mode is the reduction of anode-cathode gap only.

The diode changes to the space charge limitation mode after formation of the solid emissive layer on the cathode surface. Let us assume that: 1) the electron cur-rent is limited by the space charge in the inter-electrode gap from the first moment of voltage application to the diode, and 2) electron beam current growth until saturation

Figure 3. Oscilloscope trace of voltage (1) and impedance value (2-experiment, 3-calculation) of stripe focusing diode with self-isolation, anode-cathode gap 8 mm.



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is determined by an increase in the surface of discrete emitting centers from zero to the total geometric area of the cathode. This approach has been successfully veri-fied [14,15] by modelling the variation of the average electron beam current in a planar diode with a discrete emitting surface. Using Eq.3 the total electron current of the diode is:

2

0

3/26

e)(

)(102.33

tvd

tSUI

where S(t) - total area of plasma emissive surface on the cathode.

In modelling the law of variation of the area of a dis-crete emitting surface, we use the following assumptions [16]: 1) the emitting centers are equidistant from each other and form a uniform cellular structure on the cath-ode surface; 2) the emitting centers are formed simulta-neously and their number remains the same during the entire period of electron beam generation. Thus, the total emitting area on the cathode is:

)sin(3π)()( 21 ααtvNtS

where N - number of emitting centers; v1 - speed of ex-pansion of the explosive emission plasma across the gap, α = 2arcos(b/v1·t), b - distance between adjoining emit-ting centers.

The total number of emitting centers can be estimated as the ratio of the cathode area to the area of a hexagonal unit cell (0.865b2). Consequently, the total electron cur-rent emitted by the discrete emissive surface is:

3/22

02

02

16

calc)(

)sin3(π)(102.7U

tvdb

SααtvI

(6)

Figure 4 shows the current changing during plasma formation by explosive emission on the potential elec-trode of the focusing diode. Experimental data are com-pared with calculated data based on the assumption that emitting centers form and expand (Eq.6, v1 = 2 cm/μs, b = 11 mm); within the presence of a solid plasma surface when the voltage is applied (Eq.3). Calculations were made of the total electron current of the diode with dis-crete emissive surface based on the assumption that the speed of expansion of the explosive emission plasma across the anode-cathode gap is equal to the speed of expansion of the graphite plasma without any magnetic field. The minimum distance between individual emit-ting centers was calculated from the electric field screening around the center [20]:

cm,500 3/41/210

UIdr

The radius of screening, r, is equal to 5-6 mm if the electron current from one emitting center I1 = 90 A and the average voltage applied to the diode, during emitting center formation is 100-120 kV (see Figure 3).

Measurements taken of the spatial distribution of en-ergy density in the electron beam formed by the diode during the first pulse confirm the existence of a periodic structure in the emitting surface on the potential elec-trode.

The distribution of energy density of electrons over the cross section was measured by using a special do-simetric film. The method of measurement is described in detail in our reference [21]. Figure 5 illustrates the change of electron beam energy density across the diode.

The dosimetric film was placed in the outside of the grounded electrode. To protect it from the effects of the ion beam, the films were covered with aluminum foil with a thickness of 15 microns. Due to the screening of the dosimetric film by slits (0.5 cm × 5 cm) on the grounded electrode, the density reduces to zero at x = 10 and 70 mm.

During the process in which the discrete emissive surface spreads from separate emitting centers to a solid plasma layer that covers the cathode, a form factor must be incorporated in Eq.6 [19]. This form factor accounts for the distortion of the electric field strength near emitting

Figure 4. The diode current changing during plasma formation by explosive emission: (1) experimental data, and calculation for (2) solid and (3) discrete plasma surface on the cathode.

Figure 5. Distribution of energy density in the elec-tron beam (cross section).



424


centers [20]. The dependence of planar diode current with a discrete emissive cathode is:

FU

tvdb

StvIcalc

2/32

02

02

16

)(

)sin(3)(107.2

It has been demonstrated [22,23] that the current- voltage characteristic of a planar diode with flat elec-trodes (U = 20-40 kV) and a single emitter arising at an artificial micro-protrusion on the cathode surface (d = 0.3-1 mm) in the initial stage of emitter evolution (v·t < d/3) is well described (to within 10%) by the following relation:

2

0

3/261044.4

d

tvUI

Note that this expression is obtained from correlation (1) for a cathode with an emitting area of π(v·t)2 and F = 6. Figure 6 shows the ratio of experimental values of diode current divided by calculated values based on the correlation (6).

This ratio reflects the change in form factor during explosive emission plasma formation. There are 4 typi-cal ranges for the form factor. During the initial period of time (t < 80 ns) the value of the form factor exceeds 6, which fits with an increase in the number of emitting centers and a simultaneous increase in their sizes. Dur-ing the time that the ratio of distance between adjacent emitting centers to their radius decreases from 7 to 5 (80 < t < 100 ns, see Figure 6) the formation of additional emitting centers is suppressed by screening effects. The value of the form factor is constant (within the meas-urement accuracy) and is equal to 6. Furthermore, with the size of the emitting center increasing as neighboring centers overlap, the value of F decreases to 1. Thus, a continuous emission surface forms on the graphite cath-ode to generate the second pulse, F = 1.

Previous research has shown that the duration of solid emissive layer formation on the cathode surface (only when congruent with the duration of the pulse edge) depends on the area and material of the cathode [16]. Figure 7 gives a comparison of the duration of explosive emission plasma formation on the graphite cathode sur-face of an electron diode (400 kV, 10 kA, 80 ns).

This was done with both 45 and 60 mm diameter cathodes. The values of the duration of solid emissive surface formation on the potential graphite electrode of the ion diode (flat and focusing geometries) in the self-magnetic insulation mode (first pulse) are illustrated in Figure 7. It was shown that, in the discrete emissive surface mode, the influence of the magnetic field on the dynamics of the cathode plasma in a magnetically insu-lated diode is negligible.

5. SPACE СHARGE LIMITATION MODE

After covering the potential electrode surface with plasma, the total diode current is limited only by the electron charge in the anode-cathode gap (250 ns < t < 600 ns in Figure 3). Experimental values of the diode impedance are well described by correlation (5). Figure 8 shows the values of cathode plasma expansion speed calculated from Eq.4.

Through these experiments we have found that the rate of expansion of a graphite plasma (across the anode- cathode gap) in a magnetically insulated diode is sig-nificantly lower than the plasma rate of expansion in an electron diode with graphite explosive emission cathode [12]. This indicates the significant influence of the mag-netic field in the gap on the expansion dynamics of an explosive emission plasma. In the generation of power-ful ion beams, the reduction of the rate of expansion of the explosive emission plasma is a useful effect which decreases the possibility of the anode-cathode gap being bridged by the plasma.

Figure 6. Oscilloscope trace of voltage (1) and form-factor for the formation of the plasma surface on the cathode (2).

Figure 7. Dependence of the duration of solid emis-sive surface formation on graphite cathode surface area.



425425


Figure 8. Change of explosive emission plasma speed during beam generation in electron diode with graphite cathode (1) and in planar ion (2) and focusing ion (3) diodes with graphite potential electrode.

6. DISCUSSION OF EXPERIMENTAL RESULTS

To provide magnetic insulation to the electrons in the diode, the grounded electrode is connected to a camera only from one side. Electrons, which compose the major part of charge carriers in the diode, generate a magnetic field as they move through the electrode (or along the surface).

The magnetic field vector is perpendicular to the elec-tric field vector in the anode-cathode gap. Figure 9 shows the change in magnetic induction in the diode during the formation of the explosive-emission plasma and the generation of the ion beam (according to the Biot-Savart law). The calculation of magnetic field using the ratio, based on diode geometry [13], gives results in close agreement.

We consider an electron, emitted from the explosive emission plasma on the potential electrode at the start of the first pulse. Under the influence of the electric field, it is accelerated towards the grounded electrode. In crossed electric and magnetic fields under the influence of the Lorentz force, the electron begins to change its direction of motion from a transverse one to a longitudinal one, along the grounded electrode. The Lorentz force does no work and does not change the energy (or velocity) of the electron, but changes only the direction of motion. Elec-tron motion in crossed electric and magnetic fields can be represented as a rotation over the cyclotron circumference with the center of the circumference drifting in the direction perpendicular to vectors E and B. The drift velocity is equal to the ratio of electric field strength to magnetic induction.

At the first pulse, the electrons are emitted from the plasma surface on the potential electrode and move to-wards the grounded electrode, which is a current-car- rying conductor. The magnetic induction in the anode- cathode gap increases towards the grounded electrode.

When the electron approaches the distance to the grounded electrode, at which the magnetic induction exceeds Вcr, the electron fails to reach its surface because it is in cycloidal motion in the diode gap along the grounded electrode. However, the electron motion in vacuum in the diode gap comprises the current of the grounded electrode and generates a self-magnetic field.

The change of critical magnetic induction during for-mation of the plasma on the cathode by explosive emis-sion is shown in Figure 9.

It is obvious that during the first 200-300 ns (discrete emissive surface mode) the magnetic field induction in the whole anode-cathode gap exceeds Вcr. This points to the fact that at the first pulse, electrons move into the anode-cathode gap near the potential electrode surface.

It is evident that the influence of the magnetic field on the value of electron current (magnetic cut-off) is sig-nificant when the duration of electron drift in crossed magnetic and electric fields is comparable to the dura-tion of the voltage pulse applied to the diode. The aver-age drift velocity is equal to 12 cm/ns, and the electrons reach the end of the diode (electrode length of 25 cm) within 2 ns. Therefore the effect of magnetic insula-tion at the first pulse is insignificant. It is important to note that, at the first pulse, only a small portion of elec-trons reach the surface of the grounded electrode, which restricts the process of plasma creation near the anode surface.

7. CONCLUSIONS

An analysis of the pulsed ion diode with a passive anode in the double-pulse mode, with matching of the diode impedance to the output resistance of the nanosecond generator has shown that the effect of magnetic self- isolation is significant only during ion beam generation (second pulse). In the initial period, at the stage of forming explosive centres and developing the potential electrode emission surface of the diode, the plasma dy-namics are similar to the processes in the electronic diode

Figure 9. (1) the change of the accelerating voltage; (2) the magnetic induction close to the potential electrode; (3) critical magnetic induction.



426

cal Physics Letters, 6(22), 1404-1406. of a pulsed electron accelerator. After the solid plasma forms on the surface of the passive anode, the effect of magnetic insulation manifests itself only in a reduction of the rate of explosive emission plasma expansion across the gap. The value of electron current amounts to more than 90% of the total diode current. It is well de-scribed by the model of space charge limitation. The electron drift duration in the crossed magnetic and elec-tric fields during plasma formation in the passive anode does not exceed a few nanoseconds, and the height of the Trochoid of drift motion is close to the value of the anode-cathode gap.

[6] Logachev, E.I., Remnev, G.E. and Usov, Y.P. (1983) Generator of nanosecond pulsed. Soviet Patent SU 852149A.

[7] Pushkarev, A.I., Tarbokov, V.A., Sazonov, R.V. and Isakov, I.F. (2009) Pulsed ion generator. Russian Patent RU 86374 U1.

[8] Bystrickii, V.M. and Didenko, A.N. (1984) High-power ion beams. Energoatomizdat, Moscow.

[9] Gordon, A.M. (2006) Ph.D. Dessertation, Tomsk Poly- technic University, Tomsk.

[10] Hodgson, R.T., Baglin, J.E.E., Pal, R., Neri, J.M. and Hammer, D.A. (1980) Ion beam annealing of semicon-ductors. Applied Physics Letters, 37(2), 187-189. The results of our investigations show that the experi-

mental current-voltage characteristics of a magnetically insulated ion diode with a graphite explosive emission cathode in the initial stage (featuring a discrete emitting surface) is satisfactorily described by a modified Child- Langmuir formula, assuming that all discrete emitters are formed simultaneously, their number is constant, and the emitter radius increases at a constant rate. In the ini-tial period of time, when the emitter radius is much smaller than the distance between adjacent emitters, the form factor in the modified Child-Langmuir formula corresponds to the experimental value F = 6, obtained for a diode with a single emitting center. As the emitter area increases, the form factor decreases from F = 6 to 1, which corresponds to the current-voltage characteristic of a planar diode with a continuous emitting surface on the graphite cathode.

[11] Didenko, A.N., Kusnetsov, B.I. and Remnev, G.E. (1981) Proceedings of National Conference Application of Electron and Ion Technology in National Economy, Tbilisi.

[12] Pushkarev, A.I. and Sazonov, R.V. (2009) Research of cathode plasma speed in planar diode with explosive emission cathode. IEEE Transactions on Plasma Science, 37(10), 1901-1907.

[13] Xin, J.P., Zhu, X.P. and Lei, M.K. (2008) Initial plasma of a magnetically insulated ion diode in bipolar-pulse mode. Physics Plasmas, 15(12), 123101-123108.

[14] Belomyttsev, S.Y., Korovin, S.D. and Pegel, I.V. (1999) Current in a high-current planar diode with a discrete emitting surface. Technical Physics, 44(6), 695-699.

[15] Djogo, G. and Gross, J.D. (1997) Circuit modeling of a vacuum gap during breakdown. IEEE Transactions on Plasma Science, 25(4), 617-624.

[16] Pushkarev, A.I. and Sazonov, R.V. (2008) A planar diode operating in the regime of limited electron emission. Technical Physics Letters, 34(4), 292-295.

8. ACKNOWLEDGMENTS [17] Remnev, G.E., Isakov, I.F., Opekounov, M.S., Kotlya- revsky, G.I., Matvienko, V.M., et al. (1997) High-power ion sources for industrial application. Surface and Coatings Technology, 96(1), 103-109.

This work was supported by the Russian Foundation for Basic Re-search under project No. 08-08-12086.

[18] Remnev, G.E., Isakov, I.F., Opekounov, M.S., Matvienko, V.M., Pushkarev, A.I., et al. (1999) High Intensity pulsed ion beam sources and their industrial applications. Surface and Coatings Technology, 114(2-3), 206-212.

REFERENCES

[1] Langmuir, I. (1913) The effect of space charge and residual gases on thermionic currents in high vacuum. Physical Review, 2(6), 450-486.

[19] Pushkarev, A.I. (2008) Perveance of a planar diode with a multipoint cathode. Technical Physics, 53(3), 363-367.

[20] Mesyats, G.A. (2000) Actons in vacuum discharge: Breakdown, the spark, and the ark. Nauka, Moscow. [2] Sudan, R.N. and Lovelace, R.V. (1973) Generation of

intense ion beams in pulsed diodes. Physical Review Letters, 31(19), 1174-1177.

[21] Pushkarev, A.I. and Sazonov, R.V. (2007) Research of high-current pulsed electron beam energy distribution in depth of sheet of water. Bulletin of Tomsk Polytechnic University, 311(2), 47-50.

[3] Dreike, P., Eichenberger, C., Humphries, S. and Sudan, R. (1976) Production of intense proton fluxes in a magneti-cally insulated diode. Journal of Applied Physics, 47(1), 85-88.

[22] Mesyats, G.A. (2004) Pulsed power and electronics. Nauka, Moscow.

[23] Shubin, A.F. and Yurike, Y.Y. (1975) Current rise in the initial stages of vacuum breakdown between plane electrodes with slowly increasing voltage. Russion Physics Journal, 18(6), 870-872.

[4] Humphries, S. (1977) Self magnetic insulation of pulsed ion diodes. Plasma Physics, 19(5), 399-406.

[5] Logachev, E.I., Remnev, G.E. and Usov, Y.P. (1980) Ion acceleration from explosion-emissive plasma. Techni-


Vol.2, No.5, 427-431 (2010)doi: 10.4236/ns.2010.25052

Copyright © 2010 SciRes Openly accessible at http://www.scirp.org/journal/NS/

Natural Science

The capabilities of the calculated approach for the astroclimatic assessment in radioastronomy

Nikolay V. Ruzhentsev*, Alexander S. Mihailov

Department of Microwave Radiospectrometry, Institute of Radio Astronomy, National Academy of Sciences, Kharkov, Ukraine; *Corresponding Author: [email protected]

Received 30 December 2009; revised 3 February 2010; accepted 25 February 2010.

ABSTRACT

The work is dedicated to calculation of daily variability of monthly averaged full vertical at-mospheric absorption for six well-known mountain locations of sub-millimeter wave band ra-diotelescopes obtained with usage of chosen by authors models combination. Test locations were defined as follows: Chajnantor plateau in the Atacama mountain desert (Chile), Hanle (India), South Pole (Antarctic), Mauna Kea (Hawaii, USA), Sierra Negra (Puebla, Mexico) and El Leoncito (Argentine). The data of these calcula-tions were compared with the data of long term radiometric observations of other authors. Se- arching for new alternative places to comple-ment existing sub-millimeter telescopes loca-tions was attempted too.

Keywords: Radiotelescopes; Atmosphere Absorption; Sub-Millimeter Waves Range; Astroclimate

1. INTRODUCTION

Earth atmosphere causes considerable impediments for radioastronomical observations at millimeter and submillimeter waves bands due to atmosphere attenuation and instability its transfer function. Consequently, even a slight improvement of the transfer function and its sta-bility can lead to a tangible radioastronomical observa-tions efficiency increase, especially in the submillimeter wave range. In view of this and taking uniqueness and high cost of the radiotelescopes that are installed in various regions of the world, location astroclimatic suit-ability assessment is required. These assessments are usually experimental and consist of gathering the statis-tical information concerning the extent of atmospheric attenuation, it’s seasonal and daily unsteadiness to reveal the most favorable time and place for the observation to be performed [1-5]. A few only places with most suitable

for allocation of sub-millimeter waves radiotelescopes are known [6] to the present time in a world. It is Cha-jnantor plateau in the Atacama mountain desert (Chile), Hanle in the Chanthang mountain plateau (India), South Pole (Antarctic) Mauna Kea (Hawaii, USA). The un-questionable advantage of the experimental approach is the precise measurement of the atmospheric attenuation in the defined frequency range in the defined location. However, the most significant disadvantages of this ap-proach are the fact that only a limited set of frequencies is used for the experiment (usually 1 or 2 frequencies) [1-4], the necessity of continuous observations cycles that last several years, financial, hardware and organiza-tional efforts and expenses of this important, but yet auxiliary support for radioastronomic observation.

On the contrary, analytical astroclymatic assessment approaches could provide operative and cheap daily and seasonal unsteadiness prognostication for any millimeter and submillimeter wavelength in any location of the world. However, there was an impediment on the route to the practical implementation of this approach: There were no adequate models of global distribution of mete-orological parameters altitude profile that were required for this kind of astroclymatic assessment until present days. The purpose of this work is the finding and dem-onstrating new possibility (unavailable earlier) of ob-taining of astroclimatic estimations of atmospheric at-tenuation average values by calculated method, as well as definition new locations of radio telescopes (most favourable for radioastronomical observations) of sub- millimetre range.

As we have shown recently in [7-9] (on the example of 15 regions of Ukraine) utilization of the chosen com-bination of Liebe’s atmosphere attenuation model and the latest meteorological atmosphere standard (grounded on the ERA-15 base data) [10-12] allows us to acquire a good (within the 10% precision interval) harmony of analytical calculations and experimental data concerning the monthly attenuation average in the atmosphere above the plain-type landscape. It is also very important for certain applications that the chosen models combination

N. V. Ruzhentsev et al. / Natural Science 1 (2010) 427-431

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allows the atmospheric absorption variability prognosti-cation for different times of day. However, mentioned above application of models need to be considered for the mountainous landscape also because a great amount of radiotelescopes at millimeter range and especially submillimeter range are located up to 5 km above the sea level.

This work contains the calculation of daily variability of monthly averaged full vertical atmospheric absorption using the chosen model combination for six well-known mountain locations of submillimeter waverange radio-telescopes. Test locations were defined as follows: Cha-jnantor plain in the Atacama mountain desert (Chile, 5000 m), Hanle (India, 4500 m), South Pole (Antarctic, 2800 m), Mauna Kea (Hawaii, USA, 4100 m), Sierra Negra (Puebla, Mexico, 4600 m) and El Leonsito (Ar-gentina, 2500 m).

Data from these calculations were compared with data of long period radiometric observations (from one year up to nine years) of other authors. Searching for new, alternative or other places to complement existing submillimeter telescope locations was attempted.

2. RESULTS OF CALCULATIONS AND ITS COMPARISON WITH EXPERIMENTAL DATA

Figure 1 displays analytically acquired dependencies of monthly average full vertical atmospheric absorption values on the UT time of day for Chajnantor during January and July on the frequency of 225 GHz.

International astronomic submillimeter waverange observatory (ALMA) is located on the height of 5 km above sea level. Nine years cycle atmospheric absorption yearly observation results for the frequency of 225 GHz [1] as well as the results of our calculations are displayed on the same figure.

Figure 2 displays the calculated dependencies of the full vertical atmospheric absorption on the UT time of day for Hanle during January and July on the frequency of 220 GHz. Indian astronomic submillimeter waverange observatory (IAO) is located on the height of 4.5 km above sea level in the Hanle. Atmospheric absorption year cycle observation results for the frequency of 220 GHz taken from [2] are displayed on the same figure.

Figure 3(a) display analytically acquired dependen-cies of monthly average full vertical atmospheric ab-sorption values on the UT time of day for EI Leoncito during January, February and July on the frequency of 405 GHz. EI Leoncito is an Argentinean-Brazilian sub-millimeter wave range radiotelescope located on the height of 2.5 km above sea level. The results of one-year cycle of atmospheric absorption observation for 405 GHz frequency taken from [4] are displayed on the Fig-ure 3(b).

Figure 4(a) display analytically acquired dependen-cies of monthly average full vertical atmospheric ab-sorption values on the UT time of day for South Pole during January and July on the frequency of 225 GHz. American Antarctic remote observatory with a sub-mil-limeter wave range radiotelescope (AST-RO) was lo-cated in the South Pole on the height of 2.8 km above sea level. The results of half-year cycle of atmospheric absorption observation for the 225 GHz frequency taken from [3] are displayed on the Figure 4(b).

In the Figures 1-4 the rather close layout of data cal-culated by us and points taken from the literary data [1-5] is well visible for radio telescopes in El Leoncito Argen-tina (405 GHz), on South Pole (220 GHz), in Hanle (220 GHz), in Chajnantor (225 GHz).

The differences of calculation data and data of ex-periments, as a rule, are concentrated in the interval of

(a)

(b)

Figure 1. Calculated dependencies of the daily changing of the monthly average values of full verti-cal atmospheric absorption (a) as well as the results of our calculations (bold curve) and experimental observations [1] (square) of monthly averaged values of vertical atmospheric absorption (b) for the Cha-jnantor (225 GHz).



429429


(a)

(b)

Figure 2. Calculated dependencies of daily changing of the monthly average values of full vertical atmospheric absorption (a) as well as the results of our calculations (bold curve) and experimental observations [2] (squares) of monthly averaged values of vertical atmospheric absorption (b) for the Hanle on the frequency of 220 GHz.

10-20% in the winter and 20-30% in the summer for the year courses of monthly average values of vertical ab-sorption of atmosphere on all of selected by us radiotelescopes. (The separate summer month only takes place an exception in Chajnantor and in Hanle, when these differences are noticeably higher.) Our calculations of diurnal variability have completely qualitatively coin-ciding with experiment and differed quantitatively from experiments less than on 15-20%.

Let’s remark that it would be possible to expect a de-creasing all these 10-30% values of differences in a case of more correct comparison of calculated and experi-mental data. The absence of complete correctness in realization of such comparisons is caused by different peri-ods of averaging for different literary experimental data

and data of our calculation during definition of monthly vertical absorption values of atmosphere. The values of this parameter calculated by us were based on the data of fifteen-years meteorological observations, while the ex-perimental values reduced in the literature were obtained by averaging from radiometer data for one of separated years only.

Influence of above-mentioned cause is well visible, for example, on dispersion of experimental values of monthly average absorption defined for different years in the Chajnantor (Figure 1) or in the Hanle [2]. The small differences (less than 15%) between data of our calculations (averaged for fifteen-years term) and ex-perimental average-year values of absorption in Mauna Kea [5] (averaged for eleven-years term) as well as be-tween experimental data for Sierra Negra [13] (averaged for four-years term) shows a validity of such point of view.

Besides it is necessary to take into account that the experimental measurements were carried out not always in the conditions of completely clear atmosphere. But nevertheless a carried out qualitative and quantitative (though upper estimations) rating of errors of considered

(a)

(b)

term of observations: 28 February-1 March

Figure 3. Calculated dependencies of daily changing of the monthly average values of full vertical atmospheric absorp-tion (a) as well as the results of our calculations of monthly averaged (gray curve) and daily experimental observations (squares) values of vertical atmospheric absorption [4] (b) for EI leoncito observatory on 405 GHz.



430


(a)

(b)

Figure 4. Calculated dependencies of daily changing of the monthly average values of full vertical atmos-pheric absorption (a) as well as the results of our cal-culations of monthly averaged (gray curve) and daily experimental observations (squares) values of vertical atmospheric absorption [3] (b) for South Pole on the frequency of 220 GHz.

by us method of astroclimatic forecasting is visual and useful enough (Figures 1-4).

3. SEARCH FOR ALTERNATIVE AND ADDITIONAL RADIOTELESCOPE LOCATIONS

The results that were acquired in the previous section demonstrated an efficiency, high operability and func-tional capabilities of calculational approach to astrocly-matic assessment. At the same time, analysis of the most successful locations of operational sub-millimeter wave range radiotelescopes from the astroclymatic point of view [6] allows us to note that out of four radiotelescope locations two are situated in the southern hemisphere (one in the Andes and one in the Antarctic) and two in the tropics of the northern hemisphere (in the Hawaii and in the Indian part of Chanthang mountain plateau).

And the most notable locations of southern hemisphere are distinguished by being more seasonally stable and by a smaller optical thickness value than in the northern hemisphere. According to our calculation (Figure 5) we can note that the best location in the astroclymatic point of view would be southern pole where monthly-average atmospheric absorption values () are two-three times less than, for example, for the Mauna Kea or in the summer months for the Chajnantor.

Seasonal vertical atmospheric absorption variability is characteristic for every sub-millimeter wave range ra-diotelescope location, but in different degrees. In con-nection with that, the best radio astronomical observa-tions from November to April can be made in the South Pole, Hanle and Mauna Kea, while from May to October they can be made on the South Pole, Chajnnator and Mauna Kea (Figure 5).

Mentioned alternative locations or complementary locations (Russian Altay, Rocky Mountains in Colorado, New Earth in Russia, Greenland, Chines Gobi desert, Tibetan Chanthang plateau, etc.) were chosen by us for consideration on the common physical grounds (altitude above sea level, latitude, climate peculiarities). The comparison of result calculations that were carried out for these new places allowed us to distinguish only three astroclymatically suitable locations to compete with best known locations.

These are: Greenland (H = 2,2 km, 80 N, 40 W), Chi-nese part of Chanthang plateau (H = 5 km, 33 N, 94 E) and Altay (H = 3 km, 50 N, 88 E) (Figure 6). For exam-ple, first two locations are not inferior to and even better than the South Pole and the Hanle from November to April.

It is noticeable that their monthly-average atmospheric absorption value and seasonal variability is almost iden-tical (Figure 5 and Figure 6) between the one in the Chajnantor (ALMA) and found location on the Green- land (if corresponding seasons are compared for north-ern and southern hemispheres). Moreover, calculations

Figure 5. Annual changing for the most known locations of radiotelescopes of sub-millimetre waves.



431431

operated radiotelescopes).

REFERENCES [1] Simon, J.E. (2003) Radford (NRAO), Conditions for

observing with the ALMA at Chajnantor. http://www. tuc. nrao.edu/alma/site

[2] Ananthasubramanian, P.G., Yamamoto, S., Prabhu, T.P. and Angchuk, D. (2004) Measurements of 220 GHz at-mospheric transparency at IAO, Hanle, during 2000- 2003. Bulletin of Astronomy Society in India, 32(2), 99- 111.

[3] Richard, A., Chamberlin and Bally, J. (1994) 225-GHz atmospheric opacity of the South Pole sky derived from continual radiometric measurements of the sky-bright-ness temperature. Applied Optics, 33(6), 1095-1099.

Figure 6. Annual changing for the new found locations which are suitable for radiotelescopes of sub-millimetre waves. [4] Melo, A., Kaufmann, P., de Castro, C., Raulin, J., Levato,

H., Marun, A., Giuliani, J. and Pereyra, P. (2005) Sub- millimetre-wave atmospheric transmission at El Leoncito, Argentina Andes. IEEE Transactions, AP-53(4), 1528- 1534.

show that optical atmospheric thickness daily deviation is almost absent in the Greenland during all year while this value reaches up to 50% in summer in the Chajnan-tor (Figure 1(a)).

Comparing data on Figure 5 and Figure 6 allows us to note noticeable (almost twice) astroclymatic condi-tions increase while shifting on the Chanthang plateau from Hanle (India, H = 4.5 km, 32 N, 78 E) to Tibetan location (H = 5 km, 33 N, 94 E) which is situated on the same plateau, but on the Chinese part of it. The same situation is observed while shifting from El Leoncito (Argentine) in the Atakama to the Chajnantor in Chile. However, this case is also affected by the altitude dif-ference of these locations.

[5] Masson, C. (1990) Atmospheric opacity and water vapor. Sub-Millimetre Array Technical Memorandum, 12(1), 10-14.

[6] http://en.wikipedia.org/wiki/Submillimetre_astronomy

[7] Mihailov, A.S. and Ruzhentsev, N.V. (2007) Features of global allocation of atmospheric attenuation in the range 10-1000 GHz. Radiophysics and Radio Astronomy, in Russian, 12(1), 76-83.

[8] Mihailov, A.S. and Ruzhentsev, N.V. (2009) Research of spatial distribution of atmospheric attenuation for terri-tory of Ukraine at millimetre-waves band. Applied Ra-dioelectronics, in Russian, 2(1), 12-22.

[9] Ruzhentsev, N.V., Mihailov, A.S. and Shirin, A.M. (2007) Investigations of season-diurnal dependencies of atmos-pheric absorption with usage of model ERA-15 and its additional testing. Proceedings of Eeleventh URSI Com-mission F Open Symposium on Radio Waves Propagation and Remote Sensing, Rio de Janeiro, RS3.3-1-RS3.3-5.

4. CONCLUSIONS

Thus, for the first time is shown that usage most of modern standard of atmosphere (designed by ESA on the basis of the database ERA-15) in aggregate with МРМ model of atmospheric attenuation allows to obtain a seasonal-diurnal statistics of vertical atmospheric ab-sorption for any item of a world. Such possibility of the calculated method was shown as by comparison of the original data of calculation with the experimental data of other authors, as and by definition new astroclimatically favourable locations of sub-millimetre radiotelescopes. This new and previously unavailable ability to obtain astroclymatic assessments of ensures high operability, functional abilities increase and minimal expenditures in comparison with the traditional approach that is based on long-term experimental observations (which usually uses to choose the location of the projected radiotelescope or to specify astroclymatic assessment in the locations of

[10] Liebe, H.J. (1989) MPM-An atmosphere millimeter wave propagation model. International Journal on Infrared and Millimeter Waves, 10(6), 631-650.

[11] Martellucci, A., Rastburg, B.A., Poiares Baptista, J.P.V. and Blarzino, G. (2003) New reference standard atmos-pheres based on numerical weather products. Abstracts of International Workshop-ClimDiff’2003, Fortaleza, clim.1.

[12] Riva, C., Martellucci, A., Kubista, E., Chonhuber, M. and Luini, L. (2005) ERA-15 climatological databases for propagation modeling. Proceedings of International Con- ference-ClimDiff, Cleveland, 26-27 September 2005, clim. 12.1-12.7.

[13] The Large Millimeter Telescope (LMT) site: http:www. lmtgtm.org/site.html


http://www.tuc.nrao.edu/alma/site

http://www.tuc.nrao.edu/alma/site

http://en.wikipedia.org/wiki/Submillimetre_astronomy

Vol.2, No.5, 432-443 (2010)doi: 10.4236/ns.2010.25053


Natural Science

Kinetic spectrophotometric determination of certain cephalosporins using iodate/iodide mixture

Salwa R. El-Shaboury, Fardous A. Mohamed, Gamal A. Saleh, Azza H. Rageh*

Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt; [email protected];

[email protected]; [email protected]; *Corresponding Author: [email protected]

Received 10 December 2009; revised 28 January 2010; accepted 8 March 2010.

ABSTRACT

A simple, precise and accurate kinetic spectro-photometric method for determination of ce-fradine anhydrous, cefaclor monohydrate, ce-fadroxil monohydrate, cefalexin anhydrous and cefixime in bulk and in pharmaceutical formula-tions has been developed. The method based on a kinetic investigation of the reaction of the free carboxylic acid group of the drug with a mixture of potassium iodate and potassium io-dide at room temperature to form yellow col-oured triiodide ions. The reaction was followed up spectrophotometrically by measuring the increase in absorbance at 352 nm as a function of time. The initial rate, fixed time, variable time and rate-constant methods were adopted for constructing the calibration curves but fixed time method has been found to be more appli-cable. The analytical performance of the method, in terms of accuracy and precision, was statis-tically validated; the results were satisfactory. The method has been successfully applied to the determination of the studied drugs in com-mercial pharmaceutical formulations. Statistical comparison of the results with a well estab-lished reported method showed excellent agreement and proved that there is no significant difference in the accuracy and precision.

Keywords: Cephalosporins; Kinetic Spectrophotometry; Lodate/Lodide Mixture; Pharmaceutical Analysis

1. INTRODUCTION

Because cephalosporins are among the safest and the most effective broad-spectrum bactericidal antimicrobial agents available to the clinician, they have become the most widely prescribed of all antibiotics. All of these semi-synthetic antibiotics are derived from 7-amino-ce-

phalosporanic acid and contain a β-lactam ring fused to a dihydrothiazine ring (Table 1) but differ in the nature of the substituents attached at the 3 and/or 7-positions of the cephem ring. These substitutions affect either the pharmacokinetic properties (3-position) or the antibacte-rial spectrum (7-position) of the cephalosporins. Cepha-losporins operate by inhibiting bacterial cell wall bio-synthesis which grows actively against a wide range of both gram-positive and gram-negative bacteria. The positive results of these drugs include the resistance of penicillinases and ability to treat infections that are re-sistant to penicillin derivatives. The official methods for analyzing cephalosporins are mostly chromatographic methods [1] which are expensive. Most of the reported methods involve the cleavage of the β-lactam moiety of the cephalosporin structure. These methods include spectrophotometric [2-6] spectrofluorimetric [7-10]. and electrochemical methods [11-13]. A direct chemical analysis based on the reactivity of the intact molecule is not frequently encountered.

Kinetic spectrophotometric methods are becoming of great interest in chemical and pharmaceutical analysis [14]. The application of these methods offered some specific advantages [15,16].

1) Simplicity owing to elimination of some experi-mental steps such as filtration and extraction prior to absorbance measurements.

2) High selectivity due to the measurement of the in-crease or decrease of the absorbance as a function of reaction time instead of measuring the concrete absorb-ance value.

3) Avoiding the interference of the coloured and/or turbidity background of the samples, and possibility of avoiding the interference of the other active compounds present in the commercial product if they are resisting the established reaction conditions.

The literatures are still lacking analytical procedures based on kinetics for determination of the investigated drugs in commercial dosage forms. A kinetic spectro-photometric method has been reported for determination of cefadroxil based on its alkaline hydrolysis [17]. With



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the exception of cefadroxil, this part represents the first attempt for assaying the investigated drugs without deg-radation in pure forms and in different pharmaceutical dosage forms using kinetic spectrophotometric method. The literature reveals a kinetic spectrophotometric method for determination of ramipril [18] that based on the reaction of its carboxylic acid group with iodate/iodide mixture in aqueous medium at room temperature to form yellow coloured triiodide ions. The reaction was fol-lowed up spectrophotometrically by measuring the in-crease in absorbance at 352 nm as a function of time.

This reaction drew our attention to investigate it on our studied drugs that contain free carboxylic acid group (Table 1). Accordingly, this reaction was studied in order to find out if it would lend itself applicable to the analysis of cefradine anhydrous, cefaclor monohydrate, cefadroxil monohydrate, cefalexin anhydrous and cefixime in pure forms and in pharmaceutical for-mulations. As a result of these investigations; a simple, rapid and accurate kinetic spectrophotometric method for determination of the aforementioned cephalosporin drugs without degradation was devised. The fixed time method is adopted after full investigation and under-standing of the kinetics of the reaction. The proposed method does not require the elaboration of treatment and procedures, which are usually associated with chroma

tographic methods.

2. EXPERIMENTAL

2.1. Apparatus

Shimadzu UV-1700 PC, UV-Visible Spectrophotometer (Tokyo, Japan), Ultrasonic cleaner (Cole – Parmer, Chi-cago, USA) and Sartorious handy balance – H51 (Han-nover, Germany).

2.2. Materials and Reagents

All solvents used were of analytical-reagent grade, po-tassium iodide (El-Nasr Chemical Co. Cairo, Egypt) freshly prepared aqueous solution (1.5 M), potassium iodate (El-Nasr Chemical Co. Cairo, Egypt) freshly pre-pared aqueous solution (0.3 M), cefaclor monohydrate and cefradine anhydrous (Sigma Chemical Co., St. Louis, USA) cefadroxil monohydrate (Amoun Pharmaceutical Industries Co., APIC, Cairo, Egypt), cefalexin anhydrous (GalaxoWellcome, S.A.E., El Salam City, Cairo, Egypt) and cefixime (El-Hekma Co., Cairo, Egypt) were ob-tained as gifts and were used as supplied and pharma-ceutical formulations containing the studied drugs were purchased from local market.

Table 1. Chemical structures of the investigated cephalosporin antibiotics.

S

NR2

CO OR3

O

NC

O

R1

H

1 2

34

678 5

No. Name R1 R2 Generation

1. Cefalexin anhydrous HC

NH2

-CH3 First

2. Cefradine anhydrous HC

NH2

-CH3 First

3. Cefadroxil

monohydrate

HC

NH2

HO

-CH3 First

4. Cefaclor

monohydrate

HC

NH2

-Cl Second

5. Cefixime

S

N

H2N C

NOCH2CO2H

CH

CH2

Third



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2.3. Preparation of Standard Solutions

Stock solutions containing 1 mg mL-1 of each cepha-losporin namely, cefradine anhydrous, cefadroxil mono-hydrate, cefaclor monohydrate, cefalexin anhydrous and cefixime were prepared in methanol. Working standard solutions containing 0.1-0.5 mg mL-1 (in case of ce-fixime, working standard solutions containing 0.05-0.25 mg mL-1) were prepared by suitable dilution of the stock solution with methanol. The stock and working standard solutions must be freshly prepared.

2.4. Preparation of Sample Solutions

2.4.1. Tablets and Capsules Twenty tablets or the contents of 20 capsules were weighed, finely powdered and mixed thoroughly. An accurately weighed amount of the powder obtained from tablets or capsules equivalent to 250 mg of each drug was transferred into a 50-mL volumetric flask, dissolved in about 25 mL methanol, sonicated for 15 min, diluted to the mark with methanol, mixed well and filtered; the first portion of the filtrate was rejected. Further dilutions with methanol were made to obtain sample solution containing 0.3 mg mL-1 (in case of cefixime, further di-lutions with methanol were made to obtain sample solu-tion containing 0.15 mg mL-1) and then the general pro-cedure was followed.

2.4.2. Powder for Oral Suspension An accurately weighed amount of powder equivalent to 250 mg of each drug was transferred into a 50 mL volu-metric flask, then the procedure was followed as under tablets and capsules beginning from (dissolved in about 25 mL methanol).

2.3. General Procedure

Accurately measured one millilitre aliquot volume of the standard or sample solutions was transferred into 10- mL volumetric flask. One millilitre of 0.3 M of potassium iodate was added followed by 1 mL of 1.5 M of potas-sium iodide. The content of the flask was mixed well and diluted to volume with methanol. The increase in absorbance was measured at 352 nm against reagent blank treated similarly. The four kinetic methods namely, initial rate, fixed time, variable time and rate constant methods were used for construction of the calibration curves and determination of the studied drugs.

3. RESULTS AND DISCUSSION

3.1. Absorption Spectra

Absorption spectrum of cefradine anhydrous which was taken as a representative example for all studied drugs is shown in Figure 1. This spectrum shows no absorption at 352 nm whereas the absorbance of the reagent solu-tion (KIO3 and KI in methanol) at 352 nm is about 0.02. The wavelengths of maximum absorption of the interac-

tion coloured product of cefradine anhydrous with KIO3 and KI are at 298 and 352 nm. It is obvious that at 298 nm there is background absorption from the drug itself and from the reagent blank (Figure 1). Therefore, the absorbance measurements for the determination of the studied drugs were made at 352 nm. The equilibrium is attained in ~30 minutes. Therefore, a kinetically based spectrophotometric method was developed for the quan-titative determination of the investigated drugs by meas-uring the increase in absorbance at 352 nm as a function of time.

3.2. Optimization of Reaction Conditions

The experimental parameters affecting the reaction be-tween the investigated drugs, potassium iodate and po-tassium iodide were carefully studied and optimized. Cefardine anhydrous (30 μg mL-1) was taken as a repre-sentative example for this study. These factors include:

3.2.1. Effect of Potassium Iodate Concentration The concentration of potassium iodate, for the maximum colour development at 352 nm, was studied in the range of 0.05-0.6 M. From Figure 2, it was found that the

Figure 1. Absorption spectra of (a) cefradine anhydrous (30 μg mL-1); (b) reagent solution (0.3 M potassium iodate and 1.5M potassium iodide) and (c) the interaction coloured product of cefradine anhydrous with potassium iodate and potassium iodide.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Potassium iodate concentration (M)

Ab

so

rba

nc

e, 3

52

nm

Figure 2. Effect of potassium iodate concentration on the absorbance of the reaction coloured product at 352 nm.



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absorbance of the interaction coloured product is in-creased with increasing potassium iodate concentration. Maximum absorbance was attained by using 0.25 M; above this concentration and up to 0.6 M KIO3, the ab-sorbance remains constant. Therefore, 1 mL of 0.3 M potassium iodate was selected during subsequent work.

3.2.2. Effect of Potassium Lodide Concentration The influence of potassium iodide concentration on producing the maximum absorption intensity was inves-tigated using 0.3-2.4 M potassium iodide. Maximum absorption readings were obtained upon using 1 mL of 1.3 M potassium iodide; above this concentration the absorbance remains constant. So, 1 mL of 1.5M of KI was used for further work (Figure 3).

3.2.3. Effect of Diluting Solvent Different solvents were tested in order to select the most appropriate solvent for producing the maximum absorp-tion intensity. The results given in Table 2 show the slight effect on λmax while the absorption intensity was affected. Methanol was used throughout this work be-cause it gave the highest absorbance readings and the most reproducible results.

3.2.4. Effect of Temperature As expected from the Arrhenius equation [19], the reac-tion rate is increased with increasing temperature. So, trials have been done to carry out the reaction at higher temperatures. It was found that the studied drugs un-dergo degradation and iodine is unstable at higher tem-peratures [20]. Therefore, room temperature (25 ± 5) was recommended as the optimum temperature for this study.

3.2.5. Quantitation Methods The initial rate, fixed time, variable time and rate con-stant methods [21,22] were tested and the most suitable analytical approach was chosen regarding the applicability, sensitivity, the values of the intercept and correlation coef-ficient (r).

3.2.6. Initial Rate Method Under the optimum experimental conditions, the assay of cefradine anhydrous, cefadroxil monohydrate, cefa-clor monohydrate, cefalexin anhydrous and cefixime was performed at different concentration levels for 17 min at intervals of 2 min starting from 1 min at room temperature (25 ± 5). The absorbance at 352 nm was then recorded at each time interval. The assay was car-ried out in presence of excess concentration of potassium iodate and potassium iodide. Therefore, a pseudo-zero order reaction condition was worked out with respect to the concentration of the reagent.

The kinetic plots are all sigmoid in nature and the ini-tial rate of reaction was obtained by measuring the slopes (ΔA/Δt) of the initial tangent to the absorb-ance-time curves at different concentrations of the inves-

tigated drugs. Figure 4 shows the kinetic plot for ce-fradine anhydrous as a representative example.

The initial rate of reaction would follow a pseudo-first order and obeyed the following rate equation:

nCkt

Av '

(1)

whereas ν is the reaction rate, A is the absorbance, t is the measuring time, k' is the pseudo-first order rate con-stant, C is the concentration of the drug and n is the or-der of the reaction. The logarithmic form of the above equation is written as follows:

Cn'kt

Av loglogloglog

(2)

A calibration curve was constructed by plotting the logarithm of the initial rate of reaction (log v) versus logarithm of initial concentration of the investigated drugs (log C), which showed a linear relationship over concentration range of 2.59 × 10-5 - 1.44 × 10-4 M for cefadroxil monohydrate, cefaclor monohydrate, ce-falexin anhydrous and cefradine anhydrous (in case of cefixime, 1.10 × 10-5 - 5.51 × 10-5 M). The regression equations of log rate versus log C are given in Table 3.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.

potassium iodide concentration (M)

Ab

so

rba

nc

e, 3

52

nm

7

Figure 3. Effect of potassium iodide concentration on the absorbance of the reaction coloured product at 352 nm.

Figure 4. Absorbance-time curve for the reaction of cefradine anhydrous (μg mL-1) with potassium iodate and potassium iodide.


Copyright © 2010 SciRes http://www.scirp.org/journal/NS/Openly accessible at

436

Table 2. Effect of solvent on λmax and the absorption intensity of the reaction coloured product of the studied drugs with KIO3 and KI.

Drug Solvent

Cefradine anhydrous (30 μg mL-1)

Cefadroxil mono-hydrate (30 μg mL-1)

Cefaclor monohy-drate (30 μg mL-1)

Cefalexin anhy-drous (30 μg mL-1)

Cefixime (15 μg mL-1)

λmax (nm) Aa λmax (nm) Aa λmax (nm) Aa λmax (nm) Aa λmax (nm) Aa Water 346 0.420 346 0.394 346 0.308 347 0.331 348 0.318

Ethanol 357 0.520 358 0.410 359 0.382 358 0.410 358 0.394 Methanol 352 0.544 352 0.510 352 0.400 352 0.429 352 0.413 Acetone 359 0.400 360 0.385 359 0.294 360 0.315 360 0.303

Acetonitrile 352 0.410 356 0.390 351 0.312 351 0.322 353 0.306 Propan-1-ol 358 0.390 359 0.375 360 0.296 358 0.306 357 0.280 Propan-2-ol 361 0.434 361 0.420 361 0.327 361 0.333 360 0.300

DMF 351 0.380 355 0.360 354 0.286 351 0.290 352 0.260 DMSO 350 0.375 349 0.370 350 0.282 350 0.287 350 0.260

a Average of 3 determinations.

Table 3. Relation between reaction rates and concentrations.

log ΔA/Δt log [Drug] (M) Calibration equation log ν = log k' + n log C Correlation coefficient (r) Cefradine anhydrous

-1.577 -4.543 log ν = 2.729 + 0.956 log C 0.9867 -1.377 -4.240 -1.164 -4.066 -1.066 -3.941 -0.893 -3.844

Cefadroxil monohydrate -1.577 -4.581 log ν = 2.765 + 0.956 log C 0.9868 -1.377 -4.280 -1.164 -4.104 -1.066 -3.979 -0.893 -3.882

Cefaclor monohydrate -1.699 -4.586 log ν = 3.441 + 1.122 log C 0.9971 -1.377 -4.285 -1.164 -4.109 -1.066 -3.984 -0.893 -3.887

Cefalexin anhydrous -1.553 -4.541 log ν = 2.976 + 1.002 log C 0.9828 -1.268 -4.240 -1.155 -4.064 -1.011 -3.939 -0.801 -3.842

Cefixime -1.523 -4.958 log ν = 4.011 + 1.126 log C 0.9856 -1.314 -4.656 -1.039 -4.480 -0.905 -4.355 -0.738 -4.259

The correlation coefficients (r) of all studied drugs ranged from 0.9828 to 0.9971. The order (n) with respect to the studied drugs was evaluated by plotting the logarithm of the initial rate of reaction versus logarithm of the concentrations of the investigated drugs and was found to be approximately one which confirms the first-order reac-tion with respect to all investigated drug concentrations.

3.2.7. Fixed Time Method In this method, the absorbance changes caused by effect of drug acidity on a mixture of potassium iodate and potassium iodide were recorded at a preselected fixed time at intervals of 2 min. The change in absorbance (ΔA) between the times t1 (1 min) and t2 (3, 5, 7, 9, 11,

13, 15 and 17) was computed and plotted against the concentration of each of the studied drugs. The corre-sponding linear regression equations with correlation coefficients are summarised in Table 4. It is evident from the table that the most acceptable linearity was obtained when the calibration graphs were plotted by considering the change in absorbance between 1 and 11 min (i.e. ΔA= A11-A1). It is also clear that the slope increases with time and the most acceptable values of r and the inter-cept were obtained for a fixed time of 10 min, which was therefore chosen as the most suitable time interval for the measurement. The calibration curve was linear in the range of 10 to 50 µg mL-1 for cefadroxil



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Table 4. Calibration equations for the studied drugs of different concentrations at different time intervals using fixed time method.

Δt (min) Calibration equation ΔA = a + b C Correlation coefficient (r) Cefradine anhydrous

2 ΔA = -0.008 + 0.005 C 0.9842 4 ΔA = 0.033 + 0.008 C 0.9989 6 ΔA = 0.063 + 0.010 C 0.9991 8 ΔA = 0.871 + 0.012 C 0.9994 10 ΔA = 0.104 + 0.015 C 0.9997 12 ΔA = 0.140 + 0.016 C 0.9988 14 ΔA = 0.163 + 0.016 C 0.9980 16 ΔA = 0.196 + 0.017 C 0.9963

Cefadroxil monohydrate

2 ΔA = -0.008 + 0.005 C 0.9842 4 ΔA = 0.009 + 0.009 C 0.9975 6 ΔA = 0.026 + 0.011 C 0.9978 8 ΔA = 0.044 + 0.013 C 0.9991 10 ΔA = 0.036 + 0.016 C 0.9997 12 ΔA = 0.048 + 0.017 C 0.9995 14 ΔA = 0.062 + 0.018 C 0.9981 16 ΔA = 0.089 + 0.019 C 0.9963

Cefaclor monohydrate

2 ΔA = -0.018 + 0.005 C 0.9898 4 ΔA = -0.019 + 0.009 C 0.9955 6 ΔA = -0.017 + 0.011 C 0.9967 8 ΔA = 0.001 + 0.012 C 0.9985 10 ΔA = 0.012 + 0.013 C 0.9996 12 ΔA = 0.006 + 0.015 C 0.9984 14 ΔA = 0.009 + 0.016 C 0.9989 16 ΔA = 0.016 + 0.016 C 0.9983

Cefalexin anhydrous

2 ΔA = -0.019 + 0.006 C 0.9666 4 ΔA = -0.074 + 0.012 C 0.9852 6 ΔA = -0.048 + 0.013 C 0.9928 8 ΔA = -0.031 + 0.014 C 0.9954 10 ΔA = -0.012 + 0.015 C 0.9991 12 ΔA = -0.018 + 0.017 C 0.9987 14 ΔA = -0.014 + 0.017 C 0.9989 16 ΔA = -0.005 + 0.018 C 0.9971

Cefixime 2 ΔA = -0.038 + 0.015 C 0.09859 4 ΔA = -0.003 + 0.021 C 0.09994 6 ΔA = 0.008 + 0.024 C 0.9988 8 ΔA = 0.005 + 0.026 C 0.9982 10 ΔA = 0.017 + 0.027 C 0.9994 12 ΔA = 0.011 + 0.029 C 0.9992 14 ΔA = 0.021 + 0.031C 0.9987 16 ΔA = 0.020 + 0.033 C 0.9974

monohydrate, cefaclor monohydrate, cefalexin anhy-drous and cefradine anhydrous (in case of cefixime, 5-25 µg mL-1). The correlation coefficients (r) of all studied drugs ranged from 0.9991 to 0.9997. Reasonable values of LOD and LOQ were obtained which ranged from 0.22 to 1.10 and from 0.67 to 3.33 µg mL-1; respectively as indicated in Table 5.

3.2.8. Variable Time Method The general procedure was followed up for each of the studied drugs at different concentration levels by re-cording the time in seconds required for the absorbance to reach 0.20. This preselected value of the absorbance

was chosen as it gives the widest calibration range. The reciprocal of time (1/Δt) versus the initial concentration of the studied drugs was plotted and the equations of the calibration graphs are given in Table 6. The correlation coefficients (r) of all studied drugs ranged from 0.9646 to 0.9873.

3.2.9. Rate Constant Method Under the described experimental conditions, analysis was carried out for each of the studied drugs at different concentration levels starting from 1 min until 17 min at regular intervals of 2 min at room temperature (25 ± 5). Graphs of log absorbance change at 352 nm versus



438

Table 5. Summary of quantitative parameters and statistical data using fixed time method.

Drug Intercept (a) ±

SDa Slope (b) ±

SDa Linearity range

(μg mL-1 ) Correlation

coefficient (r)Determination coefficient (r2)

LOD (µg mL-1)

LOQ (µg mL-1)

Cefradine anhydrous

0.104 ± 0.005 0.015 ± 0.002 10-50 0.9997 0.9994 1.10 3.33


0.036 ± 0.002 0.016 ± 0.002 10-50 0.9997 0.9994 0.41 1.25


0.012 ± 0.001 0.013 ± 0.001 10-50 0.9996 0.9992 0.25 0.80

Cefalexin anhydrous

-0.012 ± 0.001 0.015 ± 0.001 10-50 0.9991 0.9982 0.22 0.67

Cefixime 0.017 ± 0.002 0.027 ± 0.003 5-25 0.9994 0.9988 0.24 0.74 a Average of six determinations.

Table 6. Calibration equations and correlation coefficients using variable time method.

Δt (min) 1/ Δt (s-1) [Drug] (M) Calibration equation1/Δt = a + b C Correlation coefficient (r) Cefradine anhydrous

7.5 2.22 × 10-3 2.86 × 10-5 1/Δt = -0.001 + 73.720 C 0.9646 5 3.33 × 10-3 5.73 × 10-5 3 5.56 × 10-3 8.59 × 10-5

2.5 6.67 × 10-3 1.15 × 10-4 1.5 11.11 × 10-3 1.43 × 10-4

Cefadro × il monohydrate

11 1.52 × 10-3 2.62 × 10-5 1/Δt = -0.001 + 85.911 C 0.9754 5 3.33 × 10-3 5.24 × 10-5 3 5.56 × 10-3 7.87 × 10-5

2.5 6.67 × 10-3 1.05 × 10-4 1.5 11.11 × 10-3 1.13 × 10-4


16 1.04 × 10-3 2.80 × 10-5 1/Δt = -0.002 + 94.370 C 0.9793 6 2.78 × 10-3 5.18 × 10-5

3.5 4.76 × 10-3 7.78 × 10-5 2.5 6.67 × 10-3 1.04 × 10-4 1.5 11.11 × 10-3 1.30 × 10-4

Cefale × in anhydrous

16 1.04 × 10-3 2.88 × 10-5 1/Δt = -0.003 + 114.352C 0.9724 6 2.78 × 10-3 5.76 × 10-5

3.5 4.76 × 10-3 8.64 × 10-5 1.5 11.11 × 10-3 1.15 × 10-4

1.25 13.33 × 10-3 14.40 × 10-4

Cefi × ime 16 1.04 × 10-3 1.10 × 10-5 1/Δt = -0.003 + 346.347C 0.9873 6 4.17 × 10-3 2.21 × 10-5

2.5 6.67 × 10-3 3.31 × 10-5 1.5 11.11 × 10-3 4.41 × 10-5 1 16.67 × 10-3 5.51 × 10-5

time in seconds for each of the studied drugs were con-structed. Pseudo first-order rate constants (k') corre-sponding to different investigated drugs concentrations (C) were calculated from the slopes, multiplied by -2.303. Pseudo first-order rate constant (k') versus the initial concentration of the studied drugs was then plot-ted and the equations of the calibration graphs are given in Table 7. The correlation coefficients (r) for all the studied drugs ranged from 0.8742 to 0.9290. These low values of r may be due to slight changes in temperature.

3.3. Method Validation Study

Fixed time method was chosen to carry out the valida-

tion study as it gives the highest values of correlation coefficients. The proposed method was validated ac-cording to ICH (International Conference on Harmoni-zation) guidelines on the validation of analytical meth-ods [23] and complied with USP 31 validation guidelines [1]. All results were expressed as percentages, where n represents the number of values. For the statistical analysis Excel 2003 (Microsoft Office) was used. A 5% significance level was selected.

3.3.1. Accuracy The accuracy of the method was determined by investi-gating the recovery of each of the studied drugs at three



439439

concentration levels covering the specified calibration range (six replicates of each concentration). The results shown in Table 8 depict good accuracy and recovery percentage ranged from 98.0 to 101.9%.

3.3.2. Precision As indicated in Table 9, the results of SD and % RSD can be considered to be very satisfactory which prove the precision of the proposed method.

3.3.3. Selectivity The selectivity of the proposed method for determination of the studied drugs in the presence of frequently en-

countered excipients such as; starch, talc, lactose, glu-cose, sucrose, magnesium-stearate and gum acacia was studied. It was found that there is no interference from these excipients and additives. So, the proposed method can be considered a selective one.

3.3.4. Robustness Robustness was examined by evaluating the influence of small variation of method variables including; potassium iodate concentration, potassium iodide concentration, measurement time on the method suitability and sensi-tivity. It was found that none of these variables signifi-cantly affected the performance of the method (Table 10).

Table 7. Values of k' calculated from slopes of log A versus t graphs multiplied by -2.303 for different concentrations of the studied drugs.

k' (s-1) [Drug] (M) Calibration equation k' = a + b C Correlation coefficient (r) Cefradine anhydrous

-1.79 × 10-3 2.86 × 10-5 k' = -0.002 + 1.942 C 0.9256 -1.77 × 10-3 5.73 × 10-5 -1.63 × 10-3 8.59 × 10-5 -1.67 × 10-3 1.15 × 10-4 -1.57 × 10-3 1.43 × 10-4

Cefadro × il monohydrate

-1.85 × 10-3 5.24 × 10-5 k' = -0.002 + 4.112 C 0.9290 -1.71 × 10-3 7.87 × 10-5 -1.72 × 10-3 9.20 × 10-5 -1.72 × 10-3 1.05 × 10-4 -1.49 × 10-3 1.31 × 10-4


-1.61 × 10-3 2.59 × 10-5 k' = -0.002 + 3.203 C 0.8731 -1.50 × 10-3 5.18 × 10-5 -1.32 × 10-3 7.78 × 10-5 -1.43 × 10-3 1.04 × 10-4 -1.23 × 10-3 1.30 × 10-4

Cefale × in anhydrous

-1.43 × 10-3 2.88 × 10-5 k' = -0.002 + 5.458 C 0.8742 -1.26 × 10-3 5.76 × 10-5 -1.41 × 10-3 8.64 × 10-5 -0.90 × 10-3 1.15 × 10-4 -0.83 × 10-3 1.44 × 10-4

Cefi × ime -1.17 × 10-3 1.10 × 10-5 k' = -0.001 + 7.215 C 0.9147 -1.14 × 10-3 2.21 × 10-5 -0.94 × 10-3 3.31 × 10-5 .00 × 10-3 4.41 × 10-5 0.84 × 10-3 5.51 × 10-5

Table 8. Accuracy of the proposed kinetic spectrophotmetric method for analysis of the studied drugs at three concentration levels.

Recovery (%) ± SDa Drug 20 µg mL-1 30 µg mL-1 40 µg mL-1

Cefradine anhydrous 99.3 ± 0.72 98.0 ± 0.40 101.9 ± 1.00 Cefadroxil monohydrate 100.3 ± 1.13 101.4 ± 1.00 98.7 ± 0.54 Cefaclor monohydrate 101.1 ± 1.14 99.0 ± 0.22 99.7 ± 1.39 Cefalexin anhydrous 98.5 ± 1.16 99.1 ± 1.25 98.6 ± 0.82

Recovery (%) ± SDa 10 µg mL-1 15 µg mL-1 20 µg mL-1

Cefixime 100.3 ± 0.91 101.6 ± 1.43 100.7 ± 0.88 a Average of six replicates.



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3.4. Applications to the Analysis of

Pharmaceutical Dosage Forms

The proposed method (fixed time) was applied success-fully for determination of the studied drugs in their pharmaceutical dosage forms. The results obtained (Ta-ble 11) were satisfactory compared to those given by a previously reported method [24]. Recovery studies were also carried out by standard addition method [25]. Good recoveries (96.3 to 102.8%) were obtained and these values confirmed the absence of interference due to common excipients (Table 12). The proposed method couldn’t be applied to pharmaceutical formulations con-taining L-arginine as it is a basic amino acid (its side chain contains a strongly basic guanidine group, pKa = 13.2 [26]) and so, interferes with iodine liberation from the studied drug.

3.5. Suggested Reaction Mechanism

It has been suggested that water-soluble acidic compounds liberate iodine from a solution containing both KIO3 and

KI according to the reaction [27];

223 3IO3H6H5IIO

Yellowing of the solution reveals the occurrence of the reaction. The yellow colour of the solution is due to the formation of I2, which immediately converted into triio-dide ions (I2 + I− → I3

−) exhibiting absorption maxima at 290 nm and 360 nm [18]. The chemical structure of in-vestigated cephalosporins contains –COOH group in its moiety and hence possibly undergo a similar reaction with iodide-iodate mixture resulting in the production of iodine. The liberated iodine immediately reacts with potassium iodide to give triiodide ions showing absorp-tion maxima at 298 nm and 352 nm. The reaction se-quence is shown in Formula (1).

OH3I6RCOOK5KIKIO6RCOOH 223 (1)

32 KIKII (2)

Formula (1) suggested reaction sequence of the proposed method.

Table 9. Intra- and inter-day precision of the proposed kinetic spectrophotometric method.

Intra-day precision Inter-day precision Drug Drug Conc. (µg mL-1)

Mean ± SDa % RSD Mean ± SDa % RSD 20 98.5 ± 0.90 0.91 99.5 ± 0.81 0.81 30 98.6 ± 1.54 1.57 99.7 ± 1.17 1.17 Cefradine anhydrous 40 99.8 ± 1.02 1.03 99.6 ± 1.48 1.48 20 99.4 ± 0.99 1.00 100.6 ± 1.63 1.62 30 98.9 ± 1.12 1.13 101.0 ± 1.27 1.26 Cefadroxil monohydrate 40 99.7 ± 0.67 0.67 100.8 ± 1.15 1.14 20 101.0 ± 1.27 1.26 100.6 ± 1.63 1.62 30 100.6 ± 1.36 1.35 100.5 ± 1.15 1.14 Cefaclor monohydrate 40 99.8 ± 1.02 1.03 100.9 ± 0.99 0.98 20 100.7 ± 1.12 1.12 99.8 ± 1.65 1.65 30 98.6 ± 0.52 0.53 101.1 ± 1.20 1.19 Cefalexin anhydrous 40 100.0 ± 1.56 1.56 98.6 ± 0.94 0.95 10 100.0 ± 1.15 1.15 100.7 ± 1.12 1.12 15 100.7 ± 0.87 0.87 99.0 ± 0.97 0.98 Cefixime 20 99.9 ± 1.65 1.66 99.8 ± 1.85 1.85

a Average of six determinations.

Table 10. Robustness of the proposed kinetic spectrophotometric method.

Recovery (%) ± SDa

Experimental parameter variation Cefradine anhy-drous (30 μg mL-1)

Cefadroxil monohydrate (30 μg mL-1)

Cefaclor mono-hydrate (30 μg

mL-1)

Cefalexin an-hydrous (30 μg

mL-1)

Cefixime (15 μg mL-1)

No variationb 97.9 ± 1.20 100.5 ± 1.23 101.5 ± 1.32 99.5 ± 0.47 99.4 ± 1.31 1 - Potassiium iodate concentration

0.28M 0.32M

98.0 ± 1.35 97.7 ± 1.65

101.9 ± 0.79 102.4 ± 0.85

99.8 ± 1.37 100.9 ± 0.99

99.4 ± 1.29 98.5 ± 0.90

101.9 ± 1.45 100.8 ± 0.88

2 - Potassium iodide concentration 1.45M 1.55M

101.4 ± 0.99 102.1 ± 1.13

98.7 ± 1.45 97.8 ± 0.64

98.7 ± 1.21 102.3 ± 1.56

99.0 ± 1.36 98.7 ± 1.23

102.1 ± 0.73 100.7 ± 0.92

3 - Measurement time 8 min 12 min

98.7 ± 1.53 101.6 ± 1.45

99.0 ± 1.75 100.0 ± 0.47

98.6 ± 0.84 99.9 ± 1.38

98.6 ± 0.64 97.6 ± 0.88

100.7 ± 0.61 97.8 ± 0.81

a Average of three determinations. b Following the general assay procedure conditions.



441441

Table 11. Determination of the studied drugs in their pharmaceutical dosage forms using fixed time method.

Recovery % ± SD

Drug Pharmaceutical product Proposed method (n = 6) Reported methoda (n = 6)

Ceclor® suspensionc 99.2 ± 0.60 t = 1.225b F = 1.778b

98.7 ± 0.80

Cefaclo monohydrate

Bacticlor® suspensiond 101.2 ± 0.60

t = 1.569 F = 1.440

100.7 ± 0.50

Duricef® tabletse 98.9 ± 0.60 t = 2.038

F = 2.250 99.8 ± 0.90

Duricef® suspensione 102.2 ± 1.60

t = 0.630 F = 1.129

101.6 ± 1.70

Duricef® capsulese 98.9 ± 1.30 t = 0.831 F = 1.173

99.5 ± 1.20

Biodroxil® capsulesf 101.4 ± 1.00

t = 1.275 F = 1.234

100.7 ± 0.90


Biodroxil® suspensionf 99.1 ± 0.90 t = 0.906 F = 2.250

98.7 ± 0.60

Ceporex® tabletsg 97.8 ± 1.10 t = 1.054 F = 1.860

98.6 ± 1.50

Ceporex® suspensiong 100.5 ± 1.20

t = 1.470 F = 1.778

99.6 ± 0.90

Cefalexin anhydrous

Ospexin® suspensionh 99.7 ± 1.50 t = 1.153 F = 3.516

100.5 ± 0.80

Velosef® capsulese 101.2 ± 1.50

t = 0.432b

F = 3.516b 100.9 ± 0.80

Cefradine anhydrous

Velosef® tabletse 99.9 ± 1.20 t = 1.307 F = 1.778

100.7 ± 0.90

Cefixime Ximacef® capsulesi 98.7 ± 0.40 t = 1.644 F = 4.000

99.0 ± 0.20

a Reference 24. b Theoretical value for t and F at 95% confidence limit, t = 2.228 and F = 5.053. c Egyptian Pharmaceuticals and chemicals industries Co., S.A.E., Bayad El-Arab, Beni Suef, Egypt.

d Pharco Pharmaceuticals, Alexandria under license from Ranbaxy UK. e Bristol-Myers Squibb Pharmaceutical Co., Cairo, Egypt. f Kahira Pharm. & Chem. Ind. Co. under license from Novartis Pharma S.A.E., Cairo, Egypt.

g GlaxoSmithKline, S.A.E., El Salam City, Cairo, Egypt. h Pharco Pharmaceuticals, Alexandria under license from Biochemie GmbH., Vienna, Austria. i Sigma pharmaceutical industries, S.A.E., Egypt.

The confirmatory test for the presence of iodine in the

final solution of the drug is established by the blue col-our, which appears on addition of starch solution. In case

of cefixime, it may be suggested that 3 mole of cefixime instead of six react with iodate/ioide mixture as it con-tains 2 carboxylic acid groups.



442


Table 12. Standard addition method for the assay of the studied drugs in their pharmaceutical dosage forms using fixed time method.

Drug Pharmaceutical formulation Authentic drug added

(μg mL-1) Authentic drug found

(μg mL-1) Recovery (%) ± SDa

10.00 9.95 99.5 ± 1.40 15.00 15.15 101.0 ± 1.10 Ceclor® suspension 20.00 19.80 99.0 ± 1.70

10.00 10.07 100.7 ± 1.10 15.00 14.95 99.7 ± 1.00


Bacticlor® suspension 20.00 20.19 100.9 ± 1.50

10.00 9.87 98.7 ± 1.20 15.00 15.25 101.7 ± 1.50 Duricef® tablets 20.00 19.60 98.0 ± 1.70

10.00 9.75 97.5 ± 1.60 15.00 14.90 99.3 ± 1.40 Duricef® suspension 20.00 20.40 102.0 ± 1.50

10.00 9.75 97.5 ± 1.20 15.00 14.85 99.0 ± 0.90 Duricef® capsules 20.00 20.30 101.5 ± 1.00

10.00 9.87 98.7 ± 1.10 15.00 14.85 99.0 ± 0.80 Biodroxil® capsules 20.00 20.21 101.1 ± 0.70

10.00 10.23 102.3 ± 1.30 15.00 15.30 101.0 ± 1.20


Biodroxil® suspension 20.00 19.85 99.3 ± 0.80

10.00 10.13 101.3 ± 0.40 15.00 14.63 97.5 ± 0.60 Ceporex® tablets 20.00 20.16 100.8 ± 1.70

10.00 9.85 98.5 ± 1.30 15.00 15.09 100.6 ± 0.90 Ceporex® suspension 20.00 19.86 99.3 ± 1.80

10.00 10.22 102.3 ± 0.70 15.00 15.11 100.7 ± 1.90

Cefalexin anhydrous

Ospexin® suspension 20.00 20.18 100.9 ± 1.50

10.00 9.90 99.0 ± 0.90 15.00 14.67 97.8 ± 1.10 Velosef® capsules 20.00 20.19 101.0 ± 0.80

10.00 10.14 101.4 ± 1.30 15.00 14.73 98.2 ± 0.60

Cefradine anhydrous

Velosef® tablets 20.00 20.26 101.3 ± 0.90

10.00 9.89 98.9 ± 0.90 12.50 12.25 98.0 ± 1.40 Cefixime Ximacef® capsules 15.00 20.15 100.8 ± 0.70

aAverage of six determination.

4. CONCLUSIONS

The developed kinetic spectrophptometric technique is precise, selective and accurate. The proposed method is applicable in aqueous medium at room temperature and thus there is no fear of decomposition of the drug due to heat, acid or base. Statistical analysis proves that the method is repeatable and selective for the analysis of ce-fadroxil monohydrate, cefaclor monohydrate, cefalexin

anhydrous, cefradine anhydrous and cefixime in bulk drug and in pharmaceutical formulations and can be used for routine quality control analyses of active drug in the laboratories of hospitals, pharmaceutical industries and research institutions.

REFERENCES [1] United States Pharmacopoeia 31 and NF 26. (2008)



443443

American Pharmaceutical Association, Washington, DC.

[2] El-Obeid, H.A., Gad-Kariem, E.A., Al-Rashood, K.A., Al-Khames, H.A., El-Shafie, F.S. and Bawaseer, G.A.M. (1999) A selective colorimetric method for the determi-nation of penicillins and cephalosporins with α-aminoa-cyl functions. Analytical Letters, 32(14), 2809-2823.

[3] Metwally, F.H., Alwarthan, A.A. and Al-Tamimi, S.A. (2001) Flow-injection spectrophotometric determination of certain cephalosporins based on the formation of dyes. Il Farmaco, 56(8), 601-607.

[4] Sastry, C.S.P., Rao, S.G., Naidu, P.Y. and Srinivas, K.R. (1998) New spectrophotometric method for the determi-nation of some drugs with iodine and wool fast blue BL. Talanta, 45(6), 1227-1234.

[5] Ivama, V.M., Rodrigues, L.N.C, Guaratini, C.C.I and Zanoni, M.V.B. (1999) Spectrophotometric determination of cefaclor in pharmaceutical preparations. Quimica Nova, 22(2), 201-204.

[6] Al-Momani, I.F. (2004) Flow-injection spectrophotomet-ric determination of amoxycillin, cefalexin, ampicillin, and cefradine in pharmaceutical formulations. Analytical Letters, 37(10), 2099-2110.

[7] Yang, J., Zhou, G.J., Cao, X.H., Ma, Q.L. and Dong, J. (1998) Study on the fluorescence characteristics of alka-line degradation of cefadroxil, cefradine, cefotaxime so-dium and amoxycillin. Analytical Letters, 31, 1047-1060.

[8] Aly, F.A., Hefnawy, M.M. and Belal, F. (1996) A selec-tive spectrofluorimetric method for the determination of some α-aminocephalosporins in formulations and bio-logical fluids. Analytical Letters, 29(1), 117-130.

[9] Yang, J.H., Zhou, G.J., Jie, N.Q., Han, R.J., Lin, C.G. and Hu, J.T. (1996) Simultaneous determination of cefalexin and cefadroxil by using the coupling technique of syn-chronous fluorimetry and h-point standard additions method. Analytica Chimica Acta, 325(3), 195-200.

[10] Yang, J.H., Ma, Q.L., Wu, X., Sun, L.M., Cao, X.H. (1999) A new luminescence spectrometry for the determination of some β-lactamic antibiotics. Analytical Letters, 32(3), 471-480.

[11] Chailapakul, O., Aksharanandana, P., Frelink, T., Einaga, Y. and Fujishima, A. (2001) The electrooxidation of sul-fur-containing compounds at boron-doped diamond elec-trode. Sensors and Actuators B, 80(3), 193-201.

[12] Chailapakul, O., Fujishima, A., Tipthara, P. and Siri-wongchai, H. (2001) Electroanalysis of glutathione and cefalexin using the boron-doped diamond thin-film elec-trode applied to flow-injection analysis. Analytical Sci-ences, 17(ICAS2001), i417-i422.

[13] Li, Q.L. and Chen, S. (1993) Studies on electrochemical

behaviour of cefalexin. Analytica Chimica Acta, 282(1), 145-152.

[14] Crouch, S.R., Cullen, T.F., Scheeline, A. and Kirkor, E.S. (1998) Kinetic determinations and some kinetic aspects of analytical chemistry. Analytical Chemistry, 70(12), 53R-106R.

[15] Perez-Bendito, D., Gomez-Hens, A. and Silva, M. (1996) Advances in drug analysis by kinetic methods. Journal of Pharmaceutical and Biomedical Analysis, 14(8-10), 917-930.

[16] Espinosa-Mansilla, A., Acedo Valenzuela, M.I., Salinas, F. and Canada, F. (1998) Kinetic determination of ansamicins in pharmaceutical formulations and human urine; manual and semiautomatic (stopped-flow) proce-dures. Analytica Chimica Acta, 376(3), 365-375.

[17] Helaleh, M.I.H. and Abu-Nameh, E.S.M. (1998) A ki-netic approach for determination of cefadroxil in phar-maceuticals by alkaline hydrolysis. Journal of AOAC In-ternational, 81(3), 528-533.

[18] Rahman, N., Ahmad, Y. and Azmi, S.N.H. (2005) Kinetic spectrophotometric method for the determination of ramipril in pharmaceutical formulations. AAPS Pharm- SciTech, 6(3), E543-E551.

[19] Neil, S.I. (1987) Physical organic chemistry. John Wiley & Sons, New York, 93.

[20] Kelly, F.C. (1953) Studies on the stability of iodine compounds in iodized salt. Bulletin of World Health Or-ganization, 9(2), 217-230.

[21] Yatsimirskii, K.B. (1966) Kinetic methods of analysis. Pergamon Press, London, 43.

[22] Laitinen H.A., Harris, W.E. (1975) Chemical analysis. 2nd Edition, McGraw-Hill, New York.

[23] (2005) Topic Q2 (R1): Validation of analytical proce-dures: text and me thodology. International Conference on Harmonization, Foster. http://www.ich.org/LOB/me-dia/MEDIA417.pdf

[24] Saleh, G.A., Askal, H., Darwish, I. and El-Shorbagi, A. (2003) Spectroscopic analytical study for the charge- transfer complexation of certain cephalosporins with chloranilic acid. Analytical Sciences, 19(2), 281-287.

[25] Harvey, D. (2000) Modern analytical chemistry. Boston, McGraw-Hill, Massachusetts, 108.

[26] The Merck index (2001) An encyclopedia of chemicals, drugs and pharmaceuticals. 13th Edition, Merck & Co., INC., New Jersey, 133.

[27] Svehla, G. (1979) Vogel’s textbook of macro and semi- micro qualitative inorganic analysis. 5th Edition, the Chaucer Press, Great Britain, 342.

http://www.ich.org/LOB/%20media/MEDIA417.pdf

http://www.ich.org/LOB/%20media/MEDIA417.pdf

Vol.2, No.5, 444-449 (2010)doi: 10.4236/ns.2010.25054


Natural Science


Application of microspectral luminescent analysis to study the intracellular metabolism in single cells and cell systems

Natalia A. Karnaukhova*, Larisa A. Sergievich, Valery N. Karnaukhov

Institute of Cell Biophysics, Russian Academy of Science, Pushchino, Russian Federation; *Corresponding Author: [email protected]

Received 4 December 2009; revised 27 January 2010; accepted 15 March 2010.

ABSTRACT

Spectral luminescent analysis of single cells and cellular systems enable us to reveal the initial changes of intracellular metabolism that can followed by human diseases or failure in biocenosis. Two cytodiagnostic systems of de-vices and techniques have been developed: 1) Microspectrofluorimeters registering the fluo-rescent spectra of individual cells or intracellu-lar organelles used for fundamental investiga-tions of cell reactions and for discovering and studying new dimensionless fluorescent char-acteristic parameters reflecting the biochemical or physiological properties of the cells; 2) Dou-ble- and multi-wave microfluorimeters for rapid registration of fluorescent characteristic parameters for many cells to obtain statistical in-formation about cell population. These techniques are useful especially in medical and eco-logical investigations.

Keywords: Intracellular Metabolism; Spectral Luminescent Analysis; Microspectrofluorimeters; Double and Multi-Wave Microfluorimeters; Cytodiagnostics; Low-Frequency Variable Magnetic Fields; Solar Activity

1. INTRODUCTION

All cells have a general physical-chemical basis of func-tioning in spite of different morphological structures and functions (synthesis of nucleic acids and proteins, ener-getic, etc.). Therefore, the success in the solution of this problem is essentially dependent on finding specific features which could form the basis of the algorithm for recognition of cells by their chemical composition. Be-sides, it is necessary to provide possibility for the mor-phological analysis of “suspicious” cells. The character-istic features common for cells of different kinds were

defined on the base of fundamental studies of intracellu-lar regulation of metabolism with microspectral lumi-nescent analysis. It formed a new trend in automation of cytodiagnostics. To study plant, animal and microorgan-ism cells both self-luminescence of some intracellular compounds (NADH, flavoproteins, for example) and the secondary luminescence induced by interaction of fluo- rochromes with the cells are used. The advance in fluo-rescent cytodiagnostics is only possible with best meth-ods and tools available for microspectral analysis which present a combination of a fluorescent microscope with a spectroanalysing instrument supplied with electronic registration and control units. Such a system satisfying all needs of cytodiagnostics would consist of the two types of instruments. The interconnection between them is determined by their functional peculiarities which will be considered below with some characteristic parameters using in medicine and ecology [1-3].

2. DEVICES AND TECHNIQUES FOR LUMINESCENT CYTODIAGNOSTICS

2.1. Microspectrofluorimeter- Microspectrophotometer

This instrument is used to study luminescent spectral characteristics of cell. The general diagram of microspectrofluorimeters is given in Figure 1. This scheme can be varied depending on concrete task. Different dis-persing elements are used as monochromator: prisms, diffraction gratings and interfering light filters of vary-ing wave length [1]. Microspectrofluorimeter MSF-1 enables registration of luminescence spectra of cells and intracellular compartments up to 0.5 µm in diameter in the range from 400 to 800 nm. The luminescence spectra of individual cells are insufficient to follow any process, and information on the behavior of the whole cell popu-lation under study is required. It is convenient for this purpose to describe the luminescence spectrum of each cell with one parameter only. Thus, microspectrofluo-


N. A. Karnaukhova et al. / Natural Science 2 (2010) 444-449


445445


rimeters registering the fluorescent spectra of individual cells or intracellular organelles are used for fundamental investigations of cell reactions and for discovering and studying of the new dimensionless fluorescent charac-teristic parameters, reflecting the biological or physio-logical properties of the cells.

2.2. Microfluorimeter DMF-2

Using microspectrofluorimeter the luminescence spectra of various types of cells have been studied and the char-acteristic parameters of the cells have been defined, there is no need to register the total spectrum for each cell. It is enough to measure the luminescence intensity of the cells in two characteristic spectral regions. Micro-fluorimeter DMF-2 (Radical) interfaced to a PC/AT compatible computer is used to measure fluorescence intensities at two separate wavelengths (Figure 2). It is based on a fluorescent microscope with a double-channel fluorescent sensor assembly. The first channel is tuned to record one fluorescence intensity and the sec-ond another fluorescence intensity of the same object. This allows measurement of the fluorescent characteris-tics of definite single cells or their compartments, the structure of which is controlled by microscopy. The fluorescence of the cells is excited by the emission of a DRSh-250-2 mercury arc lamp with chosen wavelength. The size of the photometered area corresponds to the cell size. A special program “Microfluor” made it possible to obtain the distribution histograms of the fluorescence intensities in the different regions of the spectrum as well as the distribution histograms of the characteristic parameters for 200 cells within 15-20 min with the points plotted onto the phase plane, and to perform sta-tistical analysis of the data [4,5]. These techniques are useful especially in medical and ecological investiga-tions.

3. APPLICATIONS OF DEVICES AND METHODS FOR LUMINESCENT CYTODIAGNOSTICS

3.1. Synthetic Activity of Cells

To investigate synthetic activity of cells the characteris-tic parameter α have been offered. Fluorochrome ac-ridine orange (AO) is widely used to investigate the nu-cleic acids in living as well as fixed cells. As an example of information obtained with the help of MSF-1 it can consider fluorescence spectra of AO stained blood lym-phocytes of rabbit at different stages of their immune response to exogenous protein ovalbumin [6]. The spec-tra consist of two emission bands. AO interacts with DNA and RNA by intercalation or electrostatic attraction respectively. DNA (double-helical nucleic acids) intercalated AO fluoresces green (530 nm), RNA (single-

helical nucleic acids) electrostatically bound AO fluo-resces red (640 nm).

Using the fluorescent microscope, investigator visu-ally observes the cells with colors from green through yellow, orange to red in depend of ratio in emission bands only in two spectral diapasons-in green (I530) and red (I640). And if the color in cell fluorescence can’t be

Figure 1. General scheme of MSF-1. 1-luminescent microscope; 2-probe nozzle; 3-system of monochro-mator: a-objective, b-mirror, c-diffraction grating; 4- mercury arc lamp (DRSh-250-2); 5-power supply unit; 6-photomultiplier; 7-high voltage power unit; 8-am- plifier; 9-register (X,Y-recorder).

Figure 2. General scheme of two-channel (double- wave) microfluorimeter “Radical DMF-2”. 1-luminescent microscope LUMAM; 2-probe nozzle; 3-di-chroic mirror-analyzer; 4-two-channel (double- wave) registration system;5-mercury arc lamp (DRSh-250- 2); 6-power supply unit for mercury arc lamp; 7- power supply unit for photomultipliers; 8-ADC; 9- computer.



446


used for analytical purpose, the description of cell’s color as a ratio of fluorescence intensities.

][

][

2

11

530

640

NA

NAA

I

I (1)

where А1 is the proportionality coefficient, [NA1] and [NA2] are the concentrations of single-helical (NA1) and double-helical (NA2) nucleic acids, permits to analyze quantitatively the processes in the cells.

Taking into account that in differentiated nonproli-firating cells (blood lymphocytes) the main quantity of single-helical nucleic acids is RNA, double-helical nu-cleic acids is DNA, expression (1) should be refined.

][

][1

530

640

DNA

RNAA

I

I (2)

It has been shown that under definite conditions of

cell staining with AO parameter reflects the amount of RNA per unit DNA and, hence, characterizes cell syn-thetic activity [1-3,6]. Series of α-distribution histograms is indicative of changes in synthetic activity of all popu-lation of immunocompetent cells at different stages of processes in immune system (Figure 3).

The above example demonstrates only one way of de-termining the parameter characterizing the ratio between the nucleic acids in the cell and the synthetic potentiality of the cell. Being, presumably, of greatest interest to medicine and biology [6-10], it is not the only possible method. Using the microspectral analysis of cells and a large set of luminescent dyes-labels, other useful char-acteristic parameters can be found. We further elaborate on our method for monitoring the synthetic activity of lymphocytes, testing the two-dye, three-color assay with regard to the temporal organization of the immune re-sponse. Within the same methodological framework, a good probe for protein is 1-anilino-8-naphthalene sul-fonate (ANS); we have already shown that consecutive staining of fixed cells with AO and ANS adds a third fluorescence peak in the blue region (470 nm) character-istic of protein-bound ANS [11].

470530

470

4.0][

][

II

I

DNA

proteinA

(3)

The investigations conducted on blood lymphocytes showed that parameter α is sensitive to the action of the stimulatory and damaging effects of environmental fac-tors, including electromagnetic fields and solar activity [12,13].

Under the action of low-frequency variable magnetic fields with the specified parameters, the synthetic activ-ity (parameter α) increased by 22 to 35% (Figure 4). These results and the data obtained by another methods showed that the variable magnetic fields enhances the synthetic activity of lymphocytes, improves the type of

adaptive response, and thereby increases the level of the immune resistance of the organism.

Figure 3. Distributions histograms of the parameter for rabbit blood lymphocytes in process of immune re-sponse in organism onto albumin introduction: (а) be-fore immunization; (b) on stage of the maximum activ-ity; and (c) on the later stage. Along the ordinate is the number of cells. Along the abscissa are the parameter values.

Figure 4. Diagrams of the changes in the average values of the parameters α for lymphocytes in the blood of rats as a function of frequency. * p < 0.05, ** p < 0.01 .



447447


Correlation was revealed between solar activity pa-rameters (sunspot number and the 10.7 cm solar radio flux) and the synthetic activity of blood lymphocytes in different species of animals during the same periods (January-April) of 1993, 1994, 2000, and 2002 (Table 1).

In the others seasons (April-June, August-December), the negative correlation between the studied indices was weaker and was not reliable. The data suggest seasonal regularities in the connection between the studied proc-esses.

Sign reversal of the correlation coefficient was ob-served in the second maximum of solar cycle 23. The change of sign may depend on a change in the ratio be-tween phases of the oscillatory processes studied. How-ever, the mechanism of the phenomenon observed re-mains unknown.

It was also shown that the correlation decreased under stronger internal (disease) or external (chronic gamma- irradiation) influences (Table 2).

3.2. Energetics of Animal Cells

Also the self-luminescence of living cells can be used for cytodiagnostics. For instance, it was shown [1,14,15] that the state of the mitochondrial energetic apparatus of a cell can be quantitatively characterized by the ratio of the intensities of luminescence of oxidized flavoproteins (530 nm) and reduced pyridinenucleotides (470 nm):

470

470530 5.0

I

II (4)

In this case the application of the microspectral analy-sis enabled one to find that in one cell (neurons of stretch-receptor) there are two mitochondrial pools re-sponsible for the energy supply of different functional mechanisms of the cell-membrane transport and syn-thetic processes (Figure 5). It was shown that these two mitochondrial pools are controlled by different mecha-nisms. Therefore, in one and the same cell, at one and the same instant the mitochondria belonging to different Table 1. Correlation coefficients between the 10.7 cm solar radio flux and the synthetic activity of blood lymphocytes in different years.

1993 1994 2000 2002

-0.81 ** ± 0.08 -0.63 * ± 0.17 -0.63 * ± 0.15 0.71 * ± 0.09

* p < 0.05, ** p < 0.01

Table 2. Correlation coefficients between the 10.7 cm solar radio flux and the synthetic activity of blood lymphocytes in health, in pathology, and after gamma-irradiation.

Control (healthy state)

Pathology (cholelithiasis)

Gamma - irradiation (14.4 cGy/day to 15 Gy)

-0.63 * ± 0.17 -0.24 ± 0.26 -0.45 ± 0.22

* p < 0.05

pools may be in different states of activity. It is only the method of spectral analysis that enables one to obtain such information about the state and organization of in-tracellular organelles in a functioning cell [1].

Taking into consideration the changes in the energetic apparatus of cells upon malignization found by Warburg it can be supposed that the characteristic parameter can also be used for automatic detection of cancerous ma-lignant cells in preparation [16].

470

5301 I

I

B

A (5)

3.3. Ecology and Environment Protection.

In solving the problems of environment protection, the relationship between autotrophic and heterotrophic en-ergy provision reflects the wellbeing of an autotrophic organism [2]. The energetic state of high plant cells can be determined from the ratio luminescence band intensi-ties of chlorophyll (680 nm) and oxidized flavoproteins (530 nm).

530

680

I

I

B

AX (6)

This makes it possible to detect regions with polluted atmosphere, to assess the state of the forests as well as to predict the harvest of cultivated plants [2,17]. For the same purpose, the following parameters can also be used.

645

680

I

I

B

A (7)

572

680

I

I

B

A (8)

Those characterize the state of lichens symbiotic

Figure 5. Luminescence spectra of reduced pyridi-nenucleotides (NADH, 470 nm) and oxidized flavo-proteins (FPo, 530 nm) in neuron of stretch-receptor. Ordinate-fluorescence intensity in rel. units. Ab-scissa-the wave length in nm.



448


blue-green algae which are extremely sensitive to at-mosphere pollution in places of their habitat. In the both cases, the ratio of luminescence band intensity of chlo-rophyll (680 nm) to that of phycocyanine (645 nm) or phycoerythrin (572 nm) is used [2]. It is clearly seen that the autotrophic cells having intense chlorophyll band in the luminescent spectrum (Figure 6, curve 1) later start to switch from autotrophic (algal) to heterotrophic (bac-terial) energy provision (Figure 6, curves 2, 3).

It is important that in the tropical Atlantic the majori-ties of the Cyanophyceae contain no chlorophyll and are actually heterotrophic cyanobacteria. Their typical fluo-rescence spectra (Figure 7, curve 1) have only phyco-erythrin (572 nm) and phycocyanine (645 nm) bands, though there are occasional cells that have not com-pletely lost chlorophyll (Figure 7, curves 2, 3).

Figure 6. Luminescence spectra of single cells of blue-green microorganisms in different states: 1- autotrophic (algal); 2, 3-mixed (autotrophic and heterotrophic) energy provision.

Figure 7. Luminescence spectra of blue-green mi-croorganisms in the tropical Atlantic: 1-a most typical spectrum, 2, 3-deviant spectra.

However, most interesting seems the application of the parameters φ and ψ in the cases when it is necessary to predict the onset of “blooming” of blue-green algae in water storage basins or lakes [18,19] for determining the optimal instant of treating algae population with bacte-riophages (cyanophages) in order to prevent “blooming” which causes disastrous effect. Of particular importance may be the application of the parameters φ and ψ for predicting the initiation of the disease “ciguatera”. It is the poisoning of people with usually harmless fishes and mollusks living in regions of intense “blooming” of blue-green algae in tropic zones of the oceans [19].

4. CONCLUSIONS

These few examples indicate that the methods of lumi-nescent cytodiagnostics have promising applications in different fields of biology, medicine, environment pro-tection and biotechnology. The realization of these per-spectives necessitates the development of special equipment. Two cytodiagnostic systems of devices and tech-niques have been used for the purpose:

1) Microspectrofluorimeters-microspectrophotometers registering the fluorescent spectra of individual cells or intracellular organelles are used for fundamental inves-tigations of cell reactions and for discovering and study-ing of the new dimensionless fluorescent characteristic parameters, reflecting the biochemical or physiological properties of the cells.

2) Double-and multi-wave microfluorimeters are used for the rapid registration of many cells to obtain statisti-cal information about cell population. These techniques are useful especially for medical and ecological investi-gations.

Thus, this study confirms that parameters derived from luminescent spectral analysis of fluorochromed cells (such as α and β) or self-luminescence of living cells (such as ξ, χ, φ, ψ) are valid as dynamic indices of intracellular metabolic activity in single cells and cell systems. There are broad possibilities of further devel-oping this approach and equipment into developing in-creasingly differentiated fluorescence techniques.

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[13] Karnaukhova, N.A., Sergievich, L.A., Karnaukhov, V.A. and Karnaukhov, V.N. (2004) Changes in the synthetic activity of lymphocytes under the action of physical fac-tors related to Solar activity variations. Biophysics, 49(suppl.1), 552-559.

[14] Karnaukhov, V.N., Lebedev, O.E. and Pavlenko, V.K. (1976) About two mitochondrial pools in a stretch-re-ceptor neuron. Tsitologia, 18(10), 1189-1193.

[15] Rudenko, Y.N., Bigdai, E.V. and Samoilov, V.O. (2007) Kinetics of Са2+, NADH and oxidized flavoproteids in the frog olfactory living under the effect of odorants. Biophysics, 52(1), 88-94.

[16] Thorell, B. (1981) Flow cytometric analysis of cellular endogenous fluorescence. Cytometry, 2(1), 39-43.

[17] Roshchina, V.V. (2003) Autofluorescence of plant se-creting cells as a biosensor and bioindicator reaction. Journal of Fluorescence, 13(5), 403-420.

[18] Karnaukhov, V.N., Martsenuk P.P. and Yashin, V.A. (1980) Luminescence spectral characteristics of physio-logical state of cells of blue-green algae. Fisiologia Rast, 27(1), 11-17.

[19] Karnaukhov, V.N. and Yashin, V.A. (2003) Spectral stud-ies on single cells of sea microplankton: History and prospects. Biophysics, 48(5), 940-949.

Vol.2, No.5, 450-456 (2010)doi: 10.4236/ns.2010.25055


Natural Science

Wettability alteration by magnesium ion binding in heavy oil/brine/chemical/sand systems—analysis of hydration forces

Qiang Liu1, Ming-Zhe Dong2*, Koorosh Asghari1, Yun Tu3

1Faculty of Engineering, University of Regina, Regina, Canada; 2Department of Chemical and Petroleum Engineering, University of Calgary, Calgary, Canada; *Corresponding Author: [email protected] 3Institute for Chemical Process and Environmental Technology, National Research Council, Ottawa, Canada

Received 17 November 2009; revised 13 January 2010; accepted 28 February 2010.

ABSTRACT

In laboratory sandpack tests for heavy oil re-covery by alkaline flooding, it was found that wettability alteration of the sand had a signifi-cant impact on oil recovery. In this work, a heavy oil of 14 API was used to examine the effect of organic acids in the oil and water chemistry on wettability alteration. From interfacial tension measurements and sand surface com-position analysis, it was concluded that the water-wet sand became preferentially oil-wet by magnesium ion binding. The presence of Mg2+ in the heavy oil/Na2CO3 solution/sand system in-creased the oil/water interfacial tension. This confirmed the hypothesis that magnesium ion combined with the ionized organic acids to form magnesium soap at oil/water interface. Under alkaline condition, the ionized organic acids in the oil phase partition into the water phase and subsequently adsorb on the sand surfaces. The analysis of sand surface composition suggested that more ionized organic acids adsorbed on the sand surface through magnesium ion binding. The attachment of more organic acids on the sand surface changed hydration forces, making the sand surface more oil-wet.

Keywords: Wettability Alteration; Alkaline Flooding; Magnesium Ion Binding; Interfacial Tension; Organic Acids

1. INTRODUCTION

Wettability plays an important role in determining the distribution and flow of fluids in the pores of a reservoir [1]. Whether the pore surface of reservoir rock is water-

wet or oil-wet is determined by the thickness of the wa-ter film between the rock surface and the oil [2]. For very thick films, the system is stable and remains water- wet. If it is unstable, the film will break, resulting in direct contact of oil to the rock surface and adsorption of polar components on pore walls. The stability of a thick water film is dependent on the magnitude of the disjoin-ing pressure. The disjoining pressure that tends to disjoin or separate the oil/water and water/rock interfaces are identified as a combination of van der Waals, electro-static and hydration forces. The van der Waals forces are attractive, while electrostatic forces are repulsive be-tween the interfaces. The hydration forces can be either a hydrophilic effect for a surface such as clean quartz or a hydrophobic effect for a surface with an organic coating. If the magnitude of repulsive forces is greater than the attractive forces, the water film is stable, and the surface remains water-wet.

The hydrophobic effect of hydration forces can be caused by the adsorption of polar compounds that were originally in crude oils [3-5]. These compounds have a polar end and a hydrocarbon chain. The polar end con-tacts the rock surface and the hydrocarbon chain exposes to the liquid phase, making the surface more oil-wet [6]. Some of the polar compounds are soluble in water so that they can diffuse through the thin water film to ad-sorb onto the rock surface [7]. It has been found that, even when a surface active compound has a very low solubility in water, it could reach the solid surface by diffusion through the water film [8]. This will make the attractive force greater and the water film could be drained to result in an oil-wet surface.

Kowalewski et al. [9] conducted wettability tests us-ing Berea sandstone, brine (NaCl) and n-decane with different concentrations of hexadecylamine. The wet-tability of the sandstone samples was changed from wa-ter-wet to neutral due to the adsorption of hexade-cylamine on the rock surface. Ashayer et al. [10] studied


Q. Liu et al. / Natural Science 2 (2010) 450-456


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the influence of surfactant molecules (alkyl ether car-boxylic acid) on wetting phenomena with a glass mi-cromodel. It has been found that the solid surface attracts the polar head group of the surfactant molecules and the tail of the surfactant is free at the water/glass interface. The attractive force between the hydrophobic tail of the surfactant and the oil chain causes the formation of a “hydrophobic bond”, which changes the wettability of the surface from water-wet to oil-wet. Buckley et al. [11] believed that when the brine phase contained divalent cations, wettability could be altered by ion binding mechanism. The divalent ions combined the oil with min-eral surface, making the mineral less water-wet.

Wettability alteration is one of the mechanisms of en-hanced oil recovery by alkaline flooding. Cooke et al. [12] studied wettability in alkaline flooding in glass mi-cro-model by acidic oils and formation waters. They observed that the wettability of the matrix of the glass micro-model changed from strongly water-wet to pref-erentially oil-wet after alkaline flooding. They believed that the wettability alteration was caused by the adsorp-tion of ionized acids onto the solid surface. Waterflooding of heavy oil reservoirs exhibits very poor sweep efficiency mainly due to a adverse mobility ratio and water channeling [13]. Ma et al. [14] conducted channeled sandpack flood tests of alkaline flooding for a Western Canadian heavy oil sample. It was found that wettability alteration of sand led to oil re-distribution, blockage of existing water channel in porous media and improvement in oil recovery. Liu et al. [15] studied wet-tability alteration in a heavy oil/water/sand system by analyzing the electronic forces at oil-water and water- sand interfaces through ζ-potential measurements. They found that the presence of either Na2CO3 or Mg2+ alone in the water phase could not induce wettability alteration. When the water phase contained both Na2CO3 and Mg2+, the water-wet sand became preferentially oil-wet by magnesium ion binding. The reduction in zeta ()-po-tential at both oil-water and water-sand interfaces due to the addition of Mg2+ to the heavy oil/Na2CO3 solu-tion/sand system confirmed the combination of Mg2+ and ionized organic acids at the oil/water interface. They concluded that the reduction of repulsive electrostatic forces between oil drops and sand surfaces contributed to the wettability change of the sand from water-wet to oil-wet.

The objective of this paper is to examine the contribu-tion of hydration forces at oil-water interface to the wet-tability alteration in the heavy oil/water/sand system used by Liu et al. [15]. The magnesium ion binding was investigated by measuring oil-water interfacial tension (IFT) and water surface tension and analyzing sand sur-face composition. These results provide insight into the partition of polar compounds in heavy oil/water system and their adsorption onto the sand surface as well as the

relation between the magnesium ion binding and wet-tability alteration.

2. EXPERIMENTAL

In this study, micro-slide and micro-model tests were conducted to observe wettability alteration during the oil displacement process. Heavy oil/brine interfacial ten-sions and surface tension of water phase were measured for different systems to investigate the interactions be-tween heavy oil, brine and sand. Sand surface composi-tions under different conditions were analyzed to evalu-ate the adsorption of polar substances onto sand surface after oil/water/sand interaction. All tests were conducted at ambient temperature (22 0.5C) except specified.

2.1. Materials

A heavy oil of 14API collected from a reservoir in Alberta, Canada was used in this study. The oil sample was centrifuged at 10,000 rpm at 35C for two hours to remove water and solids. The viscosity, density and acid numbers of the oil were analyzed and are shown in Ta-ble 1. The oil had a viscosity of 1,800 mPas and a den-sity of 0.964 g/cm3 at 22C.

In this study, the effect of divalent ions (mainly Ca2+ and Mg2+) on the wettability of the sand in oil/brine/sand system was examined. Solution of 1.0 wt% NaCl in de-ionized water other than the formation brine was used as water phase for the anaysis of hydration forces [15]. MgCl2 was added to adjust Mg2+ concentration in water phase. Na2CO3 was used to neutralize the organic acids (polar compounds) in the oil.

Varsol (a commercial solvent containing kerosene as the main component) and ethanol were used to clean the micromodel. The sand used in this work was from U.S. Silica Company and was originally water-wet.

2.2. Wettability Tests with Micro-Slide and Micro-Model

In this paper, two methods are employed to examine the wettability of a solid surface in porous media: mi-cro-slide test and micro-model test. For the details of the micro-slide and micro-model tests, readers are referred to a previous work by Liu et al. [15].

Micro-slide tests were conducted for observing the wettability of sands in different oil/alkaline solution sys-tems. The oil and water were equilibrated for 50 hours Table 1. Viscosity, density and acid number of the heavy oil sample.

Acid number, mg KOH per gram of sample Viscosity,

mPas Density, g/cm3

Strong Weak Total 1,800 0.964 0.89 0.43 1.32



452


and separated for micro-slide tests. Sand was added into the water phase for adsorption for 50 hours and sepa-rated for preparing the micro-slide models. Then a monolayer of the sand was sandwiched between two micro-slides and saturated with the equilibrium oil phase. The equilibrium water phase was introduced to the model for an imbibition-type displacement of oil.

A glass micro-model was used to conduct alkaline flooding. The transparent nature of the micro-model al-lows the pore-scale multi-phase displacement and wet-tability of the pore surfaces to be visually observed [16]. The displacement procedure for a micro-model test was as follows:

1) Saturate the micromodel with the water phase (1.0 wt% NaCl);

2) Inject the heavy oil or kerosene; 3) Conduct waterflood (1.0 wt% NaCl) for two pore

volumes (PV); 4) Conduct alkaline flood by injecting 0.20 wt%

Na2CO3 in brine with or without Mg2+ for one PV. Microphotographs were taken at different stages of

the displacement tests to observe the wettability of the pore surface.

2.3. IFT Measurement

The spinning drop tensiometer (Model 510, Temco, USA) was employed to measure the water surface tension and oil/water interfacial tension. For surface tension meas-urement, an air bubble was injected into a glass tube filled with a water solution; for IFT measurement, an oil droplet was injected into the glass capillary tube. The IFTs and surface tensions are determined using the fol-lowing equation:

327 )(1042694.3 Ddh L/D ≥ 4 (1)

where σ is interfacial tension (dyne/cm), h is the density of heavy (outer) phase (g/cm3), d is the density of light (drop) phase (g/cm3), is rotational velocity (rpm), D is measured drop width (diameter) (mm), and L is the length of the oil drop (mm).

2.4. Analysis of Sand Surface Composition

In the heavy oil/brine/sand systems, some of the ionized organic acids in the oil phase will partition into the water phase and subsequently adsorb on the sand surface. The adsorption of ionized organic acids on the sand surface was investigated by analyzing the surface compositions of the sand before and after it was brought to contact the water phase. Because the sand surface was easily con-taminated by oil drops in the water phase, the sand was equilibrated with the heavy oil/brine system as follows. 1) The water phase was equilibrated with the heavy oil; 2) The water phase was filtered to remove oil droplets before it was mixed with the sand; 3) The sand sample was mixed with the water phase for two weeks for ad-

sorption; 4) The sand was separated from the water us-ing a stainless steel sieve and dried in an oven at 60C for one hour. The compositions of the top 7-nm surface layer of the sand was analyzed by using a Kratos AXIS Ultra X-Ray photoelectron spectrometer (XPS), equipped with a hemispherical analyzer, a delay line detector, charge neutralizer and monochromated Al Kα X-ray source.


3.1. Onset Na2CO3 and Mg2+ Concentrations for Wettability Alteration

In the previous wettability study by Liu et al. [15], the heavy oil was equilibrated with water phases of different compositions by adding Na2CO3 or NaOH to react with the organic acids in the oil and CaCl2 and MgCl2 to ad-just Ca2+ or Mg2+ in the water phase. It was found that the presence of Na2CO3 and Mg2+ could cause wettabil-ity alteration in the heavy oil/water/sand systems. In order to examine the effect of Na2CO3 and Mg2+ on wet-tability alteration, micro-slide tests were conducted with various Na2CO3 and Mg2+ concentrations.

Table 2 shows the results of micro-slide tests at dif-ferent Na2CO3 concentrations with or without the pres-ence of Mg2+. No wettability alteration was observed for the samples of Series A, which contained only Na2CO3. In the presence of 100 mg/L Mg2+, wettability alteration occurred when Na2CO3 concentration reached a specific value; 0.10 wt% for Series B in which 100 mg/L Mg2+ was added after the water phase was equilibrated with the oil; 0.20 wt% for Series C in which 100 mg/L Mg2+ was added before the water phase was equilibrated with the oil.

The onset Mg2+ concentration for wettability altera-tion was investigated by using micro-slide tests with 0.20 wt% Na2CO3 and various Mg2+ concentrations (named as Series D in Table 3). Magnesium ions were added into the water phase before oil-water equilibration. As shown in Table 3, wettability alteration was initiated at a concentration of 50 mg/L Mg2+.

3.2. Effect of Organic Acids on Wettability Alteration

To investigate the effect of organic acids in oil on wet-tability alteration, two micro-model tests were conducted to observe wettability alteration during alkaline flooding displacement. In one test, the heavy oil was used; in the other test, kerosene was used as the oil phase which was free of organic acids. The same water phase (1.0 wt% NaCl + 0.20 wt% Na2CO3 + 100 mg/L Mg2+) was used for both tests.

Figure 1 shows the pore-level microphotographs of the micro-model taken during the test with the heavy oil,



453453

Table 2. Wettability of sand in microslide tests at different Na2CO3 concentration with or without the presence of Mg2+.

Na2CO3 concentration, wt% Test series Method of Mg2+ addition

0.020 0.050 0.10 0.20 0.50A No Mg2+ No No No No No B 100 mg/L Mg2+ added after water equilibrated with oil No No Yes Yes Yes C 100 mg/L Mg2+ added before water equilibrated with oil No No Partial Yes Yes

Note: Yeswettability alteration; Nono wettability alteration; Partialpartial wettability alteration. Table 3. Effect of Mg2+ on wettability in microslide tests, Na2CO3: 0.20 wt% (Test series D).

Concentration of Mg2+, mg/L 0 10 20 50 100 200 Wettability alteration No No No Yes Yes Yes

showing oil and water distribution at different displace-ment stages. Water films between the oil and the pore walls exist before the injection of alkaline solution. After alkaline flooding, oil films exist between the water and pore walls, indicating that the pore walls have became preferentially oil-wet. The oil/water menisci in Figures 1(b) to 1(d) are convex to the oil phase, suggesting that the glass pore is oil-wet. It is also shown from the dis-tribution of oil and water phase in the pores that the glass model has become preferentially oil-wet. The re-sults in Figure 1 are consistent with those in micro-slide tests.

Figure 2 shows the wettability of glass pores at dif-ferent stages of the micro-model test with kerosene. The glass pores remained water-wet after alkaline flooding. The difference between crude oil and kerosene is that the heavy oil contains organic acids and kerosene does not. The results of the two micro-model tests suggest that the organic acids in the oil phase are the origin of wettability alteration in alkaline flooding.


3.3. Heavy Oil/Brine/Sand Interactions

As reviewed in the introduction, hydration forces can have a hydrophobic effect for a surface with an organic coating. In this section, the effect of heavy oil/brine/sand interaction on the hydration forces is investigated. The samples of Series A through D listed in Tables 2 and 3 were used for the following measurements and tests.

3.3.1. IFT Variation Caused by the Presence of Mg2+

The combination of magnesium and ionized organic acids deactivate the ionized acids at the oil/water inter-face and, therefore, increases the oil/water interfacial tension. To see the interaction of Mg2+ and ionized or-ganic acids at oil/water interface, interfacial tensions of the heavy oil and water phase were measured for sys-tems with and without Mg2+. Figure 3 shows the inter-facial tensions as a function of Na2CO3 concentration for two water solutions: one did not contain Mg2+ and the other contained 20 mg/L Mg2+. The addition of Na2CO3 in the water phase reduced the IFT of the heavy oil and water from its original value (approximately 25 dyne/cm) to 2.13, 1.12, and 0.38 dyne/cm at 0.02, 0.05, and 0.1

wt% Na2CO3, respectively. In the presence of 20 ml/L Mg2+, the IFTs were raised to 12.0, 7.5, and 4.5 at the above three Na2CO3 concentrations, respectively, and to approximately one order magnitude higher at Na2CO3 concentrations between 0.1 and 0.5 wt%. Figure 4 shows the IFT of the heavy oil and brine at 0.20 wt% Na2CO3 and different Mg2+ concentrations. The IFT was increased dramatically with Mg2+ concentration between 0 to 20 mg/L and then increased slightly with Mg2+ con-centration in the water phase. This indicates that the sur-face activity of the ionized organic acids was decreased significantly by the presence of Mg2+. The divalent cation, Mg2+, could be concentrated at the oil/water in-terface; therefore, only 20 mg/L Mg2+ could make the ionized organic acids incapable in reducing the IFT be-tween the oil and water.

The dynamic IFTs of the heavy oil with three water samples of Series D (0, 5, and 10 mg/L Mg2+) were also

(a) (b)

(c) (d)

Figure 1. Pictures of one location of micromodel at four stages of oil displacement process. Oil phase: heavy oil, Na2CO3 concentration in alkaline slug: 0.20 wt%, Mg2+ concentration in water: 100 mg/L. (a) After water flooding; (b) after alkaline flooding; (c) 50 hours after alkaline flooding; (d) 150 hours after alkaline flooding.



454


(a) (b)

(c) (d)

Figure 2. Pictures of one location of micromodel at four stages of oil displacement process. Oil phase: kerosene, Na2CO3 con-centration in alkaline slug: 0.20 wt%, Mg2+ concentration in water: 100 mg/L. (a) After water flooding; (b) after alkaline flooding; (c) 50 hours after alkaline flooding; (d) 150 hours after alkaline flooding.

0.1

1

10

100

0 0.1 0.2 0.3 0.4 0.5 0.6

Na2CO3 concentration (wt%)

IFT

(d

yne

/cm

)

Without Mg2+

20 mg/L Mg2+

Figure 3. Interfacial tensions of heavy oil/water as a function of Na2CO3 concentration for cases of without and with 20 mg/L Mg2+ in the water phase. Na2CO3 concentration: 0.20 wt%.

0.1

1

10

100

0 50 100 150 200

Mg2+ concentration (mg/L)

IFT

(d

yn

e/c

m)

Figure 4. Interfacial tensions of heavy oil/water as a function of Mg2+ concentration. Na2CO3 concentra-tion: 0.20 wt%.

measured and are shown in Figure 5. The system with-out Mg2+ exhibited dynamic IFT behavior and the other two systems with Mg2+ did not show the dynamic be-havior within the measurement error. This indicates that the magnesium soaps were rapidly formed at the oil/water interface by magnesium ion binding.

3.3.2. Partition of Organic Acids into Water Phase

When organic acids in the oil phase are ionized in alka-line condition, they become more hydrophilic and capa-ble to partition into water phase. In the water phase, they will have opportunities to contact, attach to and change the wettability of the sand surface. The presence of Na2CO3 and/or Mg2+ can affect the partitioning of the ionized organic acids and change the surface tension of the water phase. Investigating the surface tension of wa-ter can provide useful information on the partitioning of ionized organic acids.

Surface tensions of water samples of Series A and B were measured to investigate the effect of Na2CO3 and Mg2+ on partitioning of the acids into the water phase. The results are shown in Figure 6. For Series A (without Mg2+), surface tension decreased with Na2CO3 concen-tration. Surface tension was reduced to approximately 47

0.1

1

10

100

0 10 20 30 40 50 6

Time (minute)

IFT

(dy

ne/

cm

)

0

0 mg/l Mg2+5 mg/l Mg2+10 mg/l Mg2+

Figure 5. Dynamic interfacial tensions of heavy oil/water with different Mg2+ concentrations. Na2CO3 concentration: 0.20 wt%.

40

45

50

55

60

0.01 0.1 1

Na2CO3 concentration (wt%)

Su

rface t

en

sio

n (d

yn

e/c

m)

Series ASeries B

Figure 6. Surface tensions of equilibrium water phase as a function of Na2CO3 concentration. Oil/water ratio: 1/1, Series A: no Mg2+, Series B: 100 mg/L Mg2+ in equilibrium brine.



455455

dyne/cm at 0.10 wt% Na2CO3. This is the result of the formation of ionized organic acids and their partitioning into the water phase. These ionized organic acids in the water phase could not be detected with the two-phase titration method [17], indicating that only little of ion-ized organic acids were in the water phase. For Series B, containing 100 mg/L Mg2+ in the equilibrium water phase, surface tension was higher than that of Series A at Na2CO3 concentrations higher than 0.02 wt%. This is because the ion binding that deactivated the surface ac-tivity of the ionized organic acids in the water phase.

The surface tension of 1.0 wt% NaCl brine was meas-ured to be 73.0 dyne/cm. After 1.0 wt% NaCl brine (without Na2CO3 or Mg2+) was equilibrated with the heavy oil (oil/water volume ratio = 1:1), no further re-duction in surface tension of the water phase was ob-served within the experimental error. This suggests that no organic acids in the oil phase partitioned into the wa-ter phase. This behavior can be explained by the fact that organic acids in the oil phase were not ionized and were more hydrophobic. If the organic acids in the oil do not partition into the water phase, there will be no adsorption of organic acids on the surface of sand which is wa-ter-wet and covered with water. This is why the sand surface remained strongly water-wet in Test 1 listed in Table 2.

3.3.3. Adsorption of Ionized Organic Acids on Sand Surface

In a heavy oil/brine/sand system, the adsorption of ion-ized acid onto sand surface affects hydration forces, making the sand surface more hydrophobic or more oil-wet [2]. In order to investigate the effect of the ion-ized organic acids on sand surface wettability, sand sur-face compositions were analyzed for five sand samples, one of which was the original sand. The other four sam-ples, labeled S1, S2, S3 and S4, were equilibrated with water phase of different chemical compositions before surface composition analysis as follows: the water phase with 0.02 wt% Na2CO3 in Series A was used for S1; the water phase with 0.02 wt% Na2CO3 and 100 mg/L Mg2+ in Series B was used for S2; the water phase with 0.5 wt% Na2CO3 in Series A was used for S3; and the water phase with 0.5 wt% Na2CO3 and 100 mg/L Mg2+ in Se-ries C was used for S4. For the above four sand samples, only the wettability of sand sample S4 changed from water-wet to oil-wet.

Seven elements were analyzed and the results are

shown in Table 4. The change in the element percentage on the sand surface of samples S1–S4 was the indication of the adsorbed substances. The adsorption of ionized organic acids and Na2CO3 changed the percentage of carbon (C) and oxygen (O) while the adsorption of NaCl and MgCl2 from water phase increased the percentage of chloride (Cl), sodium (Na), and magnesium (Mg).

The mole ratio of C/O on the sand surface is also listed in Table 4. The C/O mole ratio of the original sand was 0.40 and it increased from Samples S1 to S4. There are two sources for C/O ratio change on sand surface: adsorbed Na2CO3 and ionized organic acids. The C/O mole ratio for the compound of Na2CO3 is 33% which is lower than that of the original sand, showing that Na2CO3 adsorption on the sand surface from a water solution lowers the C/O mole ratio. The ionized organic acids are polar compounds with a high molecular weight and a long organic carbon chain. Because of that, they are expected to have a much higher C/O mole ratio [4, 12]. The adsorption of organic compounds on sand sur-face will provide more carbons. It is believed that the high carbon content and high C/O mole ratio on sand surface for S1 to S4 are the result of the adsorption of ionized organic acids on the sand surface.

When a higher Na2CO3 concentration is applied in the water phase that is in contact with the oil phase, more organic acids will be ionized. Some of the ionized acids will subsequently partition into water, and adsorb onto the sand surface. The adsorption of ionized organic acids on sand surface is the reason for the higher C/O mole ratio in Sample S3 (0.5 wt% Na2CO3) than in Sample S1 (0.02 wt% Na2CO3). Table 4 shows that Sample S4 has a much higher C/O mole ratio than Sample S3, indicating that much more ionized organic acids attached on the sand surface for Sample S4 than for Sample S3. There was 100 mg/L Mg2+ in water phase for Sample S4 and no Mg2+ for Sample S3. The presence of Mg2+ increased the adsorption of ionized organic acids onto the sand surface. This indicates that wettability alteration of S4 is caused by the magnesium binding mechanism in the heavy oil/brine/sand system. Through the ion binding of Mg2+, more ionized organic acids in the aqueous phase attached to the sand surface. The hydrophobic tail of the surfactant on sand surface was more easily contacted by oil. The hydration forces became unfavorable for sus-taining the water film between the oil and sand surface. It was concluded from the previous work [15] that the

Table 4. Sand surface element analysis (mole percent) and mole ratio of C/O.

Element C O Cl Si Al Na Mg C/O Original sand 20.9 51.0 N/D 18.7 7.4 0.5 N/D* 0.40

S 1 24.4 45.8 0.8 18.8 7.7 2.2 N/D 0.41 S 2 29.3 43.4 0.5 16.8 6.9 1.1 1.6 0.68 S 3 30.7 42.6 0.5 16.3 6.1 3.9 N/D 0.72 S 4 63.6 26.3 0.2 5.8 2.0 1.2 0.9 2.42

* N/Dnot detectable



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negative charges at both the oil/water and water/sand interfaces were reduced by the Mg2+ ion binding, weak-ening the repulsive forces between the two interfaces. The changes of both electrostatic forces and hydration forces contributed to the wettability alteration in the heavy oil/brine/sand system.

4. CONCLUSIONS

In this study, the mechanism of wettability alteration in a heavy oil/alkaline solution/sand system was investigated by analyzing the hydration forces, which revealed the following conclusions.

The presence of either Na2CO3 or Mg2+ alone in water could not induce wettability alteration. When water con-tained both Na2CO3 and Mg2+, wettability of the solid could be altered from water-wet to preferential oil-wet. Wettability of sand was altered from water-wet to pref-erential oil-wet by the Mg2+ ion binding mechanism. Under alkaline conditions, magnesium concentration of ~50 mg/L could cause wettability alteration.

The heavy oil-water interfacial tension was greatly increased due to the combination of Mg2+ and the ion-ized organic acids at the oil/water interface. The analysis of sand surface composition showed significant increase in carbon content and C/O ratio in sand top surface layer due to the adsorption of magnesium soap of the organic acids. These results are consistent with the reduction in surface charges at both the oil/water and water-sand in-terfaces obtained in a previous study. The magnesium ion binding reduced both electrostatic and hydration forces at the oil/water and water/sand interfaces and caused wettability alteration of sand surface.

Water phase surface tension data showed that the ion-ized organic acids can partition into the water phase. Through the Mg2+ ion binding, the ionized organic acids in the aqueous phase attached to the sand surface. The attachment of the organic acids on the sand surface de-creased the hydration forces, making the sand surface more oil-wet.

5. AKNOWLEDGEMENTS

Acknowledgment is extended to the Petroleum Technology Research Centre (PTRC), Murphy Oil Company Ltd., the Natural Sciences and Engineering Research Council (NSERC) of Canada, and the Canada Foundation for Innovation (CFI) for their financial support for this work. The authors wish to express their thanks to Murphy Oil Com-pany Ltd. for providing the oil and brine samples.

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[9] Kowalewski, E., Holt, T. and Torsaeter, O. (2002) Wet-tability alterations due to an oil additive. Journal of Pe-troleum Sciences and Engineering, 33(1-3), 19-28.

[10] Ashayer, R., Grattoni, C.A. and Luckham, P.F. (2000) Wettability changes during surfactant flooding. 6th In-ternational Symposium on Evaluation of Reservoir Wet-tability and its Effect on Oil Recovery, Socotrro.

[11] Buckley, J.S., Liu, Y. and Monsterleet, S. (1998) Mecha-nisms of wetting alteration by crude oils. Society of Pe-troleum Engineering Journal, 3(1), 54-61.

[12] Cooke, Jr.C.E., William, R.E. and Kolodzie, P.A. (1974) Oil recovery by alkaline waterflooding. Journal of Pe-troleum Technology, 26(12), 1365-1374.

[13] Miller, K.A. (2005) State of the art of western Canadian heavy oil water flood technology. Proceedings of Petro-leum Society’s 6th International Petroleum Conference (56th Annual Technical Meeting), 2005-251, Calgary.

[14] Ma, S., Dong, M., Li, Z. and Shirif, E. (2007) Evaluation of effectiveness of chemical flooding using heterogene-ous sandpack flood test. Journal of Petroleum Science and Engineering, 55(3-4), 219-228.

[15] Liu, Q., Dong, M., Asghari, K. and Tu, Y. (2007) Wet-tability alteration by magnesium ion binding in heavy oil/brine/chemical/sand systems-Analysis of electrostatic forces. Journal of Petroleum Science and Engineering, 59(1-2), 147-156.

[16] Dong, M., Foraie, J., Huang, S. and Chatzis, I. (2005) Analysis of immiscible water-alternating-gas (WAG) in-jection using micromodel. Journal Canadian Petroleum Technology, 44(2), 17-25.

[17] ASTM (1989), Standard test method for synthetic anionic ingredient by cationic titration. Annual Book of ASTM Standards, American Society for Testing and Materials, West Conshohocken, 15(4), 320-323

Vol.2, No.5, 457-463 (2010)doi: 10.4236/ns.2010.25056


Natural Science

Dicranostigma leptopodum (maxim) fedde induced apoptosis in SMMC-7721 human hepatoma cells and inhibited tumor growth in mice

Wen-Hua Zhang1, Ming-Hua Lv1, Jun Hai1, Qin-Pu Wang2, Qin Wang1*

1School of life sciences, Lanzhou University, Lanzhou, China; [email protected]; *Corresponding Author: [email protected] 2Biology Department, Tianshui Normal College, Tianshui, China

Received 10 February 2010; revised 9 March 2010; accepted 12 April 2010.

ABSTRACT

Dicranostigma Leptopodum (Maxim) Fedde (DL- F), which had been previously documented to suppress oxidative hemolysis of erythrocytes and enhance immune functions of murine peri- toneal macrophages, was investigated for its effect on anti-tumor activity. Of alkaloids ex-tracted from DLF, five have been identified with employment of chromatographic analysis. An antiproliferative role of these alkaloids was de-termined on SMMC-7721 Human Hepatoma Ce- lls in an apoptosis-inducing manner, through MTT assaying, Trypan blue exclusion assaying and cytometric analysis of cell cycle distribu-tion. To further examine their inhibitory effects on tumor progression, murine H22 cells were inoculated into Kunming mice to determine the role of these alkaloids of DLF in inhibiting tumor growth in the tumor-implanted mice. It was found that these alkaloids of DLF enhanced the tumor shrinkage effectively wherein its tumor inhibitory rate and immunohistochemistry staining of the tumor were determined and profiled, respectively.

Keywords: Dicranostigma Leptopodum (Maxim) Fedde; Anti-Tumor Activity; Apoptosis; Tumor-Growth Inhibition

1. INTRODUCTION

The medicinal use of natural products has a time-hon-ored history along with the development of human civi-lization. Throughout human history, enormous range of natural products-compounds that are derived from natu-ral sources such as plants, animals or micro-organisms- have been discovered and put into medical use, the latest version of the Dictionary of Natural Products (DNP;

http://dnp.chemnetbase.com) encompasses over 214,000 entries. These were identified as leads of drug through biological assay and became candidates for drug devel-opment. More than 60% of the marketed drugs derived from natural sources [1]. Owing to the diverse biological activities and medicinal potentials, the importance of natural products for medicine and health has been re-portedly enormous with examining the experience and knowledge accumulated of use of natural products [2]. In light of their matchless resource and biologically- synergic activities in vivo, they continue to contribute to the expansion of lead drugs and provide insights for synthesis of their non-natural analogues. Increasingly, Traditional Chinese medicine (TCM) is receiving recog-nition from modern western medicine and 908 compo-nents from Tradition Chinese Medicine Database were found structurally similar to those deposited in the Com- prehensive Medicinal Chemistry database of which 327 agents were further identified as common members of both databases [3]. Although emphasis on high-through- put screening of synthetic libraries has in part declined drug discovery research into natural products during last two decades, the potential for new discoveries of activi-ties of natural products in the long term is promising, given that the number of new natural product-derived drugs could go to zero [4].

Despite huge conceptual difference between Tradi-tional Chinese Medicine (TCM) and Modern Western Medications, the preconception-TCM can’t get them clinically approved - may be bridgeable with increased knowledge of molecular mechanisms of TCM-derived drugs [5]. By comparing 669 anti-tumor, anti-cancer or anti-neoplastic agents identified from Comprehensive Medicinal Chemistry database (CMC, containing 8659 clinically used Western drugs) to Traditional Chinese Medicine Database (TCMD, containing 10458 compo-nents), 26 pairs were found identical in structure and 20 were validated to be originally isolated from herbs [6]. With rationale borrowed from afore-mentioned discov-

http://dnp.chemnetbase.com/

W.-H. Zhang et al. / Natural Science 2 (2010) 457-463


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eries and previous findings that Dicranostigma leptopo-dum (maxim.) fedde (DLF) possessed physiological re- levance of antipyretic and analgesic, detumescence, etc. (Dictionary of Traditional Chinese Medicine 1986), we further investigated its activities implicated in the induc-tion of apoptosis of cancerous cells. The scientific name of DLF was for the first time coined and collectively classified by Harvard University Herbaria in 1987 (http://www.gbif.net/occurrences/86270582/). Enhanced effects of DLF on immune-suppression were determined in vivo [7] and its effect on suppressing oxidative hemo-lysis of erythrocytes was also validated [8]. Efforts made to separate and characterize the components of DLF have identified five crystals from DLF of which three were isocorydine, corydine and protopine [9] and five alkaloids isolated were dicranostigmine, isocorydine, corydine, protopine and sinoacutine [10]. In this study, we treated SMMC-7721Human Hepatoma Cells with extracted alkaloids of DLF, aiming to examine effect of alkaloids of DLF on inducing apoptosis of cancerous cells. Moreover, we treated H1-implanted Kunming Mice with alkaloids of DLF to have determined tumor growth-inhibiting effects of DLF.

2. EXPERIMENTAL MATERIALS AND METHOD

2.1. Materials

2.1.1. Extraction of Alakoids from DLF The powdered material of roots, stems and leaves of DLF (12.5 g) was mixed with 75% alcohol (400 mL) for 1 h in a hermetic glass container and then disrupted con-tinuously with an ultrasonic purge. The whole material was filtered in vacuum followed by a distillation process. Add alcohol to the mixture of filtrate to adjust its alcohol concentration to 85%. After 24 h, adjust the filtrate to pH 8.0 using NaOH and then filter and distill the filtrate until the alcohol is deprived. Adjust the filtrate to pH 7.0 using HCL [11].

2.1.2. Determination of Alkaloids of DLF Extracts 20 mg/mL standard DLF extracts were diluted to con-centrations of 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL and 0.7 mg/mL, respec-tively. The standard curve was made with these gradient concentrations and de-ionized water as control [12]. The separation of the alkaloids from DLF extracts was per-formed using Sephadex G-50 chromatograph analysis during which samples (50 ul) flowed through the Sepha- dex G-50 columns (0.1 mL) steadily at rate of 1 mL/min. The eluted proteins were determined at 254 nm wave-length by WD-9430D UV spectrophotometer.

2.1.3. Animals and Cells Kunming mice were purchased from the Experimental

Animal Center at Lanzhou University. The use and treatment of mice were in accordance with institutional guideline for Laboratory Animal Care. Muring H22 and SMMC-7721 Human Hepatoma cells were obtained from cell library of Institute of Cancer Biology and Drug Discovery, Lanzhou University. Cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Lanzhou Minhai Biotechnology), 2 mM l-glutamine, 100 units/mL penicillin and 100 mg/ mL streptomycin and incubated at 37 in a humidified atmosphere of 5% CO2 and 95% air.

2.2. Methods

2.2.1. MTT Assay SMMC-7721 were plated in 96-well microtiter plates at a density of 4,000/well for culture and incubated in a humidified 5% CO2-95% air atmosphere at 37 for 24 h. Then cells were treated with different concentrations of alkaloids of DLF (0-24 mg/mL) and incubated for additional 24 h, 48 h, and 72 h respectively. Cell viabil-ity was determined by MTT assay [13] whereby 20 μL of 5 mg/mL MTT was added to each well and incubated for another 4 hours at 37. The supernatant was subse-quently removed and 100 μL/well DMSO was added to dissolve the formazan crystal. After shaking plates for 1 min, the absorbance of each well was read at 570 nm wavelength with microplate reader (Bio-Rad). The vi-ability of SMMC-7721 cells was calculated employing the formula below:

Viability = (A570 of treated cells/A570 of untreated cells) × 100%

2.2.2. Trypan Blue Exclusion Assay Trypan blue dye, which would be excluded by normal cells but could diffused into cells with disrupted mem-brane integrity, was employed to determine the number of the viable cells after treatment with alkaloids of DLF [14]. 24 hours after the SMMC-7721cells were treated with PMS-1077, measurements were conducted after trypan blue (Sigma) was incubated with SMMC-7721 cells for 5 min at room temperature. At least 500 cells were counted per sample. The cell viability was calcu-lated with the following equation:

%100 cells ofnumber Total

cells blue ofNumber - cells ofnumber TotalViability

2.2.3. Morphological Analysis To determine the morphological changes of SMMC- 7721 cells upon treatment with alkaloids of DLF, SMMC-7721 cells which were treated with alkaloids of DLF for 24 h were observed under inverted dark field microscope (Nikon, Japan) and photographed after-wards.

2.2.4. Single Cell Gel Electrophoresis Assay The single cell gel electrophoresis (SCGE) assay [15]

http://www.gbif.net/occurrences/86270582/



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commonly referred to as “comet assay” allowed the very sensitive detection of DNA breakage induced by genoto- xic agents at single cell level. Thus, this method was adopted to determine the DNA damaging effects of al-kaloids of DLF on SMMC-7721 cells. Treated with dif-ferent concentrations of alkaloids of DLF for 24 h, SMMC-7721 cells were lysed by alkaline lysis solution (2.5 M NaCl, 100 mM EDTA•Na2, 10 mM Tris pH 10), 10% DMSO and 1% triton X-100 (Sigma) at 4 for 1 h. Another 20 min was allowed for the DNA to unwind in electrophoresis running buffer solution (300 mM NaOH, and 1 mM EDTA•Na2, pH 13). Electrophoresis was per-formed for 20 minutes at 50 V and 300 mA. After elec-trophoresis, the slides were gently removed and alkaline pH neutralized with 0.4 M Tris (pH 7.5). Then Ethidium bromide (75 mL of a 20 mg/mL solution, Sigma) was added to each slide and a cover glass was placed on the gel. DNA migration was analyzed on a fluorescence mi-croscope (Olympus, Japan) (Filter G-2A) and photo-graphed afterwards.

2.2.5. Flow Cytometric Analysis SMMC-7721 cells that were treated with alkaloids of DLF for 24 h were washed twice with PBS and then fixed with 70% ethanol at -20 for about 12 h. Then cells were washed again twice with PBS and suspended with 1 mL 100 μg/mL RNase (Sigma) containing 0.1% Triton-100 and 50 μg/mL propidium iodide (Sigma). Cells were stained with DNA dye for 30 min and ana-lyzed by flow cytometer (EPICS-XL, Beckman).

2.2.6. In Vivo Inhibition of Tumor Growth Six-week old inbred female Kunming mice were inocu-lated with murine Hepatoma 2.0 × 107 mg/mL H22 cells [16]. From the second day after the implantation of H22 cells, 40 tumor-bearing mice were grouped randomly into five groups as the following: 1) Blank control, 2) Model control, 3) 5-Fu positive control, 4) High dose control, 5) Low dose control. 48h after tumor implanta-tion, these mice were I.P. injected with a daily dose of 0.2 mL of 3.0 mg/mL, 0.2 mL 6.0 mg/mL, 0.2 mL, 2 mg/mL, 0.2 mL physiological saline (0.9%) and 0.2 mL physiological saline (0.9%) of alkaloids of DLF for High dose group, Low dose group, 5-Fu positive group, Blank group and Model group, respectively. Mice were exe-cuted within 24 h after the last dose of alkaloids of DLF and their tumor was obtained and analyzed. And Spleen and thymus indexes were examined. Tumor inhibitory rate (Ri) and organ index were expressed as the follow-ing formula, respectively [17]:

%m

m

tumor

tumor 100)untreated

treated1(Ri

mouse

organ

m

mIndexOrgan

2.2.7. Immunohistochemistry Analysis The tumors of mice were excised and fixed in 4% para-formaldehyde for 24 h. Paraffin sections were prepared for immunohistochemical staining and hematoxylin and cosin (H & E) staining [18,19]. Sections for immuno-histochemical staining were deparaffinized and then hy-drated by transferring them through the following solu-tions xylene bath twice for 5 min, 100% ethanol for 5 min twice, and then 90% ethanol, 80% ethanol, 70% ethanol, and PBS, for 3 min each. Subsequently, sections were placed in a microwave oven for 15 min at 100 in sodiumcitrate buffer (0.01 M, pH 5.7) to expose epitopes. After that, sections were incubated at 37 with PCNA antibody for about 1.5 h followed by the visualization using immunosystem kit (Santa Cruz, CA).

2.2.8. Statistical Analysis All experimental data were expressed as mean ± SD, and statistical analysis was performed using Student’s t-test to compare the results from the untreated group.


3.1. Extraction and Determination of Alkaloids from DLF

The alkaloids extracted from DLF in roots, leaves and whole part were 5.31%, 5.91% and 5.57%, respectively. The average content of alkaloids in whole DLF part was 5.59%. Five main alkaloids were separated using chro-matography and their contents were determined to be unevenly distributed, as indicated by the peaks of chro-matographic profile (Figure 1).

(a)

Growing Time (weeks)

(b)

Figure 1. Extraction and determination alkaloids from DLF. (a) The comparison of alkaloids content in different organs of DLF during different growing periods; (b) Separation of alkaloids from DLF extracts and the determination of their proportion.



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3.2. Anti-Proliferative Effect of Alkaloids of DLF on SMMC-7721 Cells

Treated with assigned concentrations of alkaloids of DLF for indicated time, SMMC-7721 cells exhibited compromised cell viability. A concentration-dependent anti-proliferative effect of alkaloids of DLF on SMMC- 7721cells was determined by MTT assay and Trypan Blue Exclusion assay (Figure 2).

3.3. Induction of Apoptosis of SMMC-7721 Cells by Alkaloids of DLF

Upon treatment with 8.5 mg/mL alkaloids of DLF for 24 h, SMMC-7721 cells exhibited the characteristic features of apoptosis including cell shrinkage, cell detachment and vesicle formation (Figure 3). Flow cytometric analysis demonstrated that cell cycles were arrested (Figure 4) and cell cycle distribution analysis manifested that cells were arrested predominately at G1 phase (Figure 5).

3.4. Alkaloids of DLF-Induced DNA Damage of SMMC-7721 Cells

Upon treatment with various concentrations of alkaloids of DLF, SMMC-7721 cells exhibited characteristic fea-

0

10

20

30

40

50

60

70

80

90

100

0 1.5 3 6 9 12 18 24

Via

bili

ty

Alkaloids of DLF(mg/ml)

24h

48h

72h

(a)

0

10

20

30

40

50

60

70

80

90

100

0 1.5 3 6 9 12

Via

bil

ity(%

)

Alkaloids of DLF(mg/ml)

18

SMMC-7721

(b)

Figure 2. Determination of anti-proliferative effect of DLF on SMMC-7721 cells. (a) MTT Assay: Treated with DLF for 24 h, 48 h and 72 h, respectively, cells viability was inhibited in a concentration-dependent manner; (b) Trypan Blue Ex-clusion Assay: Effect of alkaloids of DLF on inhibiting the cell viability was concentration-dependent.

tures of DNA damage. Examination of DNA damage by Single cell gel electrophoresis revealed appreciable DNA damage in terms of its length of migrating tails (Figure 6). It’s also observed that the damage was concentration- dependent.

3.5. Inhibition of Tumor Growth by Alkaloids of DLF was Determined in Vivo

Murine Hepatoma H22 cells were inoculated to six-week old Kunming mice and, from 24 h after the inoculation, a

Figure 3. Morphological analysis of Alkaloids-treated SMMC- 7721 by inverted dark field microscopy (X200). Cells were treated with different concentrations of alkaloids of DLF as indicated: (a) control; (b) 3.0 mg/ml; (c) 6.0 mg/ml and (d) 12.0 mg/ml.

Figure 4. Flow cytometric analysis of cell cycle distribution upon treatment with alkaloids of DLF. (a) 0 mg/ml; (b) 3.0 mg/ml; (c) 6.0 mg/ml; (d) 12.0 mg/ml.



461461


Figure 5. Cell cycle distribution analysis by flow cytometry.

Figure 6. Determination of DNA damage of AlkaloidsDLF- treated SMCC-7721 cells by Single Cell Gel Electrophoresis. (a) control; (b) 3.0 mg/ml; (c) 6.0 mg/ml and (d) 12.0 mg/ml. daily dosage of alkaloids of DLF was I.P. injected into these tumor-bearing mice. After 12 days, mice were ex- ecuted and their tumors, spleens and thymus were ex-cised for analysis. Alkaloids of DLF exerted a role as potent as 5-Fu in enhancing tumor shrinkage. 5-Fu in-hibited the tumor growth by 55.75% while 6.0 mg/mL alkaloids of DLF and 12.0 mg/mL alkaloids of DLF in-hibited the growth of tumor by 42.50% and 51.00%, respectively (Table 1). By examining effects of alkaloids of DLF on the growth of spleen and thymus, it revealed that DLF exerted significant effect on inhibiting the ab-errant progression thymus of tumor-bearing mice while the effects of alkaloids of DLF on the growth of spleen of tumor-bearing mice were not discernible (Table 2).

3.6. Alkaloids of DLF-Mediated Inhibition of Tumor Progression Analyzed by HE Staining and Immunohistochemistry

Histological section of tumors excised from tumor- bearing mice which were treated with 0.9% physiologi-cal saline, 5-FU, 6.0 mg/mL DLF and 12.0 mg/mL alka-loids of DLF were examined with employment of HE

staining and Immunohistochemistry (Figure 7). Both demonstrated that alkaloids of DLF exerted pronounced effects on inhibiting of the growth and progression of tumor.

3.7. Discussion

In response to the recognition that fewer side effects have been documented in phytotherapy and natural prod-uct-based therapy and therapeutic potential of natural products [5,20,21], we further explored to study the anti- tumor activities of alkaloids of DLF. In this study, approx. 5.7% of alkaloids were extracted from the whole part of DLF and five alkaloids were identified using chromatographic analysis which was in consistence with the separation of alkaloids from DLF [10]. Anti-prolif-erative effect was determined for alkaloids of DLF on SMCC-7721 cells and IC50 of alkaloids of DLF on SMCC-7721 cells was 8.5 mg/ml. Upon treatment with DLF, SMCC-7721 cells exhibited apoptotic features as a rule [11]. Flow cytometric analysis of cell cycle distribu-tion upon treatment with alkaloids of DLF revealed that cells were arrested in the G1 phase (Figure 4) during which DNA damage was determined (Figure 6). With in vitro anti-tumor activity of alkaloids of DLF validated, in vivo effect of anti-tumor of alkaloids of DLF was further explored. Tumor implantation was completed with inoculation of Muring H22 cells in Kunming mice. Inhibitory rates of 51% and 42% of tumor growth inhibi-tion were determined for 12.0 mg/ml alkaloids of DLF and 6.0 mg/ml, respectively, through examining the tu-mor weight upon treating tumor-bearing mice with alka-loids of DLF. In addition, effects of alkaloids of DLF on inhibiting the progression tumor were determined by HE staining of histological section of tumors (Figure 7). Table 1. The inhibition effect of the drug to transplanted tumor

H22. X ± s n = 8.

Group Tumor weight Inhibitory rate

Tumor control 0.40 ± 0.23 0

5-FU control 0.18 ± 0.10* 55.75%

AlkaloidsDLF 6.0 mg/ml 0.20 ± 0.09* 42.00%

AlkaloidsDLF12.0 mg/ml 0.23 ± 0.12* 51.50%

Table 2. The effects of DLF on growth of spleen and thymus

of tumor-bearing mice X ± s n = 8.

Group Spleen index (%) Thymus index (%)

Healthy group 3.48 ± 1.31* 4.26 ± 0.80

Tumor group 6.28 ± 1.08 4.88 ± 0.88

5-FU group 2.48 ± 1.08 0.98 ± 0.40*

AlkaloidsDLF 6.0 mg/ml

11 ± 1.28* 2.84 ± 0.90*

AlkaloidsDLF 12.0 mg/ml 6.66 ± 1.37 2.84 ± 0.60*



462


Figure 7. Histological Profile of tumor upon treatment. HE staining of histological section of tumors: (a) 6.0 mg/ml Alka-loidsDLF; (b) 12.0 mg/ml AlkaloidsDLF; (c) 5-FU; (d) 0.9% physiological saline. PCNA assaying of histological section of tumors; (e) 6.0 mg/ml AlkaloidsDLF; (f) 12.0 mg/ml Alka-loidsDLF; (g) 5-FU; (h) 0.9% physiological saline.

Taken together, an anti-tumor effect of alkaloids of DLF was determined by means of both in vitro and in vivo examinations. However, the elaborate molecular mechanism underlying its anti-tumor activity remains elusive for these five alkaloids. Since determining the biological properties of plants used in traditional medi-cine is helpful to drug screening, thus, further research is deserved to isolate the very compounds responsible for the observed biological activity of alkaloids of DLF.

4. CONCLUSIONS

With promising effects of alkaloids of DLF determined both in vitro and in vivo on anti-proliferating of SMCC- 7721 cells and enhancing shrinkage of tumor, DLF de-

serves further characterization of its very components entailing its anti-tumor activities and mechanisms un-derlying its anti-tumor effect. Moreover, its anti-prolif-erating effect and anti-tumor activities remains to be extended to other cancerous cell lines and tumors as well. A comprehensive understanding of its activities on a spectrum of cancerous cells and tumors will shed light on its mechanistic elucidation of anti-tumor effects.

5. ACKNOWLEDGEMENTS

This work was jointly supported by International Cooperation Pro-grams in Gansu Province (NO. 0708WCGA149) and International Science and Technology Cooperation Project (NO. 2009DFA30990).

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Vol.2, No.5, 464-468 (2010doi: 10.4236/ns.2010.25057


) Natural Science


Mesolamellar composite of TiN and CTAB using fluoride ion bridge: synthesis, mechanism & characterization

Tumbavanam Venkateswaran Anuradha

Department of Materials Engineering, Indian Institute of Science, Bangalore, India; [email protected]

Received 29 December 2009; revised 10 February 2010; accepted 5 March 2010.

ABSTRACT

In an attempt to synthesize hexagonal mesoporous titanium nitride, a mesolamellar composite based on titanium nitride and cetyltrimethylam- monium bromide (CTAB) is obtained by the sol-gel route involving templating at 80oC. The mechanism underlying the above synthesis is discussed for the first time in the literature and till date there are no reports on the synthesis of mesoporous nitrides. The above mesolamellar composite is found to form an oxide of titania (anatase) upon heat treatment at 335oC for 1h.

Keywords: Mesolamellar; Templating; Sol-Gel Method; XRD; FT-IR; SEM

1. INTRODUCTION

Mesoporous materials are generally produced by the templating approach using various surfactant species as template. The template acts like an imprint molecule which could then be extracted by either calcination or solvent extraction leaving behind pores in the regions where they are present. At the first critical micelle con-centration (cmc1), the surfactant species forms self-as-sembled aggregates with a spherical shape which changes to a rod shape on further increase in the concentra-tion of the surfactant species (cmc2). Different liquid crystalline phases with cubic, hexagonal and lamellar structures are formed by further increase in the concen-tration of the surfactant species (above cmc2) which indeed acts as the template for the production of mesoporous materials.

There are a few reports based on the synthesis of mesolamellar phases based on zirconia [1] and silica [2]. Certain species like F- ions and acetylacetone are found to favour the formation of lamellar phases when present in the reaction medium [3,4]. There are no reports on the synthesis of mesoporous materials based on TiN due to the non-availability of suitable metal-organic precursor that could be used for the sol-gel synthesis. The precur-

sors of TiN are also found to be highly oxophilic and hence there is always a possibility that they will result in the formation of oxynitrides rather than pure nitrides. The present experiments involved the formation of a mesolamellar composite based on TiN and CTAB in the medium enriched with F- ions by sol-gel route at 80oC. The mechanism underlying the above synthesis is dis-cussed in this communication in more detail for the first time in the literature.

2. EXPERIMENTAL PROCEDURES

2 g of TiN, 2 g of CTAB, 25 cc of HF-HNO3 mixture and 25 cc of distilled water were taken. TiN is dissolved in 1:1 solution of HF-HNO3 mixture (9:1) in water forming species like [Ti1-xFxNy]

3n+ to which an aqueous solution of CTAB is added to result in the yellow col-ored solution which is kept in the oven for 3 h at 80oC and then it is kept aside at RT for 24 h. The yellow solid thus obtained has the composition [CTA] (H2O)n [Ti1-x

FxNy]. Thus a mesolamellar composite based on TiN and CTAB is formed with the F- ion acting as the bridge be-tween them. The lamellar composite obtained above is characterized by x-ray diffraction (XRD) and FT-IR spectroscopy.

JEOL diffractometer (reflection type-model 8030) is used for the characterization of the materials by x-ray diffraction (XRD). Cu target is used. A voltage of 30 kV, a current of 20 mA, and a step angle of 0.020o with de-tection times of 0.5 sec at each step are used. A JEOL 2000FX-IItransmission electron microscope operating at the accelerating voltage of 200 kV is used for TEM studies. The powder samples are crushed thoroughly and dispersed in a suitable organic solvent (acetone or high purity ethanol/methanol) and subjected to ultrasonication before supporting on the carbon-coated grids for loading in the TEM equipment. FT-IR studies are carried out using Perkin Elmer FT-IR equipment. Differential Scan-ning Calorimetry (DSC) is performed with a Perkin Elmer DSC-2C calorimeter to study the thermal stability of the material. The measurements are carried out under dynamic argon flow condition. Before starting the ex-


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periment the base line was corrected and the calibration of the instrument is done using pure indium. A heating rate of 20 K/min is used.

3. RESULTS & DISCUSSION

Porous materials i.e. materials with non-lamellar mesos- tructures that are stable enough to be preserved after the removal of the structure-directing amphiphile could only be synthesized under aqueous conditions with the utili-zation of cationic surfactants [5,6] and the other attempts yielded lamellar phases only [7,8]. It is believed that a larger variety of surfactants in the alcoholic medium with a small quantity of water would result in the forma-tion of non-lamellar materials [9]. But the utilisation of alcoholic medium is avoided in this investigation since a highly acidic medium is required for the dissolution of titanium nitride. The only constraint in not making use of the alcoholic medium for the synthesis is the non-availability of a suitable organo-metallic precursor for titanium nitride. There are continuous efforts to syn-thesize mesoporous materials based on nitrides like sili-con nitride for the catalytic applications [10,11] which involves the ammonolysis of SiCl4 in organic solvent. But the handling of halides of titanium like TiCl4 or the organo-metallic precursor like (TiCl4L2) (where L = ether, amine and pyridine) is very difficult at room tem-perature since they are both air-sensitive and moisture - sensitive.

Figure 1 shows the XRD of the mesolamellar com-posite after templating TiN onto CTAB using F- ion as the bridge at 80oC. The lamellar nature of the composite is confirmed by XRD studies which is again reconfirmed using SEM studies [12,13]. The lamellar nature is found to be accompanied by atomic scale ordering and registry of the layers [14] where the first peak has appeared at d 19.4 Ao with the following peaks at d 19.4/2 Ao, 19.4/3 Ao, 19.4/4 Ao). The clearly resolved six orders of the diffraction peak as well as the plain baseline in the 20-30o 2 range suggest that the TiN layers sandwiched between the surfactants are well crystallized as in the case of lamellar TiO2 mesophase which was produced by Lin et al. [15] using CTAB as the templating surfactant for tetrabutylorthotitanate (precursor of TiO2) under ba-sic conditions. This high degree of condensation of the inorganic building units may be different from those of the silicate [16,17] and aluminophosphate phase meso- phases [18,19] whose frameworks are amorphous in nature. The reflections present in the higher 2 region are found to represent the internal structure involving the organization of the lamellae and thus could not be in-dexed to any impurities, but they could be indexed to an orthorhombic cell with a reasonable justification and this result compares very well with the mesocomposite of zinc phosphate produced by Huo et al. [20].

Figure 2 gives the FT-IR spectra of CTAB and meso-composite respectively. FT-IR studies of the composite material showed many of the vibration modes like C-H& C-N stretching and C-H bending similar to that of the template i.e. CTAB which is used as standard for com-parison, but the peaks had significantly different intensi-ties and line widths in this case and there are some addi-tional peaks which could be assumed to be due to the transition metal co-ordination with the template. FT-IR studies clearly indicated the formation of composite where CTAB is intact. The peaks appeared at 3651 cm-1 and 3587 cm-1 corresponding to the O-H stretching fre-quencies in the physisorbed water molecules and also the peak at 1627 cm-1 corresponds to the bending mode as-sociated with the physisorbed water molecules.

Differential Scanning Calorimetric (DSC) analysis in-dicated the decomposition behaviour of the lamellar

Figure 1. XRD of the mesolamellar composite of TiN and CTAB after templating at 80oC.

Figure 2. FT-IR spectrum of (a) CTAB; (b) mesolamellar composite of TiN and CTAB.



466


composite with three distinct stages of reaction. Figure 3 gives the ‘DSC’ trace of the mesolamellar composite in argon atmosphere [12] The first split peak (endother-mic) around 100oC is due to the desorption of water in-dicating the presence of two different kinds of water molecules associated with the mesocomposite structure namely, water of hydration and associated (hydrogen bonded) water. The second peak at 335oC, which is exo-thermic in nature, indicates the decomposition of the organic surfactant. But the appearance of the last two peaks at 391oC and 500oC is not completely understood. The XRD patterns of the mesocomposite after heating it at 335oC, 391oC and 500oC for 1h, are shown in Figure 4. They indicated the formation of anatase form of nano- crystalline titania (TiO2) at all the above temperatures and average crystallite sizes less than 10 nm. Figure 5 gives the bright field TEM image of anatase after heat treatment at 335oC for 1h, with its selected area electron diffraction pattern and Figure 6 shows its corresponding dark field TEM image. The dark field TEM image indi-cated the presence of particles less than 10 nm though agglomeration is also present.

Intercalated structure is indicated by TEM studies with the average particle size less than 10 nm [11] which in turn is composed of [Ti1-xFxNy]

3n+and [CTA]+ with the bridging F- ions. Hence it could be assumed that the F- ion acts as bridge between positively charged [Ti1-xFxNy]

3n+ and [CTA]+ species. The bilayers of CTAB between the layers of TiN are assumed to be interdigited since it is believed that hydrated [Ti1-xFxNy]

3n+ species is large and thus interdigitation of the surfactant tails al-lows the surfactant headgroups to be spaced well apart which compares well with the earlier report on iron ox-ide/surfactant composites [14] where the change in la-mellar ‘d’ spacing with surfactant carbon chain length for hydrated and dehydrated Fe(II) ions are studied. Figure 7 demonstrates the mechanism underlying the synthesis of lamellar mesophase based on TiN which is found to follow the counter ion (here F- ion) mediated pathway in Liquid Crystal Templating (LCT) approach. Titania is found to be stable in rutile form when the par-ticle size exceeds 14 nm and below this critical size, anatase phase is stable [21]. Titania obtained by the heat treatment of the mesolamellar composite of titanium nitride fluoride and CTAB at 335oC for 1h is found to be anatase with the average particle size less than ~10 nm.

4. CONCLUSIONS

There are no reports on the synthesis of mesostructured nitrides till date in the literature and thus it is most re-markable to produce the nanostructured mesolamellar composite of titanium nitride fluoride and CTAB with an average particle size less than ~ 10 nm by templating TiN onto CTAB in the highly acidic medium enriched

with F- ions (pH ~ 2) at 80oC. The interlamellar spacing is found to be ~ 19 Ao. The mechanism underlying the above synthesis is also discussed for the first time where F- ions act as the bridge between positively charged [Ti1-x Fx Ny]

3n+ and [CTA]+ species. The bilayers of CTAB between the layers of TiN are interdigited since that hydrated [Ti1-x Fx Ny]

3n+ species are large and thus

Figure 3. DSC trace of the mesolamellar composite of TiN and CTAB.

Figure 4. XRD pattern of the mesocomposite of TiN and CTAB, after heat treatment at (a) 335oC; (b) 391oC; (c) 500oC, for 1 h.

Figure 5. (a) The bright field TEM image of TiO2 (anatase) obtained from the mesolamellar composite after heat treatment at 335 oC for 1 h, and (b) its corresponding selected area elec-tron diffraction pattern.



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Figure 6. The dark field TEM image of TiO2 (anatase) obtained from the mesolamellar com-posite after heat treatment at 335 oC for 1 h.

Figure 7. The mechanism underlying the syn-thesis of mesolamellar composite of titanium nitride and CTAB in the medium enriched with F- ions.

interdigitation of the surfactant tails allows the surfactant headgroups to be spaced well apart. The above lamellar composite has resulted in the formation of anatase form of titania upon heat treatment at 335oC for 1h with the average particle size less than 10 nm.

5. ACKNOWLEDGEMENTS

The support of the Council for Scientific & Industrial Research (India) to T. V. Anuradha through a Senior Research Fellowship is gratefully acknowledged.

REFERENCES

[1] Huang, Y. and Sachtler, W.M.H. (1997) Preparation of mesostructured lamellar zirconia. Chemistry Communi-cation, 1181-1182.

[2] Tanev, P.T. and Pinnavaia, T.J. (1996) Biomimetic tem-plating of porous lamellar silicas by vesicular surfactant assemblies. Science, 271(5253), 1267-1269.

[3] Fernado Henrique, P.S. and Pastore, H.O. (1996) Chem-istry Communication, 7, 833-835.

[4] Feng, P., Xia, Y., Feng, J., Bu, X. and Stucky, G.D. (1997) Synthesis and characterization of mesostructured alu-minophosphates using the fluoride route. Chemistry Communication, 949-950.

[5] Luan, Z., Zhao, D., He, H., Klinowski, J. and Kevan, L. (1998) Tubular aluminophosphate mesoporous materials containing framework silicon, vanadium and Manganese. Journal of Physics and Chemistry B., 102(20), 1250- 1259.

[6] Kimura, T., Sugahara, Y. and Kuroda, K. (1999) Synthe-sis and characterization of lamellar and hexagonal mesostructured aluminophosphates using alkyltrimethy- lammonium cations as structure directing agents. Chemi-cal Materials, 11, 508-518.

[7] Froba, M. and Tiemann, M. (1998) Chemical Materials, 10(11), 3475-3483.

[8] Sayari, A., Moudrakovski, I., Reddy, J.S., Rateliffe, C.I., Ripmeester, J.A. and Preston, K.F. (1996) Chemical Ma-terials, 8, 2080.

[9] Tiemann, M., Schulz, M., Jager, C. and Froba, M. (2001) Mesoporous aluminophosphate molecular sieves synthe-sised under non-aqueous conditions. Chemical Materials, 13(9), 2885-2891.

[10] Kaskel, S. and Schlichte, K. (2001) Porous silicon nitride as a superbase catalyst, Journal of Catalysis, 201, 270-274.

[11] Kaskel, S., Farrusseng, D. and Schlichte, K. (2000) Syn-thesis of mesoporous silicon imido nitride with high sur-face area and narrow pore size distribution. Chemistry Communication, 2481-2482.

[12] Anuradha, T.V. and Ranganathan, S. (1999) A compari-son of the efficiency of three different synthetic routes viz. sol-gel method involving templating, mechano-chemical synthesis and combustion synthesis for the production of nanostructured TiO2. Nanostructured Ma-terials, 12, 1063-1073.

[13] Anuradha, T.V. and Ranganathan, S. (2000) Proceedings of International Symposium on Amorphous and Nano- crystalline Materials (Satellite Meeting of NANO-2000), Inoue, A., Ed., Japan Society for the Promotion of Sci-ence, 1.

[14] Tolbert, S.H., Sieger, P., Stucky, G.D., Aubin, S.M.J., Wu, C-.C. and Hendrickson, D.N. (1997) Control of inorganic layer thickness in self-assembled iron oxide/surfactant composites. Journal of American Chemical Society, 119(37), 8652-8661.


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[15] Lin, W., Pang, W., Sun, J. and Shen, J. (1999) Lamellar TiO2 mesophase with an unusual room temperature pho-toluminescence. Journal of Material Chemistry, 9, 641-642.

[16] Beck, J.S., Vartuli, J.C., Roth, W.J., Leonowicz, M.E., Kresge, C.T., Schmitt, K.D., Chu, C.T.-W., Olson, D.H., Sheppared, E.W., McCullen, S.B., Higgins, J.B. and Schlenker, J.L. (1992) Journal of American Chemical Society, 114(27), 10834-10843.

[17] Tanev, P.T. and Pinnavaia, T.J. (1995) A neutral templat-ing route to mesoporous molecular sieves. Science, 267(5199), 865-867.

[18] Gao, Q., Chen, J., Xu, R. and Yue, Y. (1997) Chemistry of Materials, 9, 457-462.

[19] Tiemann, M., Froba, M., Rapp, G. and Funari, S.S. (2000) Nonaqueous synthesis of mesostructured aluminophos-phate/surfactant composites: synthesis, characterization, and in-situ SAXS studies. Chemistry of Materials, 12(207), 1342-1348.

[20] Huo, Q., Margolese, D.I., Ciesla, U., Demuth, D.G., Feng, P., Gier, T.E., Sieger, P., Firouzi, A., Chmelka, B.F., Schuth, F. and Stucky, G.D. (1994) Chemistry of Materi-als, 6(8), 1176-1191.

[21] Zhang, H. and Banfield, J.F. (1998) Size dependence of the kinetic rate constant for phase transformation in TiO2 nanoparticles. Journal of Material Chemistry, 8, 2073- 2076.

Vol.2, No.5, 469-475 (2010)doi: 10.4236/ns.2010.25058


Natural Science


Extraction, identification and adsorption-kinetic studies of a natural color component from G. sepium

Konaghatta Narayanachar Vinod1, Puttaswamy1*, Kurikempanadoddi Ninge Gowda2, Rajagopal Sudhakar2

1Department of Post-Graduate studies in Chemistry, Central College Campus, Bangalore University, Bangalore, India; *Corresponding Author: [email protected] 2Department of Apparel Technology and Management, Central College Campus, Bangalore University, Bangalore, India;

Received 24 December 2009; revised 25 January 2010; accepted 23 February 2010.

ABSTRACT

The use of synthetic dyes causes environmental pollution as majority of these dyes are toxic and non-biodegradable. Natural dyes on the other hand have proved to be eco-friendly, biodegradable and highly compatible with the environ-ment. Consequently, dyes derived from natural sources have emerged as important alternatives to synthetic dyes. In the present work, the major color component isolated from the pods of G. sepium plant is morin, which is a flavonoid moiety. The dyeing behaviour of this component on silk yarn was investigated. Mordanting studies have indicated that the post-mordanting method was found to be a better method com-pared to pre-mordanting. Variation of pH on dye extract pointed out that the maximum absorb-ance was at pH 4 and hence all the dyeing studies have been carried out at that pH. Ther-modynamic parameters were determined by studying the dyeing process at different tem-peratures. Heat of dyeing was positive which indicated the dyeing process was endothermic. The adsorption process of morin on silk was tested with Langmuir, Freundlich and Tempkin- Pyzhev isotherm models. The adsorption proc-ess followed both the Langmuir and Freund- lich isotherms. The value of regression coeffi-cient, however, indicated that the Langmuir isotherm was a better fit than the Freundlich isotherm. These results signified that the adsorption of morin on silk yarn was homogeneous in nature with the formation of a monolayer. Hence, the dye obtained from the pods of G. sepium plant may be an alternative source to synthetic dye for the dyeing of silk as well as other textile fabrics.

Keywords: G. Sepium; Morin; Adsorption-Kinetics;

Silk

1. INTRODUCTION

The use of synthetic dyes causes environmental pollu-tion as majority of these dyes are toxic and non-biode-gradable. For this reason, there is a revival of interest in the non-toxic and eco-friendly natural dyes. Nature pro-vides a wealth of plants which yield color for the pur-pose of dyeing, many have been used since antiquity [1-4]. Natural dyes exhibit better biodegradability com-pared to their synthetic counterparts and generally have a higher compatibility with the environment. There are several reports in the literature pertaining to the applica-tion of natural colorants and evaluation of their dyeing properties on various fibers [5-8], but a very few reports are available on their kinetic and adsorption aspects [9-11]. G. sepium, is an important member of the family Fabaceae, which is widely naturalized in the tropical Americas, Caribbean, Africa, Asia, and the Pacific Is-lands. Since this tree possesses high nitrogen-fixing properties and also the leaves can be used for mulch and green manure, which makes it highly suitable in agro- forestry systems. G. sepium is a small, thornless, semi- deciduous tree, which yields flattened pods, 10-15 cm long, containing three to eight seeds. The abundantly available pods, unless otherwise used go as natural waste.

Keeping this in view and also due to our continued interest in the identification of colorant from natural plants, a major color component from the pods of G. sepium plant was identified. The present research was performed with the following objectives: 1) To extract and isolate the main color component from the pods of G. sepium plant, 2) To explore the dyeing properties of the color component on silk, 3) To study adsorption iso-therms of the color component on silk and 4) To evaluate the thermodynamic parameters of the dyeing process through kinetic studies.

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2. MATERIALS AND METHODS

2.1. Materials

The pods of G. Sepium were collected from the south- eastern region, Shimoga, India. The pods were washed well with tap water and dried under laboratory condi-tions. The dried pods were then finely ground to powder. Raw silk yarn of 40 denier was scoured with 2 g dm-3 non-ionic detergent and 1 g dm-3 sodium carbonate at 90 for 1 h, and rinsed with water and dried under laboratory conditions.

2.2. Isolation

The air dried powder of G. Sepium pod (550 g) was ex-tracted with 90% methanol (3 × 250 ml) at room tem-perature using a soxhlet apparatus and the procedure was repeated till the color from the extract was negligible. The extracts were combined, concentrated under reduced pressure and the residue was successively extracted with pet. ether, chloroform and ethyl acetate. The ethyl ace-tate soluble fraction was concentrated under reduced pressure and was chromatographically separated over silica gel (60-120 mesh) using methanol as eluent. The fractions obtained were combined according to TLC (silica gel, CHCl3-MeOH-H2O, 80:18:2) in increasing order of polarity to yield two fractions. Solvent was evaporated from the first fraction (major) to get yellow colored amorphous compound and it was used for fur-ther studies.

2.3. Mordanting

Pre-and post-mordantings were carried out on silk using 2, 4 and 6% tannic acid and alum (Al(NH4) (SO4)2. 12H2O) as mordants, separately. Mordanting was carried out for 30 min at 70 and the silk was then rinsed with tap water and dried.

2.4. Dyeing

Open bath beaker dyeing machine equipped with pro-grammable control of temperature and time was used to carry out all dyeing studies. The silk yarn was dyed with 4% dye solution at pH 4 with M: L ratio of 1 : 20. The dyeing was started at 40 and the temperature was gradually raised to 90 in 20 min and the dyeing proc-ess was continued for 45 min. After dyeing, samples were removed from dyeing machine and soaped at 60 for 10 min. Further, the samples were rinsed with tap water and dried. In case of post-mordanting samples, soaping was done after mordanting.

2.5. Characterization

IR spectrum of the major color component was recorded on a Perkin-Elmer 298 grating IR Spectrophotometer.

The NMR spectrum of the compound was recorded on the Brucker 400 NMR spectrophotometer. The mass spectrum was recorded on an Esquire 3000 plus spectrometer.

2.6. Absorbance and Color Strength Measurements

The dye solutions (1-5%) of the extract were prepared with M: L ratio of 1 : 20. The absorbance of the dye so-lutions was recorded prior and after the dyeing process using UV-Vis spectrophotometer. The dye exhaustion was calculated using the equation, % Exhaustion = [(Cg-Ct)/Cg] × 100, where Cg is the concentration of dye offered and Ct is the concentration of residual dye in the spent liquor.

The surface reflectance and colorimetric data (CIE L* a* b* C h) for the dyed samples were obtained by Gretag Macbath Color Eye 7000A spectrophotometer. The spectrophotometer was interfaced to a PC under illuminant D65 with a 100 standard observer. Surface color strength (K/S) was calculated from the surface reflectance values using the Kubelka-Munk equation [12]: K/S = (1-R)2/2R, where R is the reflectance, K is the absorption coeffi-cient and S is the light scattering coefficient. A higher K/S value signifies better dye receptivity of the sub-strates. The wash fastness of the dyeing was tested using ISO method 105-C10-A(1)-2006 and crocking fastness was assessed using AATCC Test Method 8-1996. Color fastness to light was evaluated as per AATCC Test Method 16-2004 option 5.

2.7. Kinetic Studies

Kinetic studies were investigated with aqueous solution of the dye extract 0.5 g dm-3 (without any further purifi-cation) prepared in an acetate buffer of pH 4. The dyeing was carried out with M:L ratio of 1:20 at 50. Known volume (5 ml each) of the dye-bath solution was pipetted into a cuvette at regular intervals of time and absorbance measurements were made at its λmax 580 nm (Figure 1). The absorbance readings of Do and Dt at the beginning and at any time interval during the dyeing process,

00.20.40.60.8

11.21.41.61.8

2

400 450 500 550 600 650 700 750 800 850

Wavelength in nm

Abs

orba

nce

pH 4 pH 7 pH 9

Figure 1. Visible Spectra of G. sepium in aqueous solution at different pH.



471471


respectively, were recorded. Plots of log (Do /Dt) versus time were made to evaluate first-order rate constants (k/ s-1). The experiment was repeated at 60, 70 and 800C and the dye uptake rate was calculated at each temperature. The energy of activation and other thermodynamic pa-rameters were deduced.

2.8. Adsorption Isotherms

The adsorption isotherm indicates how the adsorption molecules distribute between the adsorbate and adsorb-ent when the adsorption process reaches an equilibrium state. The amount of dye adsorbed at equilibrium qe (mg/g) was calculated by the following mass balance equation [13]:

MCCVq eie /)( (1)

where V is the volume of solution used in the adsorption experiment, Ci and Ce are the initial and equilibrium concentrations of the dye (mg dm-3), respectively, and M is the mass of silk (g).

In the present case, the adsorption isotherm study was carried out on Langmuir, Freundlich and Tempkin-pyzhev isotherms. The applicability of the isotherm equa-tion was compared by judging the value of regression coefficients (r). The equilibrium adsorption isotherm is fundamental in describing the interaction between ad-sorbent and adsorbate, and is also important in the de-sign of an adsorption system. A basic assumption of Langmuir theory is that in which the sorption takes place at specific homogeneous sites within the adsorbent. It is then also assumed that once a dye molecule occupies a site, no further adsorption can take place at that site. Theoretically, a saturation value is reached beyond which no further sorption can take place. The Langmuir isotherm [14] was applied for adsorption equilibrium and represented as:

00

1

Q

C

bQq

C e

e

e (2)

where Ce = Concentration of adsorbent (mg dm-3) at equilibrium; qe = Amount of dye adsorbed at equilibrium (mg/g); Qo = A constant (mg/g) which signifies the prac-tical limiting adsorption capacity when the surface is fully covered with dye molecules and it aids in the com-parison of adsorption performance and b = Langmuir constant related to the affinity of the binding sites (cm-3

mg). Freundlich isotherm model assumes heterogeneous

surface energies, in which the energy term in Langmuir equation varies as a function of the surface coverage. The well known logarithmic form of Freundlich model [15] is given by the following equation:

n

CKq e

fe

logloglog (3)

where Kf and 1/n = Freundlich constant related to ad-sorption capacity and adsorption intensity respectively obtained from the plot. In general, as the Kf value in-creases the adsorption capacity of the adsorbent for the given adsorbate increases. 1/n is the heterogeneity factor, if n is close to unity, the surface heterogeneity could be assumed to be less significant and as n approaches 10, the impact of surface heterogeneity becomes more sig-nificant [16].

Tempkin and Pyzhev [17] considered the effects of indirect adsorbate-adsorbent interactions on the adsorp-tion isotherms. As a result, the heat of adsorption of all the molecules on the adsorbent surface layer would de-crease linearly. The Tempkin isotherm can be expressed in its linear form as:

ee CBABq lnln (4)

where B and A are the Tempkin constants and can be determined by a plot of qe versus ln Ce. The constant B is related to heats of adsorption and A is the equilibrium binding constant.


3.1. Characterization of Major Color Component

Morin (2', 3, 4', 5, 7-Pentahydroxyflavone) [18-20] was isolated from the pods of G. sepium as the major color component and it was confirmed by IR, 1H-NMR, 13C-NMR and Mass spectral studies.

3.2. Effect of Dye Concentration and Color Strength

Table 1 revealed that the absorption of dye increased with increase in dye concentration (1-5%) and the maximum dye uptake was observed at 4% concentration. The values of K/S also increased with the increase in dye concentration (Table 1). Further, it was noticed that the K/S value was higher at 4% dye concentration, indicat-ing deeper shades at higher concentrations. Therefore, 4% dye concentration was fixed as optimal concentra-tion for further dyeing process. Table 1. Absorbance (%) and K/S values of different dye con-centrations at 360 nm.

Dye oncentration (%)

Absorbance Dye uptake

(%) K/S

Before dyeing after dyeing

1 0.13 0.10 23.0 4.14

2 0.27 0.19 29.6 4.28

3 0.47 0.31 34.0 4.43

4 0.61 0.33 45.9 4.50

5 0.84 0.55 34.5 4.45



472

3.3. Effect of Mordanting

The K/S values of the mordanted samples were better compared to that of the un-mordanted samples (Table 2). The K/S values and the fastness properties (Table 3) of the post-mordanted samples were better compared to pre-mordant samples. Tannic acid was found to be a bet-ter mordant with deeper shades and fastness ratings as compared to alum in pre-mordanting. But in case of post-mordanting, both the mordants showed comparable K/S values. All the color co-ordinates were positive and hence the dyed samples were located in the yellow-red quadrant of the color-space diagram.

3.4. Effect of pH on Dye Extract

The visible spectrum of the dye extract at different pH (4, 7 and 9) is illustrated in Figure 1. The λmax of the dye extract remained the same with varying pH. The ab-sorbance of the dye extract increased with an increase in pH, which may be due to the high solubility of phenolic groups in the alkaline pH.

3.5. Kinetic Studies

From the linear plot log k/ versus 1/T (r = 0.9979), the energy of activation (Ea) was found to be 61.2 kJ mol-1. Further, thermodynamic parameters such as ΔH≠, ΔG≠,

Table 2. Color co-ordinates and K/S values of the dyed silk samples.

Method Mordants K/S L* a* b* C h

Nil 4.50 63.1 15.1 17.3 23.0 48.9

Alum (2%) 4.61 69.7 11.5 14.1 18.2 50.5

Alum (4%) 4.63 69.7 11.7 13.8 18.1 49.5

Alum (6%) 4.73 69.9 11.8 13.9 18.2 49.6

Tannic acid (2%) 5.22 75.2 9.85 12.0 15.5 50.7

Tannic acid (4%) 5.27 74.3 10.1 12.8 16.3 51.6

Pre

Tannic acid (6%) 5.37 74.9 9.41 12.7 15.8 53.5

Alum (2%) 5.76 62.5 12.9 23.5 26.9 61.1

Alum (4%) 5.55 63.1 13.2 22.8 26.3 59.9

Alum (6%) 5.44 62.8 13.2 22.9 26.5 60.1

Tannic acid (2%) 5.66 68.1 12.7 17.4 21.6 53.7

Tannic acid (4%) 5.57 68.7 12.4 17.5 21.5 54.5

Post

Tannic acid (6%) 5.30 66.2 13.2 19.9 23.9 56.3

L*- Lightness, a* - (+ ve- red, - ve- blue), b* - (+ ve- yellow, - ve- green), C-Chroma and h-Hue.

Table 3. Results derived from fastness properties of dyed silk samples.

Mordants light fastness crocking fastness wash fastness

wet dry

Nil 2 4 4-5 3-4

Alum (2%) 2 4-5 4-5 4

Alum (4%) 2-3 4-5 5 4-5

Alum (6%) 2-3 4-5 5 5

Tannic acid (2%) 2-3 4-5 5 4-5

Tannic acid (4%) 2-3 5 5 5

Pre

Tannic acid (6%) 3 5 5 5

Alum (2%) 2 4-5 4-5 4

Alum (4%) 2 4-5 4-5 4

Alum (6%) 2-3 4-5 5 4-5

Tannic acid (2%) 2 4-5 4-5 4

Tannic acid (4%) 2-3 5 5 4-5

Post

Tannic acid (6%) 2-3 5 5 5



473473


ΔS≠ and log A were calculated and recorded in Table 4. The high positive value of the standard free energy (ΔG≠) indicates the spontaneous and strong adsorption of dye molecules on the surface of silk. The enthalpy of dyeing or heat of dyeing (ΔH≠) was positive indicating that the dyeing process is endothermic in nature. Further, large negative entropy (ΔS≠) indicates that the dye molecules are more systematically arranged on the surface of silk yarn.

3.6. Adsorption Isotherms

The experimental adsorption data were analyzed using Langmuir, Freundlich and Tempkin and Pyzhev isotherm models. The Langmuir constants Qo and b were deduced from the intercept and slope of the plot 1/qe versus 1/Ce. The plot was linear with a regression value of r = 0.9987 (Figure 2). The value of Qo was found to be 142 mg/g, signifies the amount of dye required to form a complete mono layer at equilibrium. The value of Langmuir con-stant b = 1.97 cm3/mg, which relates with the binding energy of dye molecules on silk. Further, the essential characteristics of the Langmuir isotherm can be ex-pressed in terms of a dimensionless constant separation factor for equilibrium parameter, RL [21-23]:

）（ oL bC

R

1

1 (5)

where Co is the initial concentration of dye (mg/dm3) and b is the Langmuir constant (mL/mg). The value of RL indicates the type of isotherm to be either irreversible (RL= 0), favorable (0 < RL< 1), linear (RL = 1) or unfa-vorable (RL > 1). In the present study, the value of RL was found to be 0.835, indicating that Langmiur adsorp-tion isotherm to be a favorable process.

A plot of log qe versus log Ce was a straight line with a regression coefficient of 0.9975 (Figure 3) for Freun- dlich adsorption isotherm. The values of Kf and 1/n, were calculated from the intercept and slope of such a plot and were found to be 2.50 and 0.84, respectively. The value of 1/n indicates that the adsorption process is homogeneous, as the value is very close to unity.

Further, Tempkin and pyzhev model was also consid-ered to correlate the experimental data. The plot of qe versus log Ce was curvilinear with regression coefficient r = 0.9560 (Figure 4).

The linear regression coefficient was normally used to decide the most fitted isotherm in adsorption process. As seen from Table 5, the Langmuir model yielded some-what better fit (r = 0.9987) than the other models fitted. The results demonstrated the formation of homogeneous monolayer. Further, the value of 1/n (0.84) in Freundlich isotherm model also supports the formation of monolayer.

4. CONCLUSIONS

The major coloring component obtained from the pods of the G. sepium was identified as morin, a flavonoid.

The dyeing behaviour of this component on silk yarn was investigated. Mordanting studies have indicated that the post-mordanting method was found to be a better method compared to pre-mordanting. For dyeing of silk, heat of dyeing (∆H≠) was positive which indicates endo-thermic nature of the process. The negative value of ∆S≠ indicates a more ordered adsorption of color component on silk. The high positive value of free energy change signifies that the color component has stronger affinity Table 4. Kinetic and thermodynamic parameters for the dyeing of coloring matter on to silk yarn.

Temperature (0C) 104 k/ (s-1)

50 2.64 60 4.79 70 9.25 80 18.4

Ea (kJ mol-1) 61.2 ΔH ≠ (kJ mol-1) 58.4 ΔG≠ (kJ mol-1) 103 ΔS≠ (JK-1 mol-1) -133

log A 6.32

Table 5. Isotherm constants for silk dyeing with pods of G. sepium extract.

Langmuir isotherm constants

Qo (mg/g) 142 b (mL/mg) 1.97

r 0.9987 Freundlich isotherm constants

Kf (mg/g) 2.50 1/n 0.843 r 0.9975

Tempkin and Pyzhev isotherm constants B (mg/g) 3.43

A (mL/mg) 1.46 r 0.9560

Figure 2. Langmiur isotherm plot at pH 4 for dyeing silk with color component of G. sepium at 80.



474


Figure 3. Freundlich isotherm plot at pH 4 for dyeing silk with color component of G. sepium at 80.

10

30

50

70

90

0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4

2 + log Ce

qe

Figure 4. Tempkin and Pyzhev isotherm plot at pH 4 for dyeing silk with color component of G. sepium at 80.

towards silk. The adsorption isotherm of morin on silk was carried out using different models: Langmuir, Freundlich and Tempkin-Pyzhev models. The adsorption of morin on silk fitted well to the Langmuir and Freun- dlich isotherm. However, Langmuir model was found to be better fit, and suggesting the formation of homoge-neous monolayer on silk.

5. ACKNOWLEDGEMENTS

One of the authors (K.N.V.) is grateful to Bangalore University, Ban-galore for awarding the Research Fellowship under the Interdiscipli-nary Collaborative Research Project.

REFERENCES

[1] Cerrato, A., Santis, D.D. and Moresi, M. (2002) Produc-tion of luteolin extracts from Reseda luteola and assess-ment of their dyeing properties. Journal of the Science of Food and Agriculture, 82(10), 1189-1192.

[2] Angelini, L.G., Pistelli, L., Belloni, P., Bertoli, A. and Panconesi, S. (1997) Rubia tinctorum a source of natural dyes: Agronomic evaluation, quantitative analysis of alizarin and industrial assays. Industrial Crops and Products, 6(3-4), 303-307.

[3] Angelini, L.G., Bertoli, A., Rolandelli S. and Pistelli, L. (2003) Agronomic potential of Reseda luteola L. as new crop for natural dyes in textiles production. Industrial Crops and Products, 17(3), 199-203.

[4] Santis, D.D. and Moresi, M. (2007) Production of aliza-rin extracts from Rubia tinctorum and assessment of their dyeing properties. Industrial Crops and Products, 26(2), 151-154.

[5] Septhum, C., Rattanaphani, S., Bremner, J.B. and Rat-tanaphani, V. (2009) An adsorption study of alum-morin dyeing onto silk yarn. Fibers and Polymers, 10(4), 481-485.

[6] Kim, S.H. (2006) Dyeing characteristics and UV protec-tion property of green tea dyed cotton fabrics. Fibers and Polymers, 7(3), 255-259.

[7] Shanker, R. and Vankar, P.S. (2007) Dyeing cotton, wool and silk with Hibiscus mutabilis. Dyes and Pigments, 74(2), 464-468.

[8] Shaukat, A., Tanveer, H. and Rakhshanda, N. (2009) Optimization of alkaline extraction of natural dye from Henna leaves and its dyeing on cotton by exhaust method. Journal of Cleaner Production, 17(1), 61-65.

[9] Gulrajani, M.L., Bhaumik, S., Oppermann, W. and Har- dtmann, G. (2002) Kinetic and thermodynamic studies on red sandalwood. Indian Journal of Fibre & Textile Re-search, 27(1), 91-95.

[10] Rattanapani, S., Chairat, M., Bremner, J.B. and Rattana-pani, V. (2007) An adsorption and thermodynamic study of lac dyeing on cotton pretreated with chitosan. Dyes and Pigments, 72(1), 88-92.

[11] Janhom, S., Griffiths, P., Watanesk R. and Watanesk, S. (2004) Enhancement of lac dye adsorption on cotton fi-bers by poly(ethyleneimine). Dyes and Pigments, 63(3), 231-235.

[12] Kubelka, P. (1954) New contributions to the optics of intensely light-scattering materials. Part II: Nonhomo- geneous layers. Journal of the Optical Society of America, 44(4), 330-334.

[13] Alkan, M., Dogan, M., Turhan, Y., Demirbas, O. and Turan, P. (2008) Adsorption kinetics and mechanism of maxilon blue 5G dye on sepiolite from aqueous solutions. Chemistry Engineering Journal, 139(2), 213-216.

[14] Langmuir, I. (1916) The constitution and fundamental properties of solids and liquids. Journal of American Chemistry Society, 38(11), 2221-2221.

[15] Freundlich, H.M.F. (1906) Over the adsorption in solution. Journal of Physics and Chemistry, 57, 385.

[16] Davila, M.M., Elizalde, M.P. and Pelaez, A.A. (2005) Adsorption interaction between natural adsorbents and textile dyes in aqueous solution. Colloids and Surfaces A, 254(1-3), 107-109.



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[17] Tempkin, M.J. and Pyzhev, V. (1940) Recent modifica-tions to Langmuir isotherms. Acta Physiochim Under-graduate Research Scholarship Scheme, 12, 217-225.

[18] Ting, Y.C., Wen, H.P., Long, H.T., Hsuan, L.Y., Rong, C.F. and Chang, W.U.Y. (2009) Flavonol glycosides from Muehlenbeckia platyclada and their anti-inflammatory activity. Chemical and Pharmaceutical Bulletin, 57(3), 280-284.

[19] Ehala, S., Vaher, M. and Kaljurand, M. (2005) Charac-terization of phenolic profiles of Northern European ber-ries by capillary electrophoresis and determination of their antioxidant activity. Journal of Agricultural and Food Chemistry, 53(6), 6484-6487.

[20] Yinon, J., Issachar, D. and Boettger, H.G. (1978) Studies

in chemical ionization mass spectrometry of some fla-vonoids. Organized Mass Spectrometry, 13(7), 167-169.

[21] Kannan, N. and Sundaram, M. (2002) Kinetics and mecha-nism of removal of methylene blue by adsorption on vari-ous carbons-A comparative study. Dyes and Pigments, 51(1), 25-29.

[22] Jain, A.K., Gupta, V.K. and Suhas, A.B. (2003) Utiliza-tion of industrial waste products as adsorbents for the removal of dyes. Journal of Hazardous Materials, 101(1), 31-35.

[23] Sivaraj, R., Namasivayam, C. and Kadirvelu, K. (2001) Orange peel as an adsorbent in the removal of Acid violet 17 (acid dye) from aqueous solutions. Waste Manage-ment, 21(1), 105-108.

Vol.2, No.5, 476-483 (2010)doi: 10.4236/ns.2010.25059


Natural Science

Evidence for the existence of localized plastic flow auto-waves generated in deforming metals

Lev B. Zuev*, Svetlana A. Barannikova

1Institute of Strength Physics and Materials Science, Siberian Branch of Russian Academy of Sciences, Tomsk, Russia; [email protected]; *Corresponding Author: [email protected]


ABSTRACT

The localized plastic flow auto-waves observed for the stages of easy glide and linear work hardening in a number of metals are considered. The propagation rates were determined experimentally for the auto-waves in question with the aid of focused-image holography. The dis-persion relation of quadratic form derived for localized plastic flow auto-waves and the de-pendencies of phase and group rates on wave number are discussed. A detailed comparison of the quantitative characteristics of phase and group waves has revealed that the two types of wave are closely related. An invariant is intro-duced for localized plastic flow phenomena occurring on the micro-and macro-scale levels in the deforming solid.

Keywords: Metallic Materials; Mechanical Testing; Optical Interferometry; Strengthening and Mechanisms; Crystal Plasticity; Fracture

1. INTRODUCTION

The experimental evidence obtained previously [1-4] suggests that the plastic deformation tends to localize in the deforming solid over the entire flow process. Plastic flow localization is most pronounced on the macro-scale level where the type of local strain pattern is governed by the law of work hardening acting at a given flow stage, i.e. ddE 1 (here Е is the elasticity modulus). The localization of plastic deformation will assume in this case the form of auto-wave1, i.e. a

self-excited process [3,4]. The occurrence of auto- wave processes by the plastic deformation is considered, e.g. in the context of gradient plasticity theory [6-8].

A considerable body of experimental and theoretical evidence pertaining to plastic flow macro-localization has been obtained thus far [1-4,6], which suggests that the macro-scale inhomogeneities of localized plastic flow have a typical scale of about 10–2 m. A characteris-tic picture is created in the deforming specimen where deformed material zones move in a concerted manner, generating thereby localized plastic flow auto-waves, which have typical wavelength of about 10–2 m. Thus a deforming body would spontaneously separate into al-ternating deformed and undeformed zones (Figure 1). Following H. Hacken [9], the spontaneous emergence of plastic flow inhomogeneities might be regarded as a manifestation of self-organization processes occurring in the deforming medium.

2. EXPERIMENTAL PROCEDURE AND MATERIALS TESTED

On the base of available experimental evidence [1-4], the quantitative characteristics of auto-wave processes were determined for a wide circle of pure metals and alloys, both single crystals and polycrystalline ones, having FCC, BCC and HCP crystal lattice. It is found that the mechanical characteristics of investigated mate-rials and the shape of plastic flow curves obtained for the test specimens would vary significantly, depending on chemical composition, grain size of polycrystalline ma-terials and extension axis orientation of single crystals. In what follows the distinctive features common to all the investigated materials are discussed. 1Autowaves are opposed to, e.g. elastic waves of the type

kxwt sin The experimental observations of localized plastic flow auto-waves [1-4] were conducted using the technique of focused-image holography related to speckle photography [10]. This technique was specially deve-

, in that they are solutions to parabolic differential

equations in the partial derivatives yDyxy ,

ycy 2[5], while

the latter obey hyperbolic equations of the type .


L. B. Zuev et al. / Natural Science 2 (2010) 476-483


477477


loped to facilitate the determination of displacement vector fields and the calculation of plastic distortion tensor components for the deforming specimen. One can visualize localized plastic flow nuclei, using the spatial distributions of plastic distortion tensor components; the kinetics of nuclei motion can be determined from the temporal evolution of nuclei.

The most interesting scenario is realized in single crystals and polycrystalline specimens tested in tension at a constant rate.

At the stages of easy glide and linear work hardening localized plastic flow auto-waves would be generated in the deforming specimen where the flow stress is re-lated to the deformation as i (here i = 1, 2 for

easy glide stage and linear work hardening, respectively, and 1

2 ). The emergent picture comprises a set of

equidistant localization zones, which moves as a whole at a constant rate, generating thereby so-called phase auto-waves (Figure 1). The nature of phase auto-waves merits special study.

The main characteristics of auto-waves, i.e. wavelength and period Т, are determined from the co-ordinates of nuclei against time (see Figure 2). Then the propagation rate of auto-waves is estimated as

kTVaw (here T 2 is the frequency

and 2k , the wave number).

1) auto-wave propagation rate; 2) dispersion relation for auto-waves; 3) change in the entropy of the system upon auto-

wave generation; 4) correspondence between the emergent pattern and

the given flow stage.

3. CHARACTERISTICS OF LOCALIZED PLASTIC FLOW AUTOWAVES: EXPERIMENTAL RESULTS

3.1. Autowave Rate

Our findings [1,2] and complementary information along these lines obtained by other workers permit the follow-ing conclusion: the propagation rate of auto-waves is a function of the work hardening coefficient, i.e.

/1~/VV 0aw (1)

where and are constants and . It is of

importance that auto-wave rates are in the range 10-5 10-4 m·s-1. Relation (1) applies to both the

easy glide and the linear work hardening stage, with the constants and having different values for the

same stages.

0V

aw

V

0V

V

0

To begin our discussion of the nature of auto-wave

processes, we must mention that plasticity waves occurring by impact loading are described in sufficient detail (see, e.g. [11]).

Plasticity wave rates are in the range 10 pwV 1/2( / )

V

102 m·s-1 (cf. m·s-1).

Apparently,

5 410 10awV

pwaw V .

Besides, the dependencies of wave rate on work hardening coefficient, V(θ), obtained for these two types

of wave differ essentially in form, i.e. 21~ pwV [11])

and (see Eq.1). The latter relation holds good

for all the investigated materials whose plastic flow curve shows easy glide and linear work hardening stages. Thus, the above quantitative analysis of the wave char-acteristics suggests that we are dealing here with two altogether different types of wave.

1~ awV

Figure 1. Auto-wave of plastic deformation localization propagating at the linear work hardening stage in the tensile single crystal of alloyed Fe; -local elongation; x and y-specimen

length and width, respectively; F-external load; -spacing of nuclei (auto-wave length); -auto-wave propagation rate.

xx

awV

Figure 2. Determination of the spatial () and temporal (T) periods of localized plastic deformation for the stages of easy glide (1) and linear work hardening (2) in single crystals of alloyed Fe; )( -stress-strain dependence; tX -variation in the co-ordinates (, and ) of localization nuclei with time.



478


3.2. Dispersion Relation for Autowaves

To gain a better understanding of the nature of auto- wave processes involved in plastic flow localization, one must consider the dispersion relation k , which is

characteristic for localized plastic flow auto-waves gen-erated at linear work hardening stage [12].

Relation has been complemented by an addi-

tional branch, which corresponds to the occurrence of a periodic localization pattern at the easy glide stage (see Figure 3(a)), i.e.

k

200 kkk (2)

where, , 0 and are constants, which depend on

work hardening stage and kind of material. Note that for easy glide, and for linear work hardening, Substituting into relation (2) of

0k

~0 and

21

00 /~ kk k sign (here ~ is the dimen-

sionless frequency and k~

is the wave number and 1sign for 0 ; 1sign for 0 is

a signum function of the term from Eq.2) yields the fol-lowing canonic formula 2 1 k (see the plot pre-sented in Figure 3(b)).

The above dispersion relation of quadratic form satis-fies the Schrödinger nonlinear equation [13,14] com-monly applied to self-organization processes occurring in active nonlinear media, which is an undeniable proof that plastic flow localization is a process involved in the self-organization of the deforming medium.

4. DISCUSSION OF RESULTS

4.1. Invariant for Deformation Processes

On the base of experimental data a close correlation has been established between the product of auto-wave macroscopic parameters, awV , and that of material

microscopic parameters, (here d is the spacing

between close-packed planes of the lattice and is the

rate of transverse elastic waves).

Vd

V

The Table 1 lists numerical data for seven metals in-vestigated. In each instance, the following equality ap-parently holds good within an acceptable range of accu-racy, i.e.

VdVaw 2/1 (3)

where the terms have the units of the diffusion coefficient . To verify relation (3), we used borrowed values of d and [15,16]. Relation (3) was

averaged for easy glide and linear work hardening stages as

12 TL

V

VdV2 aw (1.04 0.14) 1. Eq.3 was plotted

in the dimensionless co-ordinates awVVd to give

a rectilinear diagram aw7 VV22.01082.0d

(Figure 4).

(a)

– single crystals of Cu, Sn and alloyed Fe; – single crystals of alloyed Fe; – polycrystalline Al;

(b)

– easy glide stage; – linear work hardening stage.

Figure 3. A generalized dispersion curve obtained for the stages of easy glide. (1) and linear work hardening (2) in the test specimens; (a) –original data k ; (b)

–canonical form of dispersion relation in the dimen-sionless va s riable ( )k .

– easy glide stage; – linear work hardening stage.

Figure 4. Verification of the validity of relation (2) plotted for auto-waves in the dimensionless co-ordinates

awVVd .



479479

awV

and matched for easy glide and linear work hardening stages. Vd

awV

Table 1. The products


Flow stage Metal 107 (m2/s) D 1010 (m) V 10–3 (m/s) Vd 107 (m2/s)

Vd

Vaw2

Cu 1.90 2.08 2.30 4.78 0.79 Fe 3.03 2.07 3.32 6.87 0.88 Easy glide Sn 3.28 2.91 1.79 5.20 1.26 Cu 3.60 2.08 2.30 4.78 1.50 Al 7.92 2.33 3.23 7.52 2.10 Zr 1.92 2.46 2.25 5.53 0.70 V 2.80 2.14 2.83 6.06 0.92 Fe 2.55 2.07 3.32 6.87 0.74 Ni 2.10 2.03 3.22 6.54 0.64

Linear work harde-ning

Sn 2.34 2.91 1.79 5.20 0.90

Eq.3 relates the micro-scale characteristics d andV ,

which are observed for elastic waves propa-gating in crystals, to the macro-scale parameters

and ,

which are observed for elastic waves propagating in crystals, to the macro-scale parameters

awV

and ,

which are obtained for localized plastic flow auto-waves generated in the same crystals. The products of these values, and , are invariants for elastic and

plastic deformation processes, respectively (<< 1 and 1, respectively). The above regularity stems from the fact that the processes of elastic and plastic deformation are closely related. In the course of deformation the redistribution of elastic stresses occurs via micro-scale processes at the rate , while the rearrangement of

localized plastic flow nuclei involves macro-scale processes occurring at the rate , with the processes

of both types being related by Eq.3. Thus, the macro- localization phenomena must be regarded as an attribute of plastic deformation rather than a random disturbance of plastic flow homogeneity.

awV

Vd awV

V

awV

4.2. On the Physical Meaning of Relation (1)

By considering the nature of localized plastic flow auto-waves, it might be pertinent to discuss the origin of dependency and, in particular, the meaning of

deformation processes occurring in crystals is the propagation rate of elastic waves, i.e. sound rate .

For most metals 5103 m·s-1; hence

1~ awV

SVSV

1010SV .

To account for relation (1), the Dirac large-numbers hypothesis [17] was invoked. An appropriate dimensionless relation of the same order was also required which could be applied to the quantities associated with the deformation processes2. The relation of deforming medium’s viscosities defined for two limiting cases was thought to be an appropriate one, with the limiting cases being the quasi-viscous motion of dislocations, involving no interaction with local obstacles, and the break- away of dislocation segments from local obstacles. In the former case, the motion of dislocations occurs at

high acting stresses; the dislocation velocity as a function of applied stress has the form BbV disl (here

b is the Bürgers vector; B (1-3)10–4 Pas is the coefficient of dislocation drag, which characterizes the viscos-ity of phonon gas in the crystal) [18]. In the latter case, viscosity is defined from internal friction measurements to yield 3106 Pas [19]. In the case of ultrasound waves, stresses have low amplitudes; therefore, the vis-cosities observed for micro-scale plastic deformation processes might have similar values. Hence the ratio

B 1010 and, consequently, BV

V/B

S . Then one

can write

S (4)

Eq.4 can be interpreted as follows. Complex systems capable of structure formation will spontaneously sepa-rate into an information subsystem and a dynamic one [20]. It is thus assumed that the information subsystem, which is characterized by low-amplitude stresses and high viscosity, is represented by acoustic emission sig-nals whereas the dynamic subsystem, which is charac-terized by high-amplitude stresses and low viscosity, is represented by the motion of individual dislocations and dislocation ensembles, with Eq.4 formalizing the rela-tionship between the two subsystems. Thus, the former subsystem is related to the processes involving elastic wave propagation and the latter, to dislocation plasticity proper.

To better understand the physical meaning of Eq.1, one can also invoke the notions incorporated into the concept of work hardening. It is assumed that

lddV ~aw (here l is the length of slip line). Accor-

ding to the work hardening concept proposed by Seeger

[21], * * l , with and depending on ma-

terial kind; for linear work hardening, the coefficient of

work hardening

21

2~

3 nb (here b is the Bürgers

vector of dislocations and n is the number of dislocations in a dislocation pileup). In the latter case, , i.e.



480


2aw /d)/(1nbкdV (5)

where the coefficient has the units [T–1]. With depending only weakly on material type [21], Eq.1 can be derived from (5), considering that

*

/1)/( nbкVaw (6)

where . )/( nbк

The coefficient can be calculated by taking into

account that the values and depend only weakly on material kind and deformation [21]. Indeed, the deriva-tive

*

ddVaw 1.5·10–2 m·s can be estimated from the

data reported in [1,2]. For the increment in the deforma-tion 0.05, an increase in the length of slip line l is about 3·10–4 m [21]. Hence /)d/dV( awк 10 s–1.

Provided n 20 [21] and b 2·10–10 m, 8·10–7 m·s-1, which is close to the experimental value

5·10–7 m·s-1. The physical significance of the above difference lies

in the fact that localized plasticflow auto-waves belong to an altogether different class of wave phenomena, which are not identical with plasticity waves [11]. There- fore, these two types of waves cannot be grouped to-gether.

4.3. Treatment of Dispersion Relation

Dispersion relation (2) of quadratic form can be ex-plained as follows. Relation (3) can be rewritten as

kkVdVdVaw )4/(/1)2/( (7)

Let the rate of localized plastic flow auto-wave, (here Vgr is the group rate); thengraw VV dkdVaw .

Hence

dkkd (8)

Integration can be performed for Eq.8 as follows

0

0 0

kk

dkkd

(9)

to yield the dependence.

200

200 2 kkkkk , which is

identical with dispersion relation (2) derived experimentally for localized plastic flow auto-waves.

Apparently, the dispersion relation of quadratic form

, which is obtained for localized

plastic flow auto-waves occurring at the stages of easy glide and linear work hardening, follows from the equal-ity

200 kkk

VdVaw 21 , which relates the macro-scopic

characteristics of localized plastic flow auto-waves and the microscopic parameters of material crystal lattice.

The right-hand side of Eq.3 can be rewritten as

DdVd 221 (here D is the Debye frequency

and DdV 2

DBk

). Using the well-known formula

D (here is the Boltzmann constant; Bk

2h is the Planck constant and D is the tempe-

rature-ependent Debye parameter [22]), one can write

/)(2/1 2 TkdVVd DBaw (10)

Eq.10 may be useful since it predicts the temperature dependence TV Daw ~ for localized plastic flow

auto-waves [1].

4.4. Group and Phase Rates of Localized Plastic Flow Autowaves

In accordance with dispersion relation (2), the phase and group rates of localized plastic flow auto-waves can be

represented in the dimensionless variables ~ and k~

(see Figures 5(a) and 5(b)) as kkph kV~~

~~~ ~ 1

and kkddVgr

~~

~~~ , respectively (see also Figure

3(a)). From Eq.10 follows that

kkhkdkdV DBDBaw )/(/1)/( 22 (11)

The experimental evidence suggests that . The

quantity

kVgr ~

DDB dhkd 22 from (11) is taken to

be a proportionality coefficient, which can be readily calculated, using d values reported in [15] and the Debye temperatures, FeD 420 K and AlD 394 K ob-

tained for the single crystals of iron and aluminum [22]. The calculated values of the proportionality coeffi-

cient obtained for the single crystals of iron and alumi-

num are, respectively, hkd DB Fe2

Fe ≈ 3.7·10–7

m2·s–1 and hkd DB Al2

Al ≈ 4.45·10–7 m2·s–1. The

experimental values determined for the same materials

from the inclination of plots are kVgr Fe = (1 ±

0.08)·10–7 m2·s–1 and Al = (12.9 ± 0.15)·10–7 m2·s–1,

respectively. Matching of the calculated and the experi-mental values reveals a satisfactory agreement between the two sets of data.

It also follows from Figures 5(a) and 5(b) that the functions kV ph

~~ and kVgr

~~ show fundamentally

different behaviours for the stages of easy glide and lin-ear work hardening: in the former case, they would not

intersect for any k~

values, while in the latter case, they

fully coincide for ~

1k . The above difference in the function behaviours is attributable to the fact that the stage of easy glide is generally characterized by plastic

2We had to overcome a certain difficulty since Dirac’s hypothesis was initially applied to values in the ratio of about 1032.



481481


flow instabilities, while at the stage of linear work hard-ening the plastic flow will occur in a steady-state regime, with the latter case evidently corresponding to the ab-sence of dispersion, i.e.

phgr V~

V~

.

4.5. Change in the Entropy of the Deforming System by Auto-Wave Generation

The dependencies obtained for the above two types of wave are found to differ radically in form, i.e.

wV

21~ pwV and . It might be also expected that the thermodynamic properties of the medium, in par-ticular, entropy, will change in a dissimilar way by the generation of the two types of wave. It would appear

reasonable to suggest that plastic deformation

1~ awV

wV~

(here is the rate of a certain type of wave). Provided

mobile dislocation density

wV

constm , the Taylor-

Orowan equation for plastic flow rate [23]

can be applied to give

dislmVb

wdisl VV ~~

G

. For thermally ac-tivated dislocation motion, the rate is given as

(here is the Gibbs thermodynamic potential; U is the internal energy;

S is the entropy of the process;

)T/~ GVdisl exp( k B ATSU

A

(exp()/ Uk B

is the work of external stresses by the deformation and is the activa-tion volume of an elementary deformation act [23]). Hence the propagation rate is given for any type of wave as

)/)exp(~~~ TkSVV Bdislw (12)

The enthalpy

S~

wV

UH

V wln

observed for linear work hardening stage is virtually the same for most metallic materials. Consequently, taking the logarithm of (12)

evidently yields . Thus, a close correspondence is found to exist between

the rectilinear diagrams plotted in the co- ordi-

nates lnln wV for the both types of wave process (see Figure 6) on the one hand and the linear dependen-

cies lnS obtained for the same processes on the other hand. The diagrams were plotted using wave rates listed in the table for the stage of easy glide in single crystals and for the stage of linear work hardening in single crystals and polycrystalline metals and alloys (Figure 6, lines 1 and 2, respectively). The wave rates

were calculated from the expression 21

0 )/( pwV (Figure 6, line 3) and the values, from the loading curves of investigated materials; besides, borrowed

0 values were used [15].

An analysis of the dependencies wV shows that in the case of plasticity waves, an increase in the entropy of

the system would occur ( ) (see Figure 6, line 3),

which is characteristic for processes accompanied by dissipation of energy. In the case of auto-waves, the en-

tropy of the system would decrease ( ) (see Figure 6, lines 1 and 2 for easy glide and linear work hardening, respectively).

0S

0S

The above results suggest that localized plastic flow waves differ radically from other types of wave process related to plastic deformation in solids. The generation of localized plasticity waves would cause a decrease in the entropy of the deforming system, which is an indication of its self-organization (ordering) [9] since entropy is a function of the parameter of order [24]. By considering localized plastic flow waves, the coefficient of

work hardening 1 might be regarded as a para-

meter of order so that ln~S . With growing value, the entropy of the system would change linearly, with

0S corresponding to auto-waves and , to 0S

(a)

(b)

Figure 5. Wave number dependencies of phase () and group () propagation rates plotted for localized plastic flow auto-waves in the dimensionless invariables kV

~~ ; (a)

easy glide stage; (b) linear work hardening stage.



482


Figure 6. Changes in the entropies of plasticity waves (3) and of localized plastic flow auto-waves plotted for the stages of easy glide (1) and linear work hardening (2) in the co-ordinates lnln wV (see the axis ). SVw ~ln

plasticity waves (see Figure 6).

The above suggests that plasticity waves [11] are commonly known dissipative processes, while localized plastic flow auto-waves are self-organization processes that are liable to cause a decrease in the entropy of the deforming system [9].

4.6. Correspondence Between Localized Plastic Flow Patterns and Work Hardening Stages

Of particular importance is the finding that localized plastic flow patterns emerging in a deforming solid are related to the respective flow stages [21]. These stages can be readily distinguished on the flow curve of the form [21]

n 0 (13)

where 0 is the proof stress and n is the parabola ex-

ponent. The latter value will change discretely with the deformation, which enables individual stages to be sin-gled out on the flow curve.

Using this method, a correspondence rule has been established for single crystals of metals and alloys and polycrystalline materials. This holds that

For n = 0 (yield plateau) or n 0 (easy glide), a soli-tary nucleus of localized plastic flow travels along the extension axis;

For n = 1 (linear work hardening), localized plastic flow auto-waves are generated, which have wavelength and propagation rate ; awV

For n = ½ (parabolic work hardening or Tailor’s stage), a set of immobile localized plastic flow nuclei is ob-served;

For 0 < n < ½ (pre-failure stage), collapse of auto- wave takes place, which corresponds to macro-necking

[25] and For n = 0, ductile failure of material will occur. The proposed rule evidently states that typical local-

ization patterns observed on the macro-scale level are reflections of vastly different microscopic mechanisms involved in material work hardening at the different flow stages. This also testifies to the fact that the events in-volved in the deformation on the micro-scale level are directly related to those occurring on the macro-scale level in the deforming medium.

5. CONCLUSIONS

At all its stages the plastic flow involves localization processes that take the form of different types of auto- wave. The plastic flow tends to localize over the entire process; therefore, localization is taken to be its integral attribute.

The parameters of localized plastic flow evolution are found to be related to those of elastic deformation processes as VdVaw 2/1 . This suggests that the defor-

mation process will exhibit scale invariance on both the micro-scale level Vd and the macro-scale one

awV .

Due to the deformation process exhibiting invariance, the dispersion relation derived for localized plastic flow auto-waves that are generated at the stages of easy glide and linear work hardening has quadratic form, i.e.

2~1~ k .

A decrease in the entropy of the deforming medium strongly suggests that localized plasticity auto-waves are processes involved in the self-organization of the me-dium.

The localized plastic flow patterns are found to strictly correspond to the respective flow stages in single crys-tals and polycrystalline materials.

6. AKNOWLEDGEMENTS

This work was partly supported by the grant of RFBR (09-08- 00213-а).

REFERENCES

[1] Zuev, L.B. (2001) Wave phenomena in low-rate plastic flow of solids. Annals of Physics, 10(11-12), 965-984.

[2] Zuev, L.B. (2007) On the waves of plastic flow localization in pure metals and alloys. Annals of Physics, 16(1), 286-310.

[3] Zuev, L.B. and Danilov, V.I. (1999) A self-excited wave model of plastic deformation in solids. Philosophical Magazine A, 79(1), 43-57.

[4] Zuev, L.B. and Semukhin, B.S. (2002) Some acoustic



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properties of a deforming medium. Philosophical Maga-zine A, 82(6), 1183-1193.

[5] Zemskov, V.P. and Loskutov, A.Y. (2008) Oscillatory traveling waves in excitable media. Journal of Experi-mental and Theoretical Physics, 107(2), 344-349.

[6] Aifantis, E.C. (1996) Nonlinearly, periodicity and patter- ning in plasticity and fracture. International Journal of Non-Linear Mechanism, 31(8), 797-809.

[7] Aifantis, E.C. (1995) Pattern formation in plasticity. International Journal of Engineering Science, 33(15), 2161-2178.

[8] Aifantis, E.C. (2001) Gradient plasticity. In Lemaitre, J., Ed., Handbook of Materials Behavior Models, Acade- mic Press, New York, 291-307.

[9] Haken, H. (1988) Information and self-organization. A Macroscopic Approach to Complex Systems. Springer Verlag, Berlin.

[10] Zuev, L.B., Gorbatenko, V.V. and Polyakov, S.N. (2002) Instrumentation for speckle interferometry and techniques for investigating deformation and fracture. Procee- dings of SPIE, 4900, 1197-1208.

[11] Kolsky, H. (1963) Stress waves in solid. Dover, New York.

[12] Barannikova, S.A. (2004) Dispersion of the plastic strain localization waves. Technical Physics Letters, 30(4), 338-340.

[13] Scott, A. (2003) Nonlinear science. University Press, Oxford.

[14] Braun, O.M. and Kivshar, Y.S. (2004) The Frenkel- Kontorova model. Springer, Berlin.

[15] Donnay, J.D.H. (1963) Crystal data. Determinate Tables,

Williams and Heintz Map Corp., Washington.

[16] Anderson, O.L. (1965) Determination and some applications of the isotropic elastic constants of polycrystalline systems, obtained from single crystals data. In Mason, W. P., Ed., Physical Acoustics: Principles and Methods, 3B, Academic Press, New York, 43-95.

[17] Dirac, P.A.M. (1978) Directions in physics. John Wiley and Sons, New York.

[18] Al’shits, V.I. and Indenbom, V.L. (1975) Phonon and electron drag of dislocations. Soviet Physics-Uspekhi, 115(18), 3-39.

[19] Nowick, A.S. and Berry, B.S. (1972) Anelastic relaxation in crystalline solids. Academic Press, New York.

[20] Kadomtsev, B.B. (1994) Dynamics and information. Physics-Uspekhi, 164(7), 449-530.

[21] Seeger, A. (1957) Mechanism of glide and work-hardening in face-centered cubic and hexagonal close packed metals. In Fisher, J.C., Ed., Dislocations and Mechanical Properties of Crystals, John Wiley and Sons, Inc., New York, 243-329.

[22] Ashcroft, N. and Mermin, N. (1976) Solid state physics. Holt, Rinehart and Winston, New York.

[23] Aifantis, E.C. and Gerberich W.W. (1975) A theoretical review of stress relaxation testing. Materials Science and Engineering, 21(4), 107-113.

[24] White, R.M. and Geballe, T.H. (1979) Long range order in solids. Academic Press, New York.

[25] Zuev, L.B., Danilov, V.I. and Barannikova, S.A. (2008) Plastic flow, necking and failure in metals, alloys and ceramics. Materials Science and Engineering A, 483-484, 223-227.

Vol.2, No.5, 484-488 (2010)doi: 10.4236/ns.2010.25060


Natural Science

Higher dimensional bianchi type-V universe in creation-field cosmology

Kishor S. Adhav*, Shivdas D. Katore, Abhijit S. Bansod, Prachi S. Gadodia

Department of Mathematics, Sant Gadge Baba Amravati University, Amravati, India; *Corresponding Author: [email protected]

Received 16 December 2009; revised 6 March 2010; accepted 15 March 2010.

ABSTRACT

We have studied the Hoyle-Narlikar C-field cos-mology with Bianchi type-V non static space- time in higher dimensions. Using methods of Narlikar and Padmanabham [1], the solutions have been studied when the creation field C is a function of time t only as space time is non static. The geometrical and physical aspects for model are also studied.

Keywords: Bianchi Type-V Space-Time, Creation Field Cosmology, Cosmological Model of Universe, Higher Dimensions

1. INTRODUCTION

The study of higher dimensional physics is important because of several prominent results obtained in the de-velopment of the super-string theory. In the latest study of super-strings and super-gravity theories, Weinberg [2] studied the unification of the fundamental forces with gravity, which reveals that the space-time should be dif-ferent from four. Since the concept of higher dimensions is not unphysical, the string theories are discussed in 10- dimensions and 26-dimensions of space-time. Because of this, many researchers are inspired to study the higher dimensional to explore the hidden knowledge of the uni-verse. Chodos and Detweller [3], Lorentz-Petzold [4], Ibanez and Verdaguer [5], Gleiser and Diaz [6], Banerjee and Bhui [7], Reddy and Venkateswara [8], Khadekar and Gaikwad [9], Adhav et al. [10] have studied the multi-dimensional cosmological models in general rela-tivity and in other alternative theories of gravitation.

The three important observations in astronomy namely the phenomenon of expanding universe, primor-dial nucleon-synthesis and the observed isotropy of cosmic microwave background radiation (CMBR) were supposed to be successfully explained by big-bang cos-mology based on Einstein’s field equations. However, Smoot et al. [11] revealed that the earlier predictions of

the Friedman-Robertson-Walker type of models do not always exactly meet our expectations. Some puzzling results regarding the red shifts from the extra galactic objects continue to contradict the theoretical explana-tions given from the big bang type of the model. Also, CMBR discovery did not prove it to be a out come of big bang theory. Infact, Narlikar et al. [12] have proved the possibility of non-relic interpretation of CMBR. To ex-plain such phenomenon, many alternative theories have been proposed from time to time. Hoyle [13], Bondi and Gold [14] proposed steady state theory in which the uni-verse does not have singular beginning nor an end on the cosmic time scale. Moreover, they have shown that the statistical properties of the large scale features of the universe do not change. Further, the constancy of the mass density has been accounted by continuous creation of matter going on in contrast to the one time infinite and explosive creation of matter at t = 0 as in the earlier standard model. But the principle of conservation of matter was violated in this formalism. To overcome this difficulty Hoyle and Narlikar [15] adopted a field theo-retic approach by introducing a mass less and charge less scalar field C in the Einstein-Hilbert action to account for the matter creation. In the C-field theory introduced by Hoyle and Narlikar there is no big bag type of singu-larity as in the steady state theory of Bondi and Gold [14]. A solution of Einstein’s field equations admitting radiation with negative energy mass less scalar creation fields C was obtained by Narlikar and Padmanabhan [1]. The study of Hoyle and Narlikar theory [15-17] to the space-time of dimensions more than four was carried out by Chatterjee and Banerjee [18]. The solutions of Ein-stein’s field equations in the presence of creation field have been obtained for Bianchi type-V universe in four dimensions by Singh and Chaubey [19].

Here, we have considered a spatially homogeneous and anisotropic non static Bianchi type-V cosmological model in Hoyle and Narlikar C-field cosmology with five dimensions. Therefore, we have assumed that the creation field C is a function of time t only i.e.

K. S. Adhav et al. / Natural Science 2 (2010) 484-488


485485


tCtxC , . This study is important because of the fact that the

resulting cosmological model is considered to be ame-nable to the model obtained by Singh and Chaubey [19].

2. HOYLE AND NARLIKAR C-FIELD COSMOLOGY

Introducing a mass less scalar field called as creation field namely C-field, Einstein’s field equations are modified. Hoyle and Narlikar [15-17] field equations are

ij

c

ij

m

ijij TTRgR 82

1

(1)

where is matter tensor of Einstein theory and

is matter tensor due to the C-field which is given by ij

m T ij

cT

k

k

ijjiij

c CCgCCfT2

1 (2)

where and 0fii x

CC

.

Because of the negative value of , the

C-field has negative energy density producing repulsive gravitational field which causes the expansion of the universe. Hence, the energy conservation equation re-duces to

00000 TT

jji

jijc

jijm CfCTT ;;; (3)

i.e. matter creation through non-zero left hand side is possible while conserving the over all energy and mo-mentum.

Above equation is similar to

0 j

i

ij Cds

dxmg (4)

which implies that the 4-momentum of the created parti-cle is compensated by the 4-momentum of the C-field. In order to maintain the balance, the C-field must have negative energy. Further, the C-field satisfy the source

equation and ii

ii JCf ;; i

ii v

ds

dxJ , where

is homogeneous mass density.

3. METRIC AND FIELD EQUATIONS

The five-dimensional Bianchi-Type-V line element can be written as

222

4

222

3

222

2

22

1

22 dueadzeadyeadxadtds mxmxmx (5)

where , , and are functions of t only and

m is constant. 1a 2a 3a 4a

Here the extra coordinate is taken to be space like.

The above space time is non static, hence, we have assumed that creation field C is function of time t only i.e.

tCtxC , and ppppdiagT i

j

m ,,,,(6)

We have assumed that velocity of light and gravita-tional constant are equal to one unit.

Now, the Hoyle-Narlikar field Eq.1 for metric (5) with the help of Eqs.2, 3, and 6 can be written as

2

2

1

2

43

43

42

42

32

32

41

41

31

31

21

21

2

18

6

Cf

a

m

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

(7)

2

2

1

2

43

43

42

42

32

32

4

4

3

3

2

2

2

18

3

Cfp

a

m

aa

aa

aa

aa

aa

aa

a

a

a

a

a

a

(8)

2

2

1

2

43

43

41

41

31

31

4

4

3

3

1

1

2

18

3

Cfp

a

m

aa

aa

aa

aa

aa

aa

a

a

a

a

a

a

(9)

2

2

1

2

42

42

41

41

21

21

4

4

2

2

1

1

2

18

3

Cfp

a

m

aa

aa

aa

aa

aa

aa

a

a

a

a

a

a

(10)

2

2

1

2

32

32

31

31

21

21

3

3

2

2

1

1

2

18

3

Cfp

a

m

aa

aa

aa

aa

aa

aa

a

a

a

a

a

a

(11)

4

4

3

3

2

2

1

13a

a

a

a

a

a

a

a (12)

Ca

a

a

a

a

a

a

aCCf

pa

a

a

a

a

a

a

a

4

4

3

3

2

2

1

1

4

4

3

3

2

2

1

1

(13)

where dot )( indicates the derivative with respect to t.

From Eq.12, we get

432

3

1 aaaa (14)

Assume that V is a function of time t defined by

4321 aaaaV (15)

From Eqs.14 and 15, we get



486


4

1

1 Va (16)

Above Eq.13 can be written in the form

VCVdV

dVCfpV

dV

d (17)

In order to obtain a unique solution, one has to specify the rate of creation of matter-energy (at the expense of the negative energy of the C-field). Without loss of gen-erality, we assume that the rate of creation of matter en-ergy density is proportional to the strength of the exist-ing C-field energy-density. i.e. the rate of creation of matter energy density per unit proper-volume is given by

VgCpVdV

d 2222 (18)

where is proportionality constant and we have de-

fined . VgVC

Substituting it in Eq.17, we get

VgdV

dVfgpV

dV

d (19)

Comparing right hand sides of Eqs.18 and 19, we get

Vgf

gVdV

dVg 2

2 (20)

Integrating , which gives

1

1

2

f

VAVg

(21)

where is arbitrary constant of integration. 1A

We consider the equation of state of matter as

p (22)

Substituting Eqs.21 and 22 in the Eq.18, we get

12

2

1

2

2

f

VAVdV

d

(23)

which further yields

12

2

2

1

22

12

f

V

f

A

(24)

From Eq.22, we get

12

2

2

1

22

12

f

V

f

Ap

(25)

Subtracting Eq.8 from Eq.9, we get

04

4

3

3

2

2

1

1

2

2

1

1

2

2

1

1

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

dt

d

(26) Now, from Eqs.15 and 26, we get

02

2

1

1

2

2

1

1

V

V

a

a

a

a

a

a

a

a

dt

d

Integrating, which gives

V

dtxd

a

a11

2

1 exp , =constant, =constant 1d 1x

(27) Subtracting Eq.9 from Eq.10, we get

03

3

2

2

3

3

2

2

V

V

a

a

a

a

a

a

a

a

dt

d

Integrating, we get

V

dtxd

a

a22

3



04

4

3

3

4

4

3

3

V

V

a

a

a

a

a

a

a

a

dt

d

which on integration gives

V

dtxd

a

a33

4



04

4

1

1

4

4

1

1

V

V

a

a

a

a

a

a

a

a

dt

d

Integrating, we get

V

dtxd

a

a44

4

1 exp , = constant, = constant 4d 4x

(30) where 3214 dddd , 3214 xxxx and 4321 aaaaV .

Using Eqs.27, 28, 29 and 30, the values of ta1 ,

ta2 , ta3 and ta4 can be written explicitly as

V

dtXVDta 1

41

11 exp (31)

V

dtXVDta 2

41

22 exp (32)

V

dtXVDta 3

41

33 exp (33)



487487


V

dtXVDta 4

41

44 exp (34)

where the relations and14321 DDDD 21 XX

4,3,2

11

are satisfied by and

. From Eqs.10 and 31, we get

043 XX

4,3,2,1iX i

,1iDi

D

and . 01 X

Adding Eqs.8, 9, 10, 11 and 4 times Eq.7, we get

pa

m

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

a

a

a

a

a

a

a

a

3

3212

2

2

1

2

43

43

42

42

41

41

13

13

32

32

21

21

4

4

3

3

2

2

1

1

(35) From Eq.15, we have

43

43

42

42

41

41

13

13

32

32

21

21

4

4

3

3

2

2

1

1

2aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

aa

a

a

a

a

a

a

a

a

V

V

(36)

From Eqs.35, 36 and 22, we get

13

32122

1

2

a

m

V

V (37)

Substituting Eq.16 in Eq.37, we get

13

321221

2

V

m

V

V (38)


12

2

22

21

22

12

13

3212 f

V

f

A

V

m

V

V

(39)

which further gives

t

kVmV

f

fA

dV

f

1

2322

2

2

16

123

132 2

(40) where is integration constant. 1k

For 1 (Zeldovich fluid or Stiff fluid) and 01 k ,

the above equation gives 44tmV (41)


1414

1

22

ff

tmAg

(42)

Also, from equation VgVC , we get

342

3414

1

22

f

tmAC

ff

(43)

Substituting Eq.41 in Eq.24, the homogeneous mass density becomes

1818

2

1

22

2

1 ff

tfmA

(44)

Using Eq.25 and 1 , pressure becomes

1818

2

1

22

2

1 ff

tfmAp

(45)

From Eqs.38 and 39, it is observed that for ,

there is no singularity in density and pressure.

2f

Using Eq.41 in Eqs.31, 32, 33 and 34, we get

mtta 1 (46)

34

222

1

3exp

tm

XmtDta (47)

34

333

1

3exp

tm

XmtDta (48)

34

444

1

3exp

tm

XmtDta (49)

4. PHYSICAL PROPERTIES

The expansion scalar is given by

H4t

4 (50)

The mean anisotropy parameter is given by

4

14

1i

i

H

HA

68

2

4

2

3

2

2

4 tm

XXX (51)

The shear scalar is given by 2

224

1

22

2

44

2

1AHHH

ii

88

2

4

2

3

2

2

2 tm

XXX

(52) The deceleration parameter q is given by

11

Hdt

dq =0 (53)

where HHH ii and H is the Hubble parameter.



488

For large t, the expansion scalar and shear scalar tend to zero. Further, if , for large t, the model re-

duces to the vacuum case.

2f


5. CONCLUSIONS

In this paper, we have considered the space-time geome-try corresponding to Bianchi type-V in Hoyle-Narlikar [15-17] creation field theory of gravitation with five di-mensions. Bianchi type-V universe in creation-field cosmology has been investigated by Singh and Chaubey [19] whose work has been extended and studied in five di-mensions. An attempt has been made to retain Singh and Chaubey’s [19] forms of the various quantities. We have noted that all the results of Singh and Chaubey [19] can be obtained from our results by assigning appropriate values to the functions concerned.

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[3] Chodos, A. and Detweller, S. (1980) Where has the fifth dimension gone? Physical Review D, 21(8), 2167-2170.

[4] Lorentz-Petzold, K. (1985) Higher-dimensional Brans-Dicke cosmologies. General Relativity and Graviation, 17(7), 1189-1195.

[5] Ibanez, J. and Verdaguer, E. (1986) Radiative isotropic cosmologies with extra dimensions. Physical Review D, 34(12), 1202-1208.

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[7] Banerjee, S. and Bhui, B. (1990) Homogeneous Cosmo-logical Model in Higher Dimension. Monthly Notices of

the Royal Astronomical Society, 247(1), 57-61. [8] Reddy, D.R.K. and Venkateswara Rao, N. (2001) Some

cosmological models in scalar-tensor theory of gravita-tion. Astrophysics and Space Science, 277(3), 461-472.

[9] Khadekar, G. and Gaikwad, M. (2001) Higher dimen-sional Bianchi type-V cosmological model in Bimetric theory of relativity. Proceedings of Einstein Foundation International, 11, 95-100.

[10] Adhav, K., Nimkar, A. and Dawande, M. (2007) N–dimensional string cosmological model in Brans–Dicke theory of gravitation. Astrophysics and Space Science, 310(3-4), 231-235.

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[13] Hoyle, F. (1948) A new model for the expanding uni-verse. Monthly Notices of the Royal Astronomical Society, 108(1748), 372-382.

[14] Bondi, H. and Gold, T. (1948) The steady-state theory of the expanding universe. Monthly Notices of the Royal Astronomical Society, 108(3), 252-270.

[15] Hoyle, F. and Narlikar, J.V. (1966) A radical departure from the ‘steady-state’ concept in cosmology. Proeedings of Royal Society (London) A, 290(1421), 162-176.

[16] Hoyle, F. and Narlikar, J.V. (1963) Mach’s principle and the creation of matter. Proceedings of Royal Society (London) A, 273(1352), 1-11.

[17] Hoyle, F. and Narlikar, J.V. (1964) The C-field as a direct particle field. Proceedings of Royal Society (London) A, 282(1389), 178-183.

[18] Chatterjee, S. and Banerjee, A. (2004) C-field cosmology in higher dimensions. General Relativity Gravitation 36(2), 303-313.

[19] Singh, T. and Chaubey, R. (2009) Bianchi type-I, III, V, VI and Kantowski-Sachs universes in creation-field cos-mology. Astrophysical Space Science, 321, 5.

Vol.2, No.5, 489-493 (2010)doi: 10.4236/ns.2010.25061


Natural Science


Scalar-isovector δ-meson mean-field and mixed phase structure in compact stars

Grigor Bakhshi Alaverdyan

Department of Radio Physics, Yerevan State University, Yerevan, Armenia; [email protected]

Received 16 December 2009; revised 6 March 2010; accepted 15 March 2010.

ABSTRACT

The deconfinement phase transition from had-ronic matter to quark matter in the interior of compact stars is investigated. The hadronic phase is described in the framework of relativ-istic mean-field (RMF) theory, when also the scalar-isovector δ-meson effective field is taken into account. The MIT bag model for describing a quark phase is used. The changes of the pa-rameters of phase transition caused by the presence of δ-meson field are investigated. Finally, alterations in the integral and structure pa-rameters of hybrid stars due to deconfinement phase transitions are discussed.

Keywords: Neutron Stars; Equation of State; Relativistic Mean-Field; Quarks; Deconfinement Phase Transition

1. INTRODUCTION

Study of the structure characteristics and composition of the matter constituents at extremely high density region is of great interest in both nuclear and neutron star phys-ics. The RMF theory [1] has been effectively applied to describe the structure of finite nuclei, the features of heavy-ion collisions, and the equation of state (EOS) of nuclear matter. Inclusion of the scalar-isovector meson in this scheme and investigation of its influence on low density asymmetric nuclear matter was realized in [2,3]. At sufficiently high density, different exotic degrees of freedom, such as pion and kaon condensates, also deconfined quarks, may appear in the strongly in-teracting matter. The modern concept of hadron-quark phase transition is based on the feature of that transition, that is the presence of two conserved quantities in this transition: baryon number and electric charge [4]. It is known that, depending on the value of surface tension,

s , the phase transition of nuclear matter into quark

matter can occur in two scenarios [5,6]: ordinary first

order phase transition with a density jump (Maxwell construction), or formation of a mixed hadron-quark matter with a continuous variation of pressure and den-sity (Glendenning construction) [4]. Uncertainty of the surface tension values does not allow to determine the phase transition scenario, taking place in realty. In our recent paper [7] in the assumption that the transition to quark matter is a usual first-order phase transition, de-scribed by Maxwell construction, we have shown that the presence of the -meson field leads to the decrease of transition pressure , of baryon number densities 0P

Nn and . Qn

In this article we investigate the hadron-quark phase transition of neutron star matter, when the transition proceeds through a mixed phase. Influence of -meson field on such phase transition characteristics and of compact star structure is discussed.

2. NEUTRON STAR MATTER EOS WITH DECONFINEMENT PHASE TRANSITION

2.1. Nuclear Matter

For description of hadronic phase we use the relativistic Lagrangian density of many-particle system consisting of nucleons, p, n, and exchanged mesons , , , :

L ( ), ( ), ( ))

( ), ( ), ( ))

)) L ( ( )) ,

x x( ), (

L (

( (

x x

x x x

U x x

),(x

))x

(,

(1)

where

)(( xL

is the linear part of relativistic

Lagrangian density without -meson field [8], U ( )

3 4)c

( )b

(g3 4Nm g and

1L ( ) (

2

2 )m

are the -meson self-interaction term and


G. B. Alaverdyan / Natural Science 2 (2010) 489-493


490


contribution of the -meson field, respectively. This Lagrangian density (1) contains the meson-nucleon cou-pling constants, , , ,g g g g

b

( / )m

and also parameters of

σ-field self-interacting terms, and c . In our calcula-

tions we take for the2 2. m25 fa g cou-

pling constant, as in [2,7]. Also we use MeV for the bare nucleon mass, 938.93Nm

* 0.78N Nm

0 0.153n

0f K

( /a g

m

.3

(0)sym

( /a g

b c2 fm ,

for the nucleon effective mass,

fm-3 for the baryon number density at satura-

tion, MeV for the binding energy per

baryon, MeV for the incompressibility

modulus, and MeV for the asymmetry energy.

Five other constants, ,

, and , then can be numerically

determined:

16

E

m

a

300

,2)

82

32.5

9.154

2 2)a m

28 fm ,

)m

4.

( /g

aa

b c hen we

neglect the

13.621 2fm , -1654 fm , 0.01 0.01319 . W c nnel, then a and aha 0

. The knowledge of the model parameters

makes it possible to solve the set of four equations in a self-consistent way and to determine the re-denoted mean-fields,

24.794 fm

g g, ___

0 , _____

(30g

) , and _____

(3)g depending on baryon number density

and asymmetry parameter

n

) / n( n pn n . The stan-

dard QHD procedure allows to obtain expressions for energy density ( , )NM n and pressure ( ,NMP n ) [7].

In Figure 1 we illustrate the 3D-plot of the energy per baryon, ( ,E n ) ( , ) /b NM n n

n

as a function of the

baryon number density and asymmetry parameter in case of -equilibrium charged -plasma.

The curves correspond to different fixed values of the charge per baryon, . The thick one cor-

responds to

npe

( p eq n n ) n/

-equilibrium charge neutral -matter.

The lower and upper surfaces corresponds to

npe

RMF

and RMF models respectively.

Clearly, including a -meson field increases the en-ergy per baryon, and this change is greater for larger values of the asymmetry parameter. For a fixed value of the specific charge, the asymmetry parameter falls off monotonically as the density is increased.

In Figure 2 we plotted the effective mean-fields of exchanged mesons, , , and as a function of

the baryon number density for the charge-neutral β-equilibrium -plasma. The solid and dashed lines

correspond to the

nnpe

RMF and RMF models, respec-

tively.

From Figure 2 one can see that the inclusion of the scalar-isovector virtual 0( (980))a meson results in

significant changes of the and meson effective

fields. This can result in changes of deconfinement

Figure 1. Energy per baryon as a function of the baryon

number density and the asymmetry parameter bE

n in case of a -equilibrium charged -plasma. npe

Figure 2. Re-denoted meson mean-fields as a function of the baryon number density in case of a β-equilibrium charge- neutral -plasma with and without

nnpe -field.



491491


phase transition parameters and, thus, alter the structural characteristics of neutron stars. The results of our analy-sis show that the scalar - isovector -meson field in-clusion leads to the increase of the EOS stiffness of nu-clear matter due to the splitting of proton and neutron effective masses, and also due to the increase of asym-metry energy (for details see Ref. [9]).

2.2. Quark Matter

To describe the quark phase an improved version of the MIT bag model is used, in which the interactions be-tween and du， s quarks inside the bag are taken into account in the one-gluon exchange approximation [10]. We choose , and 5 MeVum 7 MeVdm 150sm

MeV for quark masses, 60B MeV/fm3 for bag pa-rameter and 0.5s for the strong interaction constant.

2.3. Deconfinement Phase Transition Parameters

There are two independent conserved charges in hadron quark phase transition: baryonic charge and electric charge. The constituents chemical potentials of the

-plasma in npe -equilibrium are expressed through

two potentials, b andel

, according to conserved

charges. The pressure NMP , energy density NM and

baryon number density NMn)

, are functions of inde-

pendent potentials, ( NMb and ( )NM

el .

The thermodynamic characteristics, pressure ,

energy density QMP

QM and baryon number density ,

are functions of chemical potentials and .

QMn( )QMel

( )QMb

The mechanical and chemical equilibrium conditions (Gibbs conditions) for mixed phase are

( ) ( )QM NMb b b , ( ) ( )QM NM

el el el , (2)

( , ) ( , )QM b el NM b elP P . (3)

The volume fraction of quark phase is

/ ( )QM QM NMV V V , (4)

where and QMV NMV are volumes occupied by quark

matter and nucleonic matter, respectively. We applied the global electrical neutrality condition

for mixed quark-nucleonic matter, according to Glenden- ning [4,8],

12 ( , ) ( , ) ( , )

3(1 ) ( , ) ( ) 0.

u b el d b el s b el

p b el e el

n n n

n n

(5)

The baryon number density in the mixed phase is de-termined as

1( , ) ( , ) ( , )

3

(1 ) ( , ) ( , )


p b el n b el

n n n n

n n

(6)

and the energy density is

( , ) ( , ) ( , )

(1 ) ( , ) ( , ) ( ).


p b el n b el e el

(7)

In case of 0 , the chemical potentials Nb and

Nel , corresponding to the lower threshold of a mixed

phase, are determined by solving Eq.3 and Eq.5. This allows to find the lower boundary parameters NP , N

and Nn . Similarly, we calculate the upper boundary

values of mixed phase parameters, , QP Q and ,

corresponding to

Qn

1 .

The system of Eqs.3, 5, 6 and 7 makes it possible to determine EOS of mixed phase between these critical states.

Note, that in the case of an ordinary first-order phase transition both nuclear and quark matter are assumed to be separately electrically neutral, and at some pressure

, corresponding to the coexistence of the two phases,

their baryon chemical potentials are equal, i.e., 0P

0( ) ( )NM QMP 0P (8)

Such phase transition scenario is known as phase tran-sition with constant pressure (Maxwell construction). Table 1 represents the parameter sets of the mixed phase both with and without -meson field. It is shown that the presence of -field alters threshold characteristics of the mixed phase. The lower threshold parameters,

, ,N N Nn P are increased, meanwhile the upper ones

, ,Q Q Qn P are slowly decreased.

In Figure 3 we plot the species number densities as a function of baryon density for Glendenning construc-

tion. Quarks appear at the critical density .

n

0.077Nn -3fm

Table 1. The mixed phase parameters with and without – meson field.

Model RMF RMF

Nn , fm-3 0.0717 0.0771

Qn , fm-3 1.0830 1.0830

N , MeV/fm3 67.728 72.793

Q , MeV/fm3 1280.889 1280.884

NP , MeV/fm3 0.336 0.434

QP , MeV/fm3 327.747 327.745



492


The hadronic matter completely disappears at 1.083Qn

, where the pure quark phase occurs. -3fmUsing the obtained EOS of nuclear matter, we have

integrated the Tolman-Oppenheimer Volkoff equations and obtained the mass M and the radius of com-pact stars for the different values of central pressure .

R

cP

Figure 4 illustrate the ( )M R dependence of neutron stars.

We can see, that the behavior of mass-radius dependence significantly differs for the two types of phase transitions. Figure 4 shows that for MeV/fm3 there are un-stable regions, where between two stable

branches of compact stars, corresponding to configura-tions with and without quark matter. In this case, there is a nonzero minimum value of the quark phase core radius. Accretion of matter on a critical neutron star configura-tion will then result in a catastrophic rearrangement of the star, forming a star with a quark matter core. The range of mass values for stars, containing the mixed phase, is . In case of Maxwellian

type phase transition, the analogous range is

60B/ cdM dP

53 ]M

0

[0.085 ;M

; 1.828 ]

1.8

[0.216 M M .

3. CONCLUSIONS

In this paper we have studied the deconfinement phase transition of neutron star matter, when the nuclear matter is described in the RMF theory with -meson effective field. We show that the inclusion of scalar – isovector -meson field terms leads to the stiff nuclear matter

Figure 3. Constituents number density vs. baryon number density in case of Glendenning construction. Vertical dotted lines represent the mixed phase boundaries.

Figure 4. The mass-radius relation of neutron star with differ-ent deconfinement phase transition scenarios. Open circles and squares denote the critical configurations for Glendenning and Maxwellian type transitions, respectively. Solid circles and squares denote hybrid stars with minimal and maximal masses, respectively. EOS. In a nucleonic star both the gravitational mass and corresponding radius of the maximum mass stable con-figuration increases with the inclusion of the -field. The presence of scalar – isovector -meson field alters the threshold characteristics of the mixed phase. The lower threshold parameters, , ,N Nn NP are increased,

while the upper thresholds, , ,Q Q Qn P , are slowly de-

creased. For EOS used in this study, the central pressure of the maximum mass neutron stars is less than the mixed phase upper threshold . The maximum mass

configuration has a gravitational mass QP

max 31.85M M

with radius 10.71mR km, and central density

g/cm3. This star has a pure strange quark

matter core with radius km, next it has a nu-

cleon-quark mixed phase layer with a thickness of

15

Qr

2.322 10c

9.43MPr

0.83

km, followed by a normal nuclear matter

layer with a thickness of km. 0.45Nr

4. ACKNOWLEDGEMENTS

The author would like to thank Prof. Yu. L. Vartanyan and Dr. G. S.

Hajyan for fruitful discussions on issues related to the subject of this

research.



493493

REFERENCES


[1] Serot, B.D. and Walecka, J.D. (1986) The relativistic nuclear many-body problem. In Adv. in Nuclear Physics, Eds., Negele, J.W and Vogt, E., 16(1), Plenum Press, New York.

[2] Liu, B., Greco, V., Baran, V., Colonna, M. and Di Toro, M. (2002) Asymmetric nuclear matter: The role of the isovector scalar channel. Physical Reviews C, 65(4), 335-345.

[3] Greco, V., Colonna, M., Toro, M.D. and Matera, F. (2003) Collective modes of asymmetric nuclear matter in quan-tum hadrodynamics. Physical Reviews C, 67(1), 015203.

[4] Glendenning, N.K. (1992) First-order phase transitions with more than one conserved charge: Consequences for neutron stars. Physical Reviews D, 46(3), 1274-1287,

[5] Heiselberg, H., Pethick, C.J. and Staubo, E.S. (1993)

Quark matter droplets in neutron stars. Physical Review Letters, 70(10), 1355-1359.

[6] Heiselberg, H. and Hjorth-Jensen, M. (2000) Phases of dense matter in neutron stars. Physics Reports, 328(5-6), 237-327.

[7] Alaverdyan, G.B. (2009) Relativistic mean-field theory equation of state of neutron star matter and a Maxwellian phase transition to strange quark matter. Astrophysics, 52(147), 132-150.

[8] Glendenning, N.K. (2000) Compact stars, Springer, Cambridge.

[9] Alaverdyan, G.B. (2009) Scalar-isovector δ meson in the relativistic mean field theory and the structure of neutron stars with a quark core. Gravitation & Cosmology, 15(1), 5-9.

[10] Farhi, E. and Jaffe, R.L. (1984) Strange matter. Physical Reviews D, 30(2), 2379-2390.

Vol.2, No.5, 494-505 (2010)doi: 10.4236/ns.2010.25062


Natural Science


Stability analysis of primary emulsion using a new emulsifying agent gum odina

Amalesh Samanta, Durbadal Ojha, Biswajit Mukherjee*

Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India; Corresponding Author: [email protected]

Received 1 February 2010; revised 10 March 2010; accepted 18 March 2010.

ABSTRACT

Gum odina and various parts of the plant Odina wodier are traditionally used in Indian folk medicine. Here an effort was made to investigate the efficacy of gum odina as new pharmaceuti-cal excipients, in particular, as an emulsifying agent. Primary emulsion was prepared using wet gum method taking oil: water: gum (4:2:1) with gum acacia powder as an emulsifying agent. This was used as a standard control formulation. In case of experimental emulsions the primary emulsion was prepared by same wet gum technique taking oil: water: gum (4:2:0.5) (gum content was just a half of gum acacia) by using gum odina powder as an emulsifier. The gum odina as emulsifying agent provided a sta-ble emulsion at a very low concentration as compared to the amount required for other conventional natural emulsifying agents. Stability studies of the emulsion were made as per the ICH guideline to study thermal stability, photo- sensitivity, pH related stability and stability in presence of oxygen. The emulsion type was identified by staining techniques (dye test by using Sudan III) as o/w type preparation without creaming or cracking even after long storage for 24 months at 25°C. It was found that the emul-sion containing gum odina produced more sta-ble emulsion at a much lower amount as com-pared to the emulsion stabilized by gum acacia.

Keywords: Emulsifying Agent, Gum Odina, Odina wodier

1. INTRODUCTION

Use of various gums as pharmaceutical excipients is nothing new. As a stabilizer and thickening agent, use of natural gum has been found in the literature about five thousand years back [1]. Some natural or induced-exuda-

tion of normally neutral or slightly acidic complex of polysaccharides or partially acetylated polysaccharide or heterogeneous polysaccharide are obtained as a mixture with calcium, potassium and magnesium salts [2-3]. As a natural defense mechanism to prevent infection or dehy-dration many trees and shrubs are known to produce an aqueous thick exudation when the plants bark is injured [4]. Eventually the solution dries up in contact with sunlight and air and a hard transparent brown-tint glass like mass is formed. This solid exudation is commonly known as natural gum [4-5]. Some of the gums used frequently now-a-days as pharmaceutical excipients and /or in food industry are gum acacia, gum tragacanth, gum Karaya etc. Gum acacia is mainly used in the con-fectionary industry. Traditionally it is used in candies to provide the appropriate texture so that they do not ad-here to the teeth. Gum acacia is used in chewing gum as a coating agent [6-7] and is also used as emulsifier in soft drink industries [8]. Pharmaceutically gum acacia is still used as a suspending agent, emulsifier, adhesive and tablet binding agent [9-11]. In cosmetic industry it is used as a stabilizer in lotions and protective creams, where it increases viscosity, imparts spreading properties and maintains a protective coating [4].

Gum tragacanth is used in ice creams to provide tex-ture to the product [12] and acts as a thickener and pro-vides texture for chewy sweets such as lozenges [13]. Gum tragacanth is widely used in pharmaceutical indus-try as an effective suspending agent. Gum tragacanth is used as a stabilizer in dermatological creams and lotions and it also provides a protective coating [14-15]. Sus-pending properties are used in jellies and tooth paste giving spreadibility and a shiny creamy appearance [16-17].

Gum Karaya is well-studied for stabilizing low pH

emulsion such as sauces [18]. Due to the water binding capacity of Gum karaya it extends the shelf-life of baked goods. It is widely used as stabilizer, thickener, texturiser and emulsifier in foods. Powdered Gum karaya is widely applied on dental plates as an adhesive [19]. It is used as a bulk laxative, and also used as an adhesive in leak-

A. Samanta et al. / Natural Science 2 (2010) 494-505


495495


proof sealing rings for post surgical drainage pouches or osotomy bags and in skin lotions [20-22].

In recent past we described the use of gum odina (Figure 1(a)) as an excellent substitute of starch paste as a tablet binder [23]. Odina wodier, Roxb. family Ana-cardiaceae is a large tall tree (Figure 1(b)) found in de-ciduous forest in India, Myanmar, Srilanka, China, Ma-laysia, Cambodia and Philippine Islands [24]. It is popu-larly known as Kashmala, Odimaram, Jiol in local lan-guage and in English it is called Rhus olina [25]. Various parts of this plant have been found to be used as medi-cines in Ayurveda. The leaves have been reported to use in Elephantiasis of the legs [25]. Juice of green branches is used as an emetic in case of coma or insensibility pro-duced by narcotic. The dried and powdered bark is found to use as tooth powder by poor villagers [24]. The bark extract has been reported to be useful in vaginal trouble, curing ulcer, heart diseases etc. [26].

In the presence study we investigated and compared the emulsifying property of the gum odina (obtained from Odina wodier, Roxb. Family Anacardiaceae) with

respect to that of a well-known natural gum emulsifier (gum acacia) and the stability aspects of emulsion pre-pared with the gum.

1.1. Materials and Methods

Chemicals procured for preparing emulsion were cod liver oil (E. Merck Ltd, Mumbai, India) and acacia pow-der (E. Merck Ltd, Mumbai, India). All other chemicals were of analytical grade and used as received if not oth-erwise mentioned.

1.2. Collection of Gum Odina

Gum was collected from the tree Odina wodier, Roxb., family Anacardiaceae during Autumn in the month of August from the Mandal Ghat of Jalpaiguri, West Bengal, India. The gum was the natural exudates on the bark of the tree. It was collected in a dry condition. After collec-tion of the gum, the entire work was carried out in the Department of Pharmaceutical Technology, Jadavpur University.

(a) (b)

Figure 1. (a) Transparent reddish brown needle shape gum liberating from the bark of the plant; (b) Tree of Odina wodier, Roxb., family Anacardiaceae.



496

2. FORMULATION DEVELOPMENT

2.1. Formula for Preparation of Primary E

mulsion

Formulation was developed by conventional “wet gum” technique [27]. Formula for primary emulsion was pre-pared using “wet gum” method taking oil: water: gum (4:2:1) with gum acacia powder as an emulsifying agent. This was used as standard control formulation. In case of experimental emulsions (test sample) the primary emul-sion was prepared by the same “wet gum” technique taking oil: water: gum (4:2:0.5) (gum content is just a half of gum acacia) by using gum odina powder as an emulsifier (Table 1).

2.2. Procedure for the Preparation of Emulsion

3.75 gm of experimental gum (gum odina) was taken in a mortar and thick mucilage was prepared by taking 15 ml of water using a pestle. Then to it, required volume (30 ml) of cod liver oil was added drop-wise with constant and uniform clockwise trituration to make a primary emul-sion [27]. Final volume was adjusted to 90 ml with water (Table 1).

2.3. Stability Study of Emulsion

2.3.1. FTIR Study IR grade KBr with a drop of respective emulsion was compressed into pellets by applying 5.5 metric tons of pressure in a hydraulic press and scanned over a wave number of 4000 cm-1 - 400 cm-1 in a FTIR spectropho-tometer 8400S Shimadzu.

2.3.2. Thermal Stability Prepared emulsions were kept (test and control) at dif-ferent temperatures namely 20°C, 40°C and 60°C for one month by following ICH guideline [28]. Samples were taken out and FTIR spectroscopy was done.

2.3.3. Stability at variable pH

Initial pH of the prepared emulsion was 4.75. To de-termine the stability at different pH values, the emul-sion were adjusted at different pH conditions namely 2, 7.4 and 10 by using 0.1(N) HCl and 0.1(N) NaOH as applicable and kept for one month both for test and control. Then the FTIR spectroscopic studies of the sample were done. Table 1. Composition of primary emulsion.

Control Experimental

Cod-liver oil-30 ml. Water q.s - 90 ml Acacia powder-7.5 g

Cod-liver oil-30 ml. Water q.s - 90 ml Gum odina powder - 3.75 g

2.3.4. Photo-Stability To determine the photo-stability, the formulations (the test and control samples) were exposed to 40 watt (2216.16 CP), 60 watt (3656.66 CP) and 100 watt (5540.4 CP) using electric bulbs. That was adjusted 6 inches above the formulations kept in transparent glass bottle capped tightly in a closed chamber for one month. Following this study FTIR spectra were determined and compared with the samples not exposed to light (i.e. kept in a dark place at 4°C) [29]. 2.3.5. Oxygenation of Emulsion and FTIR Study To analyze the susceptibility of the prepared emulsion containing gum to oxidation, 30 ml of emulsion in a glass bottle of 50 ml capacity was continuously exposed to a stream of O2 (5 L/min) for 1h and the samples were capped tightly and kept for 15 days before being ana-lyzed by studying their FTIR spectra.

30 ml of emulsion in a glass bottle of 50 ml capacity was continuously exposed to inert environment by using a stream of N2 (5 L/min) (considered as control against oxidation) for 1 h and the samples were kept for 15 days before being analyzed by studying their FTIR spectra.

3. CHARACTERIZATION OF EMULSION

3.1. Viscosity of Emulsion

Dynamic viscosity of the prepared emulsion was measured using Brook-field rotational viscometer TV-10 (Toki San-gyo Co. Pvt.Ltd, Tokyo, Japan) rotated at 60 rpm for one minute. The length and diameter of the cylinders were 10.5 cm and 3 cm respectively. Length and diameter of the spindles were 6.4 cm and 1.8 cm respectively. 3.2. Test for Identifying Emulsion Type (Dye

Test)

Several tests are available for distinguishing between o/ w and w/o type emulsions. They include tests of misci-bility, dye test, electrical conductivity measurements etc. We adopted for dye test here. 3.3. Dye Test

Prepared emulsion (10 ml) was triturated with Sudan III (0.05 g) and a drop of it was placed on a microscope slide, covered with a cover-slip and examined under a microscope. 3.4. Cracking of Emulsion

This involves coalescence of the dispersed globules and separation of the disperse phase as a separate layer. Re-dispersion cannot be achieved by shaking and the ad-



497497


vantages of emulsification are lost and accurate dosage is impossible. Simple visual observation (of the stored samples about 24 months) was the means to detect cracking.

3.5. Creaming of Emulsion

Creaming may be defined as the formation of a layer of relatively concentrated emulsion and this conditions favours breakdown of the interface and consequent coa-lescence of the oil globules and therefore, the emulsion may eventually crack. By shaking, creaming may disap-pear in many cases. Simple visual observation technique (of the stored sample about 24 months) was the method adopted here to determine creaming.

4. RESULTS

The various parts of Odina wodier have been used in Ayurveda and traditional Indian folk medicine (24). We have recently reported the gum of this plant as a tablet binder, effective at a much lower concentration as com-pared to the other available natural binders and further, the gum is devoid of toxicity [23]. In the present study

we have mainly focused on the utility of the gum as an emulsifying agent of natural origin and the stability as-pects of emulsions prepared using this emulsifying agent.

The dynamic viscosity of prepared emulsion (4:2:0.5) was measured using Bookfield type rotational viscome-ter TV-10, rotated at 60 rpm for one minute and the vis-cosity was 14 centipoises.

Several tests are available for the differentiation of types of primary emulsions i.e. o/w and w/o type emul-sions. These tests are miscibility with water, Dye test and Electrical conductivity measurements etc. [27]. Dye test is a very common test to determine the types of emulsion. The dispersed globules were appeared ‘red’ due to oil soluble dye Sudan III and the continuous phase was ‘colourless’ (Figure 2) in the present study.

Stability of emulsion was analyzed by comparing the FTIR spectra of the freshly prepared experimental emul-sion (Figure 3) and the stored (24 months) experimental emulsion (Figure 4). Physical interactions were detected between wave number 700 cm-1 and 600 cm-1 upon pro-longed storage as compared to the freshly prepared samples.

Figure 2. Determination of o/w type of emulsion i.e. dispersed oil globules appeared ‘red’ and continuous phase ‘col-ourless’.



498

Figure 3. FTIR spectra of freshly prepared experimental emulsion.

F igure 4. FTIR spectra of stored (24months) experimental emulsion kept at room temperature.




499499

Thermal stability of emulsion was analyzed by study-

ing the FTIR spectra of the emulsions stored at 20°C, 40°C and 60°C for 30 days (Figures 5-7). Interactions were detected in the wave numbers between 2700 cm-1

and 720 cm-1 for the samples stored at 40°C and 20°C; and wave number at 3461 cm-1, 2099 cm-1, 1646 cm-1, 718 cm-1 in case of the sample stored at 60°C (Figures 7-9). The types of interaction have been discussed in details in discussion section.


The impacts of variable pH on emulsion stability were detected by changing the pH of emulsion at 2, 7.4 and 10; these were stored for 30days at room tem-perature. This was followed by the FTIR spectroscopy and the data indicate that there were variations in wave numbers in the range between 3500 cm-1 and 2600 cm-1 and also at 1744 cm-1 at pH 7.4 (Figures 8-10).

For studying the photostability, the emulsion was ex-posed to 60 Watt (3656.66 CP) for 30 days and the FTIR

spectra were compared with the experimental emulsion stored in the dark for the same period. There were physi-cal interactions detected in the range of wave numbers between 3600 cm-1 and 2800 cm-1 (Figure 3 and Figure 11) and also in the range between 2400 cm-1 and 1700 cm-1. Otherwise no predominant variations in the FTIR spectra were detected when photo-exposed samples were compared.

To know the stability of emulsion exposed to oxida-tion, samples were exposed either to oxygen or nitrogen (which was used as control) as specified earlier. No in-teractions were detected upon oxygenation except some minor peak variation at the wave numbers 2665 cm-1, 3457 cm-1, 1437 cm-1, 1148 cm-1 (Figures 12 and 13). However, all the characteristic peaks of the gum were present.

Creaming and cracking of the stored emulsions (24 months) were also visually observed but no such phe-nomenons were detected.

Figure 5. FTIR spectra of thermal stability of emulsion at 20ºC.



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Figure 8. FTIR spectra of emulsion with pH 2.

Figure 9. FTIR spectra of emulsion with pH 7.4.




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Figure 10. FTIR spectra of emulsion with pH 10.

Figure 11. FTIR spectra of photo stability of emulsion at 60 Watt. (3656.66 CP).




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Figure 12. FTIR spectra of oxygenated emulsion.

Figure 13. FTIR spectra of nitrogenated emulsion.




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5. DISCUSSION In this study capability of gum odina as an emulsifying agent was investigated. Viscosity of the experimental emulsion was found to be 14 cP which suggests that it was a thicker emulsion and the emulsion would remain stable for a longer period.

Dye test with Sudan III (oil soluble dye) showed that dye was distributed in the form of droplets throughout the colourless continuous phase. This proves that oil formed the dispersed phase and water was the continu-ous phase and it was an o/w type of emulsion. Thus o/w emulsion may be prepared using gum odina as emulsi-fying agent.

When the spectra were compared, in some cases peak height varied. It may be due to the presence of variable amounts of ingredients present in the pellet. There were interactions detected in the wave range numbers between 700 cm-1 and 600 cm-1. This zone is the known stretching vibration zone of CH-alkane and OH (H bonded and normally out of plane). Hydrogen of the fatty acid might have formed weak hydrogen bond or bond due to Van der Waal force or dipole moment with OH- group of water predominantly upon the long storage.

In the cases of thermal stability analysis, samples were kept at 20°C, 40°C and 60°C for 1 month (Figures 5-7) and compared with the freshly prepared sample (Figure 3). Changes in peaks in wave numbers between 2700 cm-1 and 720 cm-1 may be possibly due to interaction between ketonic and aldehyde groups in the fatty acids by forma-tion of H-bonding or weak bondings such as Van der Waal forces or dipole moments, since the zone between 2690 cm-1 and 2840 cm-1 are the medium intensity and 1720 cm-1 - 1740 cm-1 and 1710 cm-1 - 1720 cm-1 are strong intensity carbomile stretching vibration zone and 720 cm-1 - 725 cm-1 is the weak intensity bending vibra-tion zone of CH2 rocking [30]. Reaction at the 1646 cm-1 and 718 cm-1 might be due to the opening of α and β un-stauration and interaction with OH-group or H due to heating or CH2 rocking. Possible weak bond formation between OH-group and carbonyl might have taken place at the wave range between 3200 cm-1 - 3550 cm-1 as this zone is popularly known for variable and strong OH free and OH bonded stretching vibration zone.

Emulsion adjusted at different pH conditions (2, 7.4 and 10) and stored for 30 days were analyzed using FTIR spectroscopy and data were prepared with the freshly prepared emulsion (pH -4.5). There were interac-tions between wave number 3500 cm-1 and 2600 cm-1 and at 1744 cm-1 at pH 7.4. The vibration at 3500 cm-1, 2600 cm-1, 1744 cm-1 may be explained due to the weak bond formation of OH and carbonyl group present in water and fatty acid or between carbonyl or CH group of fatty acid and OH of water since these zones are the

known stretching vibration zones of OH and C=O group. By studying the FTIR spectra of the samples kept at dif-ferent pH and the freshly prepared sample (Figures 8-10) it may be stated that emulsions at pH 7.4 was more sta-ble as compared to pH 2 and pH 10, considering the in-teraction patterns. Least interactions comparing to the freshly prepared and stable emulsion, was detected in case of emulsion with pH 7.4.

In case of photo-stability study, interactions might be due to the formation of weak bonds such as hydrogen bonds or bonds due to Van der Waal force or dipole moment between OH and C=O, since in the interaction zone predominant functional groups were OH and C=O. It suggests that the emulsion is photo-stable.

Peak variations at 2665 cm-1 and 3457 cm-1 of samples exposed to oxygen were probably due to the possible weak bond formation between OH and COOH of water and fatty acid respectively, since these are the known stretching vibration zones of OH and C=O [30]. Peak variations at 1437 cm-1 and 1148 cm-1 could be due to α- CH2 bending or C-C-C bending of fatty acid carbons, since, 1400 cm-1 - 1450 cm-1 are the bending vibration zone of α-CH2 and 1148 cm-1 is the medium intensity C-C bending vibration zone [30]. Thus, upon FTIR spectrum analysis, it may be stated that the emulsion is not susceptible to oxidation, since the reactions were due to physical bond formations. However, oxygenenation might have a role to induce such bond formations as they were not noticed in case of the samples exposed to N2.

Cracking may be caused by any chemical, physical or biological effect that changes the nature of the interfacial film that exists between oil and water [31]. These tend to make it less stable. But here, after long storage of pre-pared emulsion for 24 months, no coalescence of dis-persed globules of oil was noticed. Hence, no cracking was observed in the said period.

Creams may be formed as a layer of relatively con-centrated emulsion and this condition favors breakdown of the interface and consequent coalescence of the oil globules and therefore, the emulsion may eventually crack [31]. After a long storage of the emulsion for 24 months there were no cream formations on the upper surface of emulsion.

When experimental emulsions were compared with the prepared acacia emulsion (considered here as con-trol), it was found that requirement of gum odina was 50% of the amount of acacia required for preparation of primary emulsion. Further, gum odina produces a stable emulsion which can be stored at least for 2 years.

Thus, gum odina may be used as an emulsifying agent to prepare o/w primary emulsion.

6. ACKNOWLEDGEMENTS

The study was supported financially by Dr. V. Ravichandran Endow-ment Trust, Jadavpur University, Kolkata, India.



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REFERENCES

[1] Verbeken, D., Dierckx, S. and Dewettinck, K. (2003) Exudates gums: Occurrence, productions and applica-tions. Applied Microbiology and Biotechnology, 63(1), 10-21.

[2] De Paula, R.C.M., Santana, S.A. and Rodrigues, J.F. (2001) Composition and rheological properties of Albizia lebbeck gum exudates. Carbohydrate Polymers, 44(2), 133-139.

[3] LeCerf, D., Irinei, F. and Muller, G. (1990) Solution properties of gum exudates from Sterculia urens (Karaya gum). Carbohydrate Polymers, 13(4), 375-386.

[4] Whistler, R.L. (1993) Exudate gums. In Whistler, R.L. and Berniller, J.N., Eds. Industrial Gums: Polysaccha-rides and their Derivatives. Academic Press, San Diego, 318-337.

[5] Philips, G.O. and Williams, P.A. (2001) Tree exudates gums: Natural and versatile food additives and ingredi-ents. Food Ingredients Analysis of International, 23, 26-28.

[6] Cherukuri, S.R., Friello, D.R., Parker, E., Hopkins, W. and Mackay, D.A.M. (1983) Stable liquid red beet color and chewing gum containing same. U.S. patent 4, 371, 549.

[7] Huzinec, R.J. and Graff, A.H. (1987) Coatings for chew-ing gums containing gum arabic and a soluble calcium salt. U.S. patent 4, 681, 766.

[8] Eng, J.L. and Mackenzie, K.M. (1984) Glyceride fat based clouds for ready to drink beverages. US patent 4, 479, 971.

[9] Ferdinand, G. and Kruger, W. (1986) Vitamin E efferves-cent tablets. German patent 3, 517, 916.

[10] Millard, R. and Balmert, C.A. (1961) Effervescent com-positions. U.S. patent 2, 985, 562.

[11] Tame-said, J.I. (1997) Toothpaste and mouthwash in tablets. W.O. Patent 9, 719, 668.

[12] Weiping, W.T. and Karaya (2000) In Philips, G.O., Wil-liams, P.A., Eds., Handbook of Hydrocolloids, Woodhead, Cambridge, 155-168.

[13] Dziezak, J.D. (1991) A focus on gums. Food Technology, 45(3), 116-132.

[14] Leupold, C.W., Kellner, W. and Hellmuth, J. (1962) Ma-terial with deodorizing action. G.B. patent 901, 554.

[15] Smith, G.R. and Wands, R.C. (1966) Compositions pro-

viding a protective coating for the skin. G.B. patent 1, 049, 063.

[16] Grossmith. F. (1956) Process for the production of jellies or viscous solutions. G.B. patent 750, 126.

[17] Nebergall, W.H. (1956) Dentrifice preparations. G.B. patent 746, 550.

[18] Partyka, A. (1963) Salad dressing. G.B. patent 936, 531. [19] Steinhardt, A. and Goldwater, F.A. (1962) Gelatin adhe-

sive pharmaceutical preparations. U.S. patent 3, 029, 187.

[20] Carpenters. (1979) Seals for colostomy or like bags. G.B. patent 2, 017, 501.

[21] Marsan, A.E. (1967) Sealing pad for a post-surgical drainage pouch. U.S. patent 3, 302, 647.

[22] Sanderson, G.R. (1996) Gums and their use in food sys-tems. Food Technology, 50(3), 81-84.

[23] Mukherjee, B., Samanta, A. and Dinda, S.C. (2006) Gum odina-a new tablet binder. Trends in Applied Sciences Research. 1(4), 309-316.

[24] Chidanbarathanu, S. (1995) Index of herbs in languages. Siddha Medical Literature Research centre, Madras.

[25] Kiritikar, K.R. and Basu, B.D. (1935) Indian Medicinal Plants, 2nd Edition, International Book Distributors, Book Sellers and Publishers, Dehradun.

[26] Kiritikar, K.R. and Basu, B.D. (1987) Indian Medicinal Plants, International Book Distributors, Book Sellers and Publishers, Dehradun.

[27] Carter S.J. (1987) Dispensing for Pharmaceutical Stu-dents, 12th Edition, CBS publishers and Distributors, Delhi.

[28] Grimm, W. (1998) Extension of the international confer-ence on harmonization tripartite guideline for stability testing of new drug substances and products to countries of climatic zones III and IV. Drug Development and In-dustrial Pharmacy, 24(4), 313-325.

[29] Yoshiok, S., Ishihara, Y., Terazone, T. and Tsunakawa, N. (1994) Quinine actinometry as a method for calibrating ultraviolet radiation intensity in light-stability testing of pharmaceuticals. Drug Development and Industrial Pharmacy, 20(13), 2049-2062.

[30] Williams, G.P. (2002) Synchrotron and free electron laser sources of infrared radiation. Chalmers, J.M. and Grif-fiths, P.R., Eds., Handbook of Vibrational Spectroscopy, Wiley, Chichester, 341-342.

[31] Osol, A. (1980) Remington Pharmaceutical Sciences, Mack Publishing Company, Easton.

Vol.2, No.5, 506-514(2010)doi: 10.4236/ns.2010.25063


Natural Science


Investigation of airborne fungi at different altitudes in Shenzhen University

Li Li, Chao Lei, Zhi-Gang Liu*

College of Life Science, Shenzhen University, Shenzhen, China; *Corresponding Author: [email protected]

Received 15 December 2009; revised 24 February 2010; accepted 18 March 2010.

ABSTRACT

Aim: To investigate the richness of species or genera of airborne fungi, the amount of airborne fungi, and its seasonal variation at different al-titudes in Shenzhen University. The effect of meteorological factors on airborne fungi was also analyzed. Methods: Slide-exposure method and open-plate method were used. Re-sults: There were 27 genera or species of fun-gus spores identified. Among the identified fungal genus, Cladosporium, Ustilago, Alter-naria, Helminthsporium and Uredinales were more prevalent. There were 18 genera of fungi colonies identified. Among which Penicillium, non-sporulating fungi, Aspergillus, Saccharo-myces and Cladosporium were more common. The airborne fungal spores were present in the atmosphere of Shenzhen University all year round. The peaks of airborne spores appeared during April and October, while the lowest numbers were observed during January, July and December from March 2005-Febrary 2006. The highest volumes of fungi colonies were observed during April, October and September, while the lowest numbers were detected during in January, July and December or May from March 2005-Febrary 2006. The meteorological factors had no relationship between the total monthly spore count at 10 and 30 meter height. At 70 meter, the total spores count was nega-tively correlated with solar radiation. Conclu-sions: Most of the fungi spores decreased along with the increase of altitudes.

Keywords: Airborne Fungi; Open-Plate Method; Slide-Exposure Method

1. INTRODUCTION

Airborne fungi are one of the common allergens that

induce respiratory hypersensitivity reaction [1-3]. The major allergic symptoms include asthma, rhinitis, bron-chopulmonary mycoses and hypersensitive pneumonitis [4]. Airborne fungi also act as an indicator for the at-mospheric bio-pollution. The presence of fungal propagules, volatiles and mycotoxins in the air can pose a health hazard in all segments of the population [5]. Fungi variety and concentration depends on various fac-tors, including topography, time of day, meteorological parameters, seasonal climatic variation and type of vegetation [6-7]. Extensive investigations of airborne fungi had been done in many parts of the world [8-15]. In China, such studies had been done in different prov-inces [16-25].

But to our knowledge there are no published data on the airborne fungi at different altitudes in one place. Shenzhen city is located at 22°27'-22°52' N and 113°46'- 114°37' E. The weather in Shenzhen is associated with high temperature and humidity throughout the year, which suits for the reproduction of airborne fungi. As a well developed city, Shenzen has a large number of tall buildings and mansions providing both working and living spaces. The aim of this work was to determine the concentration of airborne fungi present at different alti-tudes and the effect of seasonal variations, which will provide useful information on the air quality of residen-tial areas.

2. METHODS

2.1. Slide-Exposure Method

The slide-exposure method based on the protocol adapted from Ye (1992) was used to determine the fungal spores [26]. Briefly, the slides with vaseline for spore sampling were set on the second floor (10 meter above ground level), the seventh floor (30 meter above ground level) and the sixtieth floor (70 meter above ground level) of the technological building in Shenzhen University (22°54′37'' N and 113°93′77'' E), respectively. Two slides were collected daily from March, 2005 to Febru-ary, 2006. The slides were stained by basic fuchsin solu-

L. Li et al. / Natural Science 2 (2010) 506-514


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tion. Fungal spores were enumerated and identified by using a light microscope (OLYMPUS BH-2, Japan). The average spore counts of the two samples in each altitude were taken.

2.2. Open-Plate Method

The open-plate method was adapted based on Ye (1992) to determine the fungal colonies [26]. Briefly, the plates with Peptone Dextrose Agar, Potato Dextrose Agar and Czapek Dox Agar respectively were set on the same places as described above once during each month (total 12 times in one year). Three plates were opened for a period of 5 min at various altitudes, and then were incu-bated at 28 ± 1 for up to 5 days. The colonies were stained by lactic acid methylene blue solution and iden-tified by colony and microscopic morphology. Colony counts were then converted to the colony forming units/m3, CFU/m3 = 5000N/A t (N: colony count; A: square of plate; t: exposure time) [27]. The total colony forming units of three plates at each altitude were added to obtain the total colony forming units at each altitude.

2.3. Statistical Analysis

The relationship between the monthly total spores count at various altitudes, the most common fungi and the monthly meteorological factors (average temperature: X1; average relative humidity: X2; average atmospheric pressure: X3; average wind speed: X4; rainfall: X5 and solar radiation: X6) were established respectively by means of stepwise multiple regression method (The data were analyzed using SPSS Version 12.0). The value of p < 0.05 was considered statistically significant. The meteorlogical data were obtained from Shenzhen me-teorlogical administration (Table 1).

3. RESULTS

3.1. Airborne Fungal Spore Count at Three Different Altitudes

During the entire year, there were 2190 slides collected at three different altitudes. The total fungal spore counts at 10, 30 and 70 meters height were 4,102, 3,540 and

2,929.5 respectively. There were 27 genera or species identified belonging to the subphylum Zygomycotina family Mucoraceae: Rhizopus (0.17%); subphylum As-comycotina family Sphaeriaceae: Chaetomium (0.39%); subphylum Basidiomycotina family Ustilaginaceae: Usti-lago (13.04%), Uredinales (9.54%); subphylum Deutero-mycotina family Moniliaceae: Cladosporium (16.45%), Aspergillus (0.59%), Geotrichum (0.52%), Botrytis (0.31%), Trichothecium (0.09%), family Dematiaceae: Alternaria (11.41%), Helminthosporium (11.37%), Curvularia (8.41%), Stachybotrys (5.38%), Stemphylium (4.58%), Nigrospora (1.93%), Heterosporium (1.02%), Acrothe-cium sp. (0.53%), Papularia (0.31%), Clavispora (0.22%), Cercospora sp. (0.09%), Wardomyces (0.05%), family Tuborculariaceae: Fusarium (0.85%), Epicoccum (0.48%), family Sphaeropsidaceae: Hendersonia sp. (4.18%), As-cochyta sp. (4.04%), Diplodia sp. (3.65%), Sphaeropsis sp. (0.20%) and unidentified spores (0.20%) (Tables 2-4).

At three different altitudes, the majority of spores were Cladosporium, Ustilago, Alternaria, Helmintho- sporium and Uredinales.

3.2. Fungal Colony Forming Units at Three Different Altitudes

During the entire year, the total of 53,473, 50,962 and 49,543 colony forming units at 10, 30 and 70 meters, respectively were collected, enumerated and then char-acterized into 18 genera or species. The common fungi belonged to the subphylum Zygomycotina family Mu-coraceae: Mucor (0.31%), Rhizopus (0.31%); subphylum Ascomycotina family Sphaeriaceae: Chaetomium (0.51%), family Saccharomyetaceae: Saccharomyces (12.67%); subphylum Deuteromycotina family Moniliaceae: Peni-cillium (24.11%), Aspergillus (15.63%), Cladosporium (11.85%), Trichothecium (0.71%), Trichoderma (0.72%), Botrytis (0.51%), Geotrichum (0.31%), family Dema-tiaceae: Alternaria (5.11%), Curvularia (5.00%), Hel- minthosporium (1.22%), Nigrospora (0.51%), Stachy-botrys (0.51%), family Tuborculariaceae: Fusarium (0.71%), family Sphaeropsidaceae: Phoma sp. (0.61%), non-sporulating fungi (18.28%) and unidentified colony (0.41%) (Tables 5-7).

Table 1. Averages of meteorological measurements: temperature (T), relative humidity (RH), atmospheric pressure (P), wind speed (WS), rainfall (R), and solar radiation (SR) in Shenzhen from March 2005-Febrary 2006.

Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb

T (ºC) 17.2 23.1 27.2 27.8 29.4 28.5 28.8 26.2 22.9 16.7 16.2 17.8

RH (%) 71 74 76 78 71 79 72 64 67 50 69 72

P (hPa) 1016.6 1011.9 1005.6 1002.4 1004.3 1002.8 1007.2 1012.8 1014.4 1019.2 1016.4 1017.6

WS (m/sec) 2.1 1.8 1.8 1.7 1.7 1.9 2.2 2.4 2.2 2.7 2.6 2.3

R (mm) 48.3 42.9 379.2 469.9 326.6 587.3 231.8 21.3 14.0 9.0 20.6 48.0

SR (h) 759 881 1194 756 2264 1639 1495 1946 1798 1549 1202 912

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Table 2. The identified airborne fungi genera or species and slide fungal spores count at 10 meter during 12 months.

Genera monthor species

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total spores

Rhizopus 0 0 0 2 0 0 2.5 0 0 0 0 0 4.5 Chaetomium 0 2 1 0 2.5 0 2 4.5 1.5 1.5 1 4 20 Ustilago 23.5 67.5 80.5 37.5 56.5 22.5 15 31 81 57.5 23.5 42.5 538.5 Uredinales 40.5 36 40 29 26.5 30.5 32 30.5 30 32.5 47.5 15.5 390.5 Cladosporium 47 21.5 19 90.5 103 52 32.5 52.5 15.5 97.5 72.5 22 625.5 Aspergillus 2.5 3 0 3 1 1 3 0 2 3.5 2.5 0 21.5 Geotrichum 6.5 0 0 2 0 0 2.5 0 3 0 2 1.5 17.5 Botrytis 0 2 1.5 0 3 2 0 1.5 3.5 0 0 5.5 19 Trichothecium 0 1 0 0 0 0 0 0 0 0 0 3 4 Alternaria 42 26.5 33.5 81 46.5 37 34.5 52 40 37 30 25.5 485.5 Helminthosporium 24 40.5 68 22.5 42 48 24.5 30 34 58 31.5 34 457 Curvularia 24 34 24.5 36.5 31.5 29 15 15.5 30.5 44 39.5 23 347 Stachybotrys 18 20 13 33 15 14.5 9.5 20 20.5 24.5 18 23.5 229.5 Stemphylium 15 15.5 15 18.5 16 14.5 13 15.5 23.5 17.5 21.5 20 205.5 Nigrospora 6.5 10.5 15 6.5 4 5.5 10 5 3.5 7.5 6 6.5 86.5 Heterosporium 5.5 0 1.5 6 2 2 1.5 0 5.5 5.5 0 0 29.5 Acrothecium sp. 2 0 2 3 3 3 2.5 3 4.5 0.5 1 4.5 29 Papularia 0 0 0 0 0 1.5 0 0 3.5 5 4.5 0 14.5 Clavispora 1 0.5 1 0 0.5 0 0 1 0.5 0 0 0 4.5 Cercospora sp. 0 0 2 0 0 0 1 0 0 0.5 0 1 4.5 Wardomyces 0 0 0 0 0 0 1 0 0 0 0 0.5 1.5 Fusarium 4 1.5 1 2.5 1.5 2 0 0 2.5 2.5 2.5 0 20 Epicoccum 2 2.5 1 2.5 0 1.5 0 2 2.5 5.5 0 2.5 22 Hendersonia sp. 15 16 27 18.5 15 9.5 13 10.5 19.5 16.5 9 24.5 194 Ascochyta sp. 9 7.5 11 16.5 7.5 10.5 15 15 17 15.5 19.5 23 167 Diplodia sp. 20 7.5 16 16.5 11.5 8 11 8 15.5 12 12 7 145 Sphaeropsis sp. 2.5 0 5 0 0 0 0 1 0 0 0 1.5 10 Unidentified spores 2.5 0.5 1 0 0 0 1 0.5 0 1 0.5 1.5 8.5 Total spores 313 316 379.5 427.5 388.5 294.5 242 299 359.5 445.5 344.5 292.5 4102


Genera month or species

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total spores

Rhizopus 0 0 1.5 2.5 0 0 1 0 0 0 0 4 9 Chaetomium 0.5 1.5 2.5 0 1.5 0 1.5 2.5 0.5 1.5 0.5 1.5 14 Ustilago 26.5 33.5 59 47 26 22.5 16.5 25.5 68 62.5 24.5 32.5 444 Uredinales 34.5 30.5 31.5 32.5 15.5 24 26 23 23 40.5 31 22.5 335 Cladosporium 21.5 28.5 29.5 86 56.5 46 32.5 43 22.5 85.5 54 38.5 544 Aspergillus 1.5 2 2 2.5 2 3 0 2 3 1.5 0 2 21.5 Geotrichum 2 0 1 3 2 0 1.5 0 3.5 0 1 1.5 15.5 Botrytis 0 1.5 0.5 0 1.5 2.5 0 1 1 0 0 3 11 Trichothecium 0 0.5 0 0 2 0 0 0 0 0 0 1 3.5 Alternaria 37.5 23.5 21 56 40.5 28.5 31.5 42 23.5 34.5 24.5 29 392 Helminthosporium 16 32 62 45.5 36.5 43 14 27 27.5 44.5 24 24.5 397 Curvularia 20.5 24.5 32 36 33.5 36 17 17 23.5 38.5 34.5 16 329 Stachybotrys 12 17.5 18 18 20.5 16 12 17.5 15.5 25.5 19 7.5 199 Stemphylium 8 12.5 11 14 20.5 19.5 10 12 20.5 14 17 15 174 Nigrospora 8 7.5 10 10 3.5 4.5 7 4 1.5 9.5 4 8.5 78 Heterosporium 3.5 0.5 1.5 5.5 3.5 2.5 1 0 4.5 5.5 0.5 2 30.5 Acrothecium sp. 1 0 0 1.5 1.5 1 1.5 2.5 3.5 0 1.5 2 16 Papularia 0 1 0 0 1.5 2.5 0 0 2 3 2.5 0 12.5 Clavispora 1 1.5 0.5 1 0 0.5 0 0 1 1.5 1 1 9 Cercospora sp. 0 0 0.5 0 0.5 0 0 0 0 0 0 0 1 Wardomyces 0 0 0 0 0.5 0.5 0 0 0 0 0 0 1 Fusarium 2 2.5 2.5 4 2.5 4 1 0 4 4.5 1.5 0 28.5 Epicoccum 1 1 1 0 0 3.5 1.5 3.5 1 4.5 0 0 17 Hendersonia sp. 19 8.5 22 15.5 11 9 10 8 15 13.5 6 14.5 152 Ascochyta sp. 13 17.5 25.5 17 9 7.5 13 12 12 12 9.5 14.5 163 Diplodia sp. 13.5 14 21.5 12 7 6 6 10.5 10.5 12.5 9.5 5 128 Sphaeropsis sp. 0 0 2.5 0 2.5 0 0 0 1.5 0 0 2.5 9 Unidentified spores 1 1 0.5 0 1 1 0.5 0 0 0 0.5 2 7.5 Total spores 243.5 263 359.5 409.5 302.5 283.5 205 253 288.5 415 266.5 250.5 3540




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Genera month or species Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Total spores

Rhizopus 0 0 0 3 0 0 0 0 0 0 1.5 0 4.5 Chaetomium 0 2 0.5 1 2 0 0 0.5 0 0 0 1 7 Ustilago 18.5 46.5 30.5 44.5 23.5 32 21 30.5 74.5 26.5 31 16.5 395.5Uredinales 13 19 40.5 43.5 19.5 14.5 20.5 15 12 19.5 35.5 30 282.5Cladosporium 29.5 16.5 95.5 88.5 33 36 19.5 36 20.5 65 65 65 570 Aspergillus 2.5 0 1.5 3.5 3 1.5 0 2.5 2 1.5 0 1 19 Geotrichum 3.5 0 1.5 5 5 0 0.5 0 3 0 1 2 21.5 Botrytis 0 0 0.5 0 0.5 2 0 0 0 0 0 0 3 Trichothecium 0 0 0 0 2.5 0 0 0 0 0 0 0 2.5 Alternaria 27 35 40.5 44.5 30 27 20 24 15.5 19.5 15 31 329 Helminthosporium 24.5 38 39 55 29.5 32.5 18.5 36 19 32 12 12 348 Curvularia 6 28.5 10 25 23 18.5 12.5 22 19.5 21 18.5 9 213.5Stachybotrys 0 12 9.5 13.5 18.5 15.5 10 15 8.5 16.5 10.5 10.5 140 Stemphylium 4 6 8 9.5 12.5 10.5 6 7 12.5 9 10.5 9 104.5Nigrospora 3 4.5 9 3.5 2 2 6 2 0.5 1.5 2 3.5 39.5 Heterosporium 8.5 1.5 2.5 2 6.5 5 0 1.5 7.5 6.5 2.5 4 48 Acrothecium sp. 1 0 1 1 1.5 0 0.5 1 2 2 0.5 1 11.5 Papularia 0 2 0 0 1 1.5 0 0 1.5 0 0 0 6 Clavispora 4 1 1.5 0.5 1 1 0 0 0 0 0 1 10 Cercospora sp. 0 0 3.5 0 0.5 0 0 0 0 0 0 0 4 Wardomyces 0 0 0 0 0 0 2 0 0.5 0 0 0 2.5 Fusarium 6 3.5 2 3 2.5 4.5 2.5 2.5 3 3.5 3.5 5 41.5 Epicoccum 0 2 2.5 0 0 1.5 1.5 1.5 0 0 3 0 12 Hendersonia sp. 13 6 11 8 7.5 7 7 5 10 8.5 4 8.5 95.5 Ascochyta sp. 5.5 7 13 8 16 5.5 8 9.5 5.5 8.5 6 5 97.5 Diplodia sp. 8 18.5 12.5 9.5 8.5 11 6.5 11.5 7.5 6.5 6.5 6.5 113 Sphaeropsis sp. 0 0 1.5 0 0 0 0 0 1 0 0 0 2.5 Unidentified spores 0 2 1 1 0 0 1 0 0 0 0 0.5 5.5 Total spores 177.5 251.5 338.5 373 249.5 229 163.5 223 226 247.5 228.5 222 2929.5

Table 5. The identified airborne fungi genera or species and colony forming count at 10 meter during 12 months.

Genera month

or species Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Total colony

Mucor 0 157 157 0 0 0 0 0 0 0 0 0 314Rhizopus 0 0 0 0 157 0 0 0 0 0 0 0 157Chaetomium 0 0 0 0 0 157 0 0 157 0 0 0 314Saccharomyces 157 472 786 944 472 629 157 472 315 315 472 472 5663Penicillium 157 944 1258 2674 472 786 315 315 2202 1887 1415 472 12897Aspergillus 315 472 315 2045 315 1573 315 315 1101 1887 472 157 9282Cladosporium 786 1258 472 315 472 315 315 472 629 315 315 629 6293Trichothecium 0 157 0 0 0 0 0 157 0 0 0 0 314Trichoderma 0 315 0 0 315 0 0 0 0 0 0 0 630Botrytis 0 0 157 0 0 0 0 0 0 157 0 0 314Geotrichum 0 0 0 0 0 0 0 157 315 0 0 0 472Alternaria 0 0 629 157 157 0 0 315 315 157 157 0 1887Curvularia 0 0 315 0 157 0 0 0 157 629 0 157 1415Helminthosporium 0 157 157 0 0 315 0 157 0 0 157 0 943Nigrospora 0 0 0 0 0 0 0 0 157 0 0 0 157Stachybotrys 0 0 157 0 0 0 0 0 0 157 0 0 314Fusarium 0 157 0 157 0 0 0 0 157 0 0 0 471Phoma sp. 0 0 157 0 0 0 0 157 0 0 0 0 314Non-sporulating fungi

0 629 629 2045 629 1101 157 944 1887 1415 786 786 11008

Unidentified fungi 157 0 0 0 0 0 0 0 0 0 157 0 314

Total colony 1572 4718 5189 8337 3146 4876 1259 3461 7392 6919 3931 2673 53473



510


Genera month


Totalcolony

Mucor 0 0 0 0 0 157 0 0 0 0 0 0 157

Rhizopus 0 0 0 0 0 157 0 0 0 0 0 0 157

Chaetomium 0 157 0 0 0 0 0 0 0 0 0 0 157

Saccharomyces 157 629 472 1101 629 315 629 315 315 315 472 315 5664

Penicillium 315 1101 1573 2045 629 629 472 629 2045 2359 1101 472 13370

Aspergillus 472 472 1573 1730 315 315 629 472 629 629 315 315 7866

Cladosporium 472 472 315 472 315 1101 0 629 315 315 157 157 4720

Trichothecium 0 0 157 0 0 0 0 0 0 315 0 0 472

Trichoderma 0 0 0 0 0 157 0 0 0 0 0 0 157

Botrytis 0 157 0 0 0 0 0 0 0 0 0 0 157

Geotrichum 0 0 0 0 0 0 0 0 0 0 0 0 0

Alternaria 0 315 0 157 0 472 0 157 944 315 157 157 2674

Curvularia 0 157 315 0 0 157 0 629 315 786 472 472 3303

Helminthosporium 0 157 0 0 0 0 157 0 0 157 0 0 471

Nigrospora 0 0 0 0 0 0 0 0 315 0 0 0 315

Stachybotrys 0 157 0 0 0 0 0 0 0 0 0 0 157

Fusarium 0 0 0 0 0 157 0 0 0 0 0 0 157

Phoma sp. 0 0 0 0 0 0 0 0 157 157 0 0 314

Non-sporulating fungi 472 315 1258 2516 315 629 0 786 1415 1730 472 629 10537


Total colony 1888 4089 5663 8021 2360 4246 1887 3617 6450 7078 3146 2517 50962


Genera month


Total colony

Mucor 0 0 0 0 0 0 0 0 0 0 0 0 0

Rhizopus 0 157 0 0 0 0 0 0 0 0 0 0 157

Chaetomium 0 157 0 0 0 0 0 0 0 157 0 0 314

Saccharomyces 472 1258 786 786 315 629 315 786 786 944 472 629 8178

Penicillium 472 315 1730 2045 944 944 472 472 629 1415 944 472 10854

Aspergillus 472 315 472 1258 315 786 472 472 786 944 629 0 6921

Cladosporium 786 472 1101 629 472 629 315 472 786 472 629 472 7235

Trichothecium 0 0 0 0 0 0 0 0 157 157 0 0 314

Trichoderma 0 0 0 157 0 0 0 0 0 0 157 0 314

Botrytis 0 0 0 0 0 157 0 0 0 0 157 0 314

Geotrichum 0 0 0 0 0 0 0 0 0 0 0 0 0

Alternaria 0 472 472 472 157 315 0 472 315 157 157 315 3304

Curvularia 0 0 0 157 157 472 0 157 786 944 0 315 2988

Helminthosporium 0 0 0 157 157 0 0 0 157 0 0 0 471

Nigrospora 0 0 0 0 0 0 0 0 315 0 0 0 315

Stachybotrys 0 0 0 0 0 157 0 0 0 0 157 0 314

Fusarium 0 157 315 0 0 0 0 0 0 0 0 0 472

Phoma sp. 0 0 0 0 0 0 0 0 0 0 157 157 314

Non-sporulating fungi 0 315 629 1573 786 786 472 315 315 629 315 472 6607


Total colony 2202 3618 5505 7234 3303 4875 2046 3146 5032 5976 3774 2832 49543



511511

3.3. Effect of Seasonal Variations on the

Total Airborne Fungi in One Year

The airborne fungal spores were present in the air around Shenzhen University throughout the year. The distribution curve of total airborne fugal spores peaked in April and October, and dropped to the lowest values in January, July and December (Figure 1). The distribution curve of the total colony forming unit spiked in April, October and September, and fell to the lowest values in January, July and December (Figure 2). Months

3.4. The Effect of Seasonal Variations on Figure 2. The distribution of colony forming count during a 12 month period. Airborn Fungi at Three Different Altitudes

At 10, 30 and 70 meters height, increased levels of total airborne fugal spores was observed during April and October, while lower levels were observed during Janu-ary, July and December (Figure 3). The concentration of fungal spores decreased with respect to the increase in the altitude. The number of spores at 70 meters was lower compared to the levels observed at 30 and 10 me-ters during September and October.

The distribution curve of fungal colony at 10, 30 and 70 meters height indicated a sharp increase during four months from January to April. The distribution curve of the fungal colonies at 10 and 70 meters reached to its lowest point during July, January, December, and at 30 meter height similar levels were observed in July, Janu-ary and May (Figure 4).

Months

Figure 3. The distribution of slide spore count at three alti-tudes during a 12 month period.

3.5. The Relationship between the Airborne Fungal and Meteorological Factors

The monthly meteorological measurements: average temperature, average relative humidity, average atmospheric pressure, average wind speed, rainfall and solar radiation had no observable relationship between the total spore count at 10 and 30 meters height. At 70 meters height, the total spore count was negatively correlated with solar radiation (Y = 342.191 – 0.718 X6, r = 0.602, p < 0.05).

Months

Figure 4. The distribution of colony forming count at three altitudes during a 12 month period.

The total monthly spore count of Cladosporium, Usti-

lago, Alremaria had no relationship between the mete-orological factors. The spore counts of Holminthsporium was negatively correlated with solar radiation (Y = 156.026 – 0.409 X6, r = 0.602, p ＜ 0.05). The spore count of Uredinales was negatively correlated with av-erage wind speed and rainfall (Y = 176.894 – 35.314 X4 – 0.099 X5, r = 0.805, p ＜ 0.05).

Months

Figure 1. The distribution of slide spore count during a 12 month period.


http://dict.cnki.net/dict_result.aspx?searchword=%e9%99%8d%e4%bd%8e&tjType=sentence&style=&t=decreased



512

4. DISCUSSION

4.1. Comparison of Airborne Fungi in Shenzhen University and Other Regions

The most common fungi documented in many Chinese districts were Alremari, Cladosporium, Ustilago, Ure- dinales, Aspergillus, Penicillium, Holminthsporium, Fu- sarium and Saccharomyces [16-25]. Similar observa-tions were documented in many other countries as well [7-9,12,14,28,29]. The top five genera of fungi spores in our study were Cladosporium (16.45%), Ustilago (13.04%), Alremaria (11.41%), Holminthsporium (11.37%) and Uredinales (9.54%). While the top five genera of fungi spore in Guangzhou were Alremaria (27.49%), Ustilago (17.26%), Uredinales (6.95%), Hol-minthsporium (6.09%) and Fusarium (4.99%). Clado- sporium only took up 0.77% in Guangzhou [16]. The top five genera of fungal colony in our study were Penicil-lium (24.11%), Aspergillus (15.63%), Saccharomyces (12.67%), Cladosporium (11.85%) and Alremaria (5.11%). The top five genera of fungi colony in Guang-zhou were Cladosporium (21.60%), Penicillium (19.71%), Alremaria (5.54%), Rhizopus (5.05%) and Aspergillus (4.43%). The non-sporulating fungi in our study area and in Guangzhou were 18.28% and 30.79%, respectively [16]. This indicates that the fungal spore count differs according to time and place.

The concentration of fungal spores changed with sea-sonal variation in many districts in China during April to October [16-25]. The concentration of fungal spores in Guangzhou was high during April, September and Oc-tober, and the lower during January, July and December [16]. Similar results were observed in our study.

4.2. Comparison of Airborne Fungal Spores at Different Altitudes

Our results showed that the total spore count decreased with the increase in height. However, the total genera of fungi did not decrease with the height. The fungi with larger spore sizes, such as Holminthsporium, Alremaria, Ustilago and Curvularia were concentrated at 10 meters height. While the fungi with smaller spore sizes such as Cladosporium was concentrated at 70 meters. The count of Aspergillus didn’t show much difference at 30 or 70 meters height. Chakraborty (2001) [13] reported that the smaller spores were dominant at greater heights and lar-ger spores and conidia were more prevalent at lower levels. Furthermore, the distribution of fungal spores at different altitude was influenced by their shapes [30].

4.3. The Relationship between the Airborne Fungi and Meteorological Factors

The distribution of airborne fungi spores can be affected by various factors including meteorological factors. The effect of meteorological factors on the count of airborne

fungal spores varied from one fungal taxon to another. Most airborne fungi have a strong relationship with temperature; however, Aspergillus/Penicillium hyphal fragments were positively correlated with wind speed. In comparison with other airborne fungi, Leptosphaeria and unidentified Ascomycetes were more closely corre-lated with rain and relative humidity during the growing season [31]. Alternaria and Cladosporium are positively correlated with temperature and duration of sunlight. However, Ustilago indicated a positive correlation with relative humidity and negative correlation with wind speed [32]. Alternaria and Cladosporium showed a posi-tive association with temperature, duration of sunlight and accumulated rainfall, but negatively correlated with daily rainfall [33].

In our results, the meteorological factors had no ob-servable relationship between the total monthly spore count and altitude. At 70 meters, the total spore count was negatively correlated with solar radiation. The total monthly spore count of Cladosporium, Ustilago, Alre-maria had no observable relationship between the mete-orological factors. The spore count of Holminthsporium was negatively correlated with solar radiation, while the spore count of Uredinales was negatively correlated with average wind speed and rainfall. In this study, the tem-perature, relative humidity, atmospheric pressure did not affect the total spore concentrations. It may be due to the subtropical climatic location of Shenzhen. The meteoro-logical data showed that the temperature, relative hu-midity, atmospheric pressure did not change drastically in Shenzhen during the entire year. Therefore, seasonal variations did not affect the distibution of the fungal spores significantly.

5. CONCLUSIONS

There were 27 genera or species of fungus spores and 18 genera of fungi colonies identified in a given year. The airborne fungal spores were present in the atmosphere of Shenzhen University all year round. The peaks of air-borne spores appeared during April and October, while the lowest numbers were observed during January, July and December. The highest volumes of fungi colonies were observed during April, October and September, while the lowest numbers were detected during in Janu-ary, July and December or May. The meteorological factors had no relationship between the total monthly spore count at 10 and 30 meter height. At 70 meter, the total spores count was negatively correlated with solar radiation. Most of fungi spores decreased along with the increase of altitudes.

6. ACKNOWLEDGEMENTS

This study was financially supported by the natural science foundation of Guangdong Province (No. 04300891). The authors would like to gratefully acknowledge Guo- qiang Xiong and Yan Ying in center for



513513


disease control of Nanchang who helped with the indentification of the fungi. The authors would also thank Shenzhen meteorlogical admini-stration for providing the meteorlogical data.

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[33] Sabariego, S., Guardia, C.D. and Alba, F. (2004) Aerobi-ological study of Alternaria and Cladosporium conidia in

the atmosphere of Almeria (SE Spain). Revista Ibero- americana de Micología, 21(3), 121-127.

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Vol.2, No.5, 515-518 (2010)doi:10.4236/ns.2010.25064


Natural Science

Effect of latex conversion on glass transition temperature

Shao-Xiang Li1, Ying-Dong Guan1*, Lu-Mei Liu2

1Department of Micropolymer Materials of Science and Engineering, Qingdao University of Science & Technology, Qingdao, China; *Corresponding Author: [email protected] 2Department of Materials Science and Engineering, Qingdao University of Science & Technology, Qingdao, China

Received 29 January 2010; revised 1 March 2010; accepted 25 March 2010.

ABSTRACT

We have synthesized styrene-acrylic latex and investigated the effect of such reaction condi-tions as the dosage of initiator, surfactant and stirring speed on monomer conversion and glass transition temperature (Tg) of polymer by means of orthogonal experiment, then we get the best reaction conditions. Test results prove that the glass transition temperature of the polymer is directly related to the monomer conversion. The improvement of monomer conversion can make the glass transition temperature close to the theoretical value. In the case of high final conversion, we can predict the glass tran-sition temperature of the polymers of different composition according to the theoretical rela-tion effectively.

Keywords: Monomer Conversion; Orthogonal Experiment; Glass Transition Temperature

1. INTRODUCTION

Styrene-acrylic latex is made of styrene and acrylate monomers, which has many advantages. For example, it has wide source of raw materials, high function/price ratio, simple synthetic process and the latex has out-standing water resistance, alkali resistance, scrub re-sistance and also the paint film has good outdoor du-rable, adhesive attraction. So the styrene-acrylic latex has been widely used in building coating, metal sur-face coating and so on. Many researchers [1-5] have studied styrene-acrylic latex. Climates are usually di-verse across countries, even in one country. Therefore, a single recipe cannot satisfy different needs in the different climate. In order to adapt to different envi-ronment, especially the temperature environment, it requires the minimum film-forming temperature can not only has an unchangeable temperature. Scholars in this area had focused mostly on performance optimi-

zation but ignored the investigation of minimum film- forming temperature. In fact, there is a big difference between actual minimum film-forming temperature and theoretical minimum film-forming temperature, which brings polymer designers difficulties in pre-dicting glass transition temperature and designing the hardness of the polymer, at the same time, brings users a lot of inconvenience in use. There are many reasons for the difference between actual minimum film- forming temperature and theoretical minimum film- forming temperature, one of the most important is the monomer conversion. Due to the minimum film- forming temperature has a good corresponding relation with the glass-transition temperature [6], so this paper mainly investigates the glass-transition temperature by means of optimizing the latex’s polymerization condi-tions. We obtain latex with high conversion, thus we can solve the above problems in polymerization tech-nology aspect and obtain the latex recipe of different glass transition temperature under the guidance of the theoretical relation.

2. EXPERIMENTAL

2.1. Materials

Butyl acrylate (BA, 96%), Styrene (St, 97%), Methyl Methacrylate (MAA, 96%) and Diacetone acryl amide (DAAM) were purchased from Qingdao Reagent Com-pany. The anionic surfactant sodium dodecyl sulfate (SDS), nonionic surfactant nonylphenol polyoxyethyl-ene ether (OP-10) and ammonium persulfate (APS) were purchased from Qingdao Chemistry Reagent Company. All materials were used without further puri-fication.

2.2. Preparation of Styrene-Acrylic Latex

All emulsifier and deionized water were feeded into four-necked flask and stirred at high speed first, then feed monomer mixtures slowly to obtain the before-hand latex. Take part of beforehand latex for seed latex, when temperature was wormed up to 75 ± 1, feed

S.-X. Li et al. / Natural Science 2 (2010) 515-518


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part of initiator solution. After the blue seed latex formed, the remaining latex was fed gradually, and par-tially drop initiator, beforehand latex and initiator were added respectively in 3.5 h and 4 h. Then the tempera-ture was heated to 85 ± 1 a nd kept this temperature for 1 h, then cooled, adjusted PH = 7-8, Filtered and Collected latex at last.

2.3. Characterization

The test of solid content:

1

0

100%m

sm

where s is the solid content of latex, m1 is the weight of the latex after dried at 80 in vacuum drying oven, m0 is the weight of the latex.

The test of monomer conversion: We calculate the conversion by below relation

%100(%)0

4321

W

WWWSWC

where W1 is the whole output of latex, W2 is the amount of gel, W3 is the amount of initiator, W4 is the amount of emulsifier, W0 is the amount of whole monomers, S is the solid content of latex.

The theoretical value of copolymer’s glass transition temperature:

Using the following FOX relation, we can get the composition of copolymers which have an expectable Tg.

31 2

1 2 3

1...... n

n

W WW W

Tg Tg Tg Tg Tg

where Tg is the glass transition temperature of co-polymers, Tgl, Tg2, Tg3, Tgn are the glass transition temperature of the respective homopolymers and Wl, W2, W3, Wn are the weight fraction of the respective groups.

2.4. Differential Scanning Calorimeter (DSC) Analysis

Tg was measured by the DSC method in a NEYZSCH 204F1 type differential scanning calorimeter for polymer samples of ~20 mg. DSC condition measurement: hold for 1.0 min at –100 , heat from –80 to 100 at 10 min –1 and with nitrogen protection.


3.1. Choice of Variables and Level of the Orthogonal Experiment and its Results

During the experiment we found that there is a big dif-ference between measured value and theoretical value of Tg (the theoretical value is calculated by FOX relation).

After analysis, the author believes that the main reason of this phenomenon is due to a lower conversion of po-lymerization, the system was not polymerized according to the expectable proportion, so we do the experiment to optimize the process parameters of polymerization by orthogonal experimental firstly in order to obtain the latex with high monomer conversion.

Based on a large number of references and many re-peated experiments, we consider that the dosage of ini-tiator (A), Emulsifier (B) and stirring speed (C) are the main factors of polymerization, and have designed L9 (33) orthogonal table (three variables, three levels Or-thogonal design), the results are shown in Table 1.

3.2. The Analysis of Orthogonal Experiment Results

The weighted average (K) and range (R) are given in Table 2.

Table 2 shows that the sequence of the effect of vari-ous factors on conversion is emulsifier > initiator > stir-ring speed, the best condition is A3B2C2: initiator: 0.8%, emulsifier: 4%, stirring speed: 180 rpm. But we can see that the difference between k2 (88.033) and k3 (89.000) is very small, and as we know the conversion increase with the increase of the initiator, but the gel will increase ob-viously and the polymerization will become unstable at the same time, so we choose A2B2C2: initiator: 0.6%, emulsifier: 4%, stirring speed: 180 rpm at last.

Table 1. Test results.

Test NO

A %

B %

C rpm

Conversion %

1 1(0.4) 1(2) 1(140) 82.6

2 1 2(4) 2(180) 90.0

3 1 3(6) 3(240) 78.3

4 2(0.6) 1 2 87.6

5 2 2 3 92.5

6 2 3 1 84.0

7 3(0.8) 1 3 90.0

8 3 2 1 91.0

9 3 3 2 86.0

Table 2. The analysis of experiment results.

Test Indicators A B C

k1 83.633 86.733 85.867

conversion k2 88.033 91.167 87.867

k3 89.000 82.767 86.933

R 5.367 8.400 2.000

Table 3. Properties of the latex under the condition of A2B2C2.

solid content (%) gel (%) water absorption(%) Conversion(%)

48.6 3.5 7 94



517517

0


As shown in Tables 1 and 2, the final conversion can reach 94% under the condition of A2B2C2 and it is higher than the others, in addition, some other properties are ideal too.

3.3. DSC Test Analysis

In this paper, the initial composition of monomer is BA: MMA:St = 33:19:18, the theoretical value of Tg which is calculated by FOX relation is 8 . But when we adopt the flowing factor: initiator: 0.6%, emulsifier: 4%, speed: 180 rpm, the final conversion reaches 94% and the meas-ured value of Tg achieved by DSC test is 9.8 (Figure 1), the difference between them is small. That is to say at the condition of high conversion, the measured value of Tg is very close to its theoretical value and so we can design the hardness of copolymers according FOX relation.

3.4. The Relationship of Monomer Conversion and Glass Transition Temperature

Figure 2 shows us the relation between Tg and mono-mer conversion (BA:MMA:St = 33:19:18) and we can

-80 -60 -40 -20 0 20 40 60 80 100 12

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Glass Transition:Mid: 9.8

DS

C /

(mW

/mg)

Temperature /

Figure 1. DSC curve of Styrene-acrylic latex (BA:MMA:St = 33:19:18).

82 84 86 88 90 92 94

8

10

12

14

16

18

20

Tg

/

Monomer conversion /%

Figure 2. The relation between Tg and monomer conversion.

see from it that with the increase of the monomer con-version, the glass transition temperature of the latex de-crease gradually, when the final conversion is over 90%, Tg reaches a plateau and the value is about 10.0 . The mainly reason is that during the radical copolymerization, when the final conversion is low, the monomer with a strong conjugacy is easier to polymerize than the others, styrene is such a hard monomer and the glass transition temperature of its homopolymer is 105 , so the Tg of copolymer will be a little higher than usual; on the con-trary, when the final conversion is high, the system is able to polymerize according to the expectable propor-tion, the measured value and theoretical value of Tg match very well. However, the conversion of polymeri-zation can not reach 100%. In addition, Tg will increase because of the hydrogen bonds formed between the Components [7]. Some references [8-11] introduce that some additives and functional monomers will have a certain impact on glass transition temperature, therefore, there will be a difference between measured and ex-pected value of Tg inevitably.

3.5. The Latex Recipe of Different Tg

In this paper, the total mass of the monomer is fixed at 70 g and the ratio of two hard monomer (St and MMA) will not change at about 1:1, we changes the proportion of soft and hard monomer only. Table 4 shows the latex recipe with different Tg which are obtained at the condi-tion of A2B2C2.

Table 4 shows that when the final conversion is at a high level, the measured value and theoretical value of Tg matches very well, thus researcher can be able to forecast the Tg of polymer according to the proportion monomers.

4. CONCLUSIONS

1) The results of the orthogonal experimental shows that emulsifier > initiator > stirring speed in terms of their effects on conversion. And we get the best condition: emulsifier 4%, initiator 0.6%, stirring speed 180 rpm. Table 4. Latex recipe with different Tg.

BA(g) MMA(g) St(g) theoretical value of Tg

measured value of Tg

1# 48 11 11 –20.0 –18.3

2# 43 14 13 –10.0 –9.3

3# 38 16 16 –3.0 –2.2

4# 33 19 18 7.8 9.8

5# 28 21 21 20.0 20.4

6# 26 22 22 24.0 25.2

7# 24 23 23 30.0 32.7

8# 20 25 25 40.0 42.7

9# 17 27 26 45.0 46.9



518

2) There is a direct relationship between conversion and glass transition temperature, the improvement of the final conversion has made the measured value close to the theoretical value.

[5] Joensson, J.L., Hassander, H. and Jansson, L.H. (1991) Morphology of two-phase polystyrene/poly (methyl methacrylate) latex particles prepared under different polymerization conditions [J]. Macromolecules, 24(1), 126-131.

3) At the condition of high conversion, the measured value and theoretical value of Tg matches very well. [6] Chen, R. (2007) Minimum temperature and polymer

glass-transition temperatures [J]. Chemical Industry of Acrylic and Application, 20(1), 17-19.

REFERENCES [7] Gómez-Carracedo, A. and Alvarez-Lorenzo, C. (2003) Chemical structure and glass transition temperature of non-ionic cellulose ethers DSC, TMDSC [J]. Journal of Thermal Analysis and Calorimetry, 73(2), 587-596.

[1] Yu, Y.B. and Zhang, Y.H. (1996) Studying progress on styrene-acrylate emulsion polymerization[J]. Chemical Industry and Engineering Progress, 2, 36-39. [8] Luo, H. and Wei, Z.G. (1997) Study on Tg of acrylic

emulsion copolymer [J]. Fine Chemicals, 69, 42-45. [2] Wang, W.F., Li, S.X. and Liu, L.M. (2007) Preparation of low VOC waterborne epoxy-acrylate hybrid emulsion for wood coating [J]. Coating Industry, 37(11), 30-33.

[9] Xu, J. and Chen, B. (2005) Prediction of glass transition temperatures of OLED materials using topological indi-ces [J]. Journal of Molecular Modeling, 12(1), 24-33. [3] Pan, G.R., Wu, L.M. and Zhang, Z.Q. (2002) Synthesis

and characterization of epoxy-acrylate composite latex [J]. Journal of Applied Polymer Science, 83(8), 1736-1743.

[10] An, J., Li, X.Y. and Zhu, X.W. (2007) Segment distribut-ing of copolymer form microemulsion copolymerization of styrene and butyl acrylate [J]. Chinese Journal of Colloid & Polymer, 25 (1), 1-2.

[4] Chen, C.F., Lee, K.H. and Chiu, W.Y. (2007) Synthesis and characterization of poly (butyl acrylate-methyl methacrylate)/polyaniline core-shell latexes [J]. Journal of Applied Polymer Science, 104(2), 823-830.

[11] Wang, C.C. and Bao, Q.Y. (2005) Acrylate coatings [M]. Chemical and Industry Publishing Company, Beijing.


Vol.2, No.5, 519-525 (2010)doi: 10.4236/ns.2010.25065


Natural Science

Genomic data provides simple evidence for a single origin of life

Kenji Sorimachi

Educational Support Center, Dokkyo Medical University, Tochigi, Japan; [email protected]


ABSTRACT

One hundred and fifty years ago, Charles Darwin’s on the Origin of Species explained the evolution of species through evolution by natural selection. To date, there is no simple piece of evidence demonstrating this concept across species. Chargaff’s first parity rule states that comple-mentary base pairs are in equal proportion across DNA strands. Chargaff’s second parity rule, in-consistently followed across species, states that the base pairs are in equal proportion within DNA strands [G ≈ C, T ≈ A and (G + A) ≈ (C + T)]. Using genomic libraries, we analyzed the extent to which DNA samples followed Chargaff’s second parity rule. In organelle DNA, nucleotide relationships were heteroskedastic. After classifying organelles into chloroplasts and mitochondria, and then into plant, vertebrate, and invertebrate I and II mito-chondria, nucleotide relationships were ex-pressed by linear regression lines. All regres-sion lines based on nuclear and organelle DNA crossed at the same point. This is a simple dem-onstration of a common ancestor across species.

Keywords: Evolution; Origin of Species; Darwin; Genome; Chargaff’s Parity Rules; Organelle; DNA; Linear Formula

1. INTRODUCTION

On the Origin of Species was published in 1859, stem-ming from observations Charles Darwin made during a voyage on HMS Beagle. According to his theory, all organisms have a common ancestor and a single origin. Since publication, evidence for this theory has accumu-lated. Although molecular clock research—using amino acid or nucleotide replacement rates [1]—has enabled scientists to draw a phylogenetic tree representing bio-logical evolution [2-7], the “Origin of Life” has not yet been drawn using these methods. During the past two

decades, advances in genomics have enabled the se-quencing of entire genomes [8,9]; the first complete ge-nome to be sequenced was that of Haemophilus influen-zae [10]. The complete human genome was sequenced early this century by two groups [11,12] and to date, more than 2,000 species’ genomes have been completely sequenced. Based on complete genome data, codon evo-lution has been precisely analyzed [13], and organisms have been consequently classified [14].

The double-stranded DNA structure is the principle information-containing component of the genome [15]. Based on structural knowledge alone, Chargaff’s first parity rule [16] [G = C, A = T and (G + A) = (T + C)] makes intuitive sense. However, Chargaff’s second par-ity rule [17], in which the same nucleotide relationships are retained within single DNA strands, makes less intui-tive sense. The biological significance of Chargaff’s second parity rule has not been elucidated because of its unclear logical foundation. In the 40 years since its pub-lication, researchers have not known whether Chargaff’s second parity rule is relevant to biological evolution. However, a recent publication has solved this historic puzzle [18]. The solution is based on the facts that ge-nome structure is homogeneous regarding nucleotide composition over the genome [19], and that both for-ward and reverse strands are almost the same [20]. Using the complementary relationship between the two strands, both G and C contents are mathematically expressed by the same G + C formula in a single strand, and eventu-ally G ≈ C and T ≈ A [18]. Thus, the first parity rule comes from the inherent characteristics of nucleotides, and the second from the similarities of nucleotide com-position between forward and reverse strands. These two rules represent different phenomena. The former is mathematically definitive and independent of biological significance, and the latter is less definite, and may or may not have biological significance.

Recently, Mitchell and Bridge examined a wide selec-tion of biological DNA samples to determine whether they fitted Chargaff’s second parity rule [21] (1,495 viral, 835 organelle, 231 bacterial and 20 archaeal genomes; and 164 sequences from 15 eukaryotes). Only single

K. Sorimachi / Natural Science 2 (2010) 519-525


520


DNA strands that formed genomic double-stranded DNA obeyed Chargaff’s second parity rule; organelle DNA and single viral DNA strands did not [21]. Nikolaou and Almirantis reported that mitochondrial DNA could be classified into three groups based on the proportions of G-C and A-T content [22]. They found that mitochon-drial DNA deviated from Chargaff’s second parity rule, and that chloroplasts shared the same relative nucleotide compositions as bacterial genomes [22]. Similar devia-tions from Chargaff’s second parity rule were reported by Bell and Forsdyke [23]. My research group previ-ously examined nuclear and organelle DNA nucleotide correlations, and found that nucleotide contents are cor-related with each other in coding, non-coding, and com-plete nuclear DNA [20]; consistent results were obtained from chloroplast and plant mitochondrial DNA, and only homonucleotide contents are correlated with each other between the coding or non-coding regions and the single DNA strand in animal mitochondria [24]. These results indicate that biological evolution can be expressed by linear formulae [20]. If evolutionary processes are ex-pressed by a single equation, it would suggest that evo-lutionary processes proceeded under the same rule. However, if this is the case, we cannot determine whether evolution diverged from a single or multiple origins, because all species are located on the same sin-gle line. If multiple equations are required, the position of the regression lines would either indicate a single or multiple evolutionary origins.

2. MATERIALS AND METHODS

Genome data were obtained from the National Center for Bio- technology Information (http://www.ncbi.nlm.nih.gov/sites) (NCBI). Chloroplast, plant mitochondria and animal mitochondria were examined. The list of organelles ex-amined has been described in our previous paper [24]. Using the same species, we examined newly collected data alongside previous data [24]. For animal mitochon-dria, classified species are as follows: Group I inverte-brates contained echinodermata (starfish), mollusca (oc-topus and squid) and arthropoda (insects); group II in-vertebrates contained cnidaria (coral), porifera (sponge) and protozoa (flagellate). All calculations were carried out using Microsoft Excel 2003 (Microsoft, Redmond, WA, USA).

3. RESULTS

3.1. Chloroplasts

After normalization, the four nucleotide contents can be expressed by the following equation: G + C + T + A = 1. The nucleotide content of each species was expressed by a linear formula, y = ax + b, where “y” and “x” are the

nucleotide contents, and “a” and “b” are constant values (expressing the nucleotide alternation rate among species and original nucleotide content at the vertical intercept). In our previous study [20], this linear formula was shown to be applicable across species. Nucleotide con-tents based on the complete chloroplast genome were plotted against C content (Figure 1, upper panel).

Two lines representing G/C content and C/C content overlapped, as did lines representing T/C, and A/C con-tent. These relationships obeyed Chargaff’s second par-ity rule. Thus, in chloroplast evolution, the G/C content alternations obey the same rule against C content, as does T/A content. This shows that G ≈ C and T ≈ A, and that the four kinds of nucleotide alternations occur syn-chronously. The former (G and C) alternation is attrib-uted to the latter (T and A) alternation in normalized values. G and C exchanges or T and A exchanges do not occur simultaneously under this rule. The equations, represented by regression lines and regression coeffi-cients, are shown in Table 1. Each regression coefficient is close to 0.9 or more than 0.9. This demonstrates an almost complete correlation between nucleotide content. The slopes in the equations were close to 1 and –1, and the constant values at the vertical intercept were close to 0 and 0.5, respectively.

Figure 1. Nucleotide relationships in normalized values. up-per panel, chloroplast; lower panel, plant mitochondria. Blue diamonds, G; pink squares, C; red triangles, T; and green triangle, A. Each nucleotide was plotted against C content. The vertical axis represents four nucleotide contents, the horizontal axis represents C content.



521521

3.2. Plant mitochondria

Plotting nucleotide contents against C content, the C/G and A/T lines almost overlapped (Figure 1, lower panel). This demonstrates that the alternations of the four nu-cleotide contents occurred synchronously. G/C content alternations obey the same rule in plant mitochondrial evolution, as do T/A alternations.

The characteristics representing linear equations are shown in Table 2. The absolute values of the slope were close to 1 in many equations, whereas that of line T ex-pressed by A was 0.576; line A expressed by T was 0.708. In these two equations, the correlations were slightly reduced and the regression coefficients were 0.67. Figure 2. Ratios of nucleotide contents in plant mito-

chondrial genomes. The horizontal axis represents the number of total nucleotides and the vertical axis represents the ratios (G/C and A/T). Red squares, G/C; and blue diamonds, A/T.

The characteristics representing linear equations are shown in Table 2. The absolute values of the slope were close to 1 in many equations, whereas that of line T ex-pressed by A was 0.576; line A expressed by T was 0.708. In these two equations, the correlations were slightly reduced and the regression coefficients were 0.67.

Plotting the ratios of C/G or T/A against the genome size in plant mitochondria, deviations from 1 were observed in the small genomes (less than 1 × 105 nucleotides), while the ratios were fixed to 1 in the larger genome sizes (more than 1 × 105 nucleotides); this rule was followed without exception in the data we used (Figure 2). 3.3. Animal Mitochondria Relationships between nucleotide contents were also ex-amined in animal mitochondria including vertebrates and invertebrates (Figure 3). The relationships were notably heteroskedastic. The values obtained from plotting G con-tent against C content was classified into two groups by line C, which represents y(C) = x(C). The two groups

Figure 3. Nucleotide relationships in animal mito-chondria. Nucleotide contents were normalized, and G content was plotted against C content. Red squares represent C content against C content. Vertical axis represents G and C content and the horizontal axis represents C content.

Table 1. Regression lines based on chloroplasts.

Sample Vs. pyrimidine R Vs. purine R C = C G = 0.902 C + 0.014 T = –0.889 C + 0.484 A = –1.013 C + 0.502

1 0.96 0.95 0.98

C = 1.024 G – 0.001 G = G T = –0.972 G + 0.495 A = –1.052 G + 0.506

0.96 1

0.97 0.95 Chloroplasts

(97) C = –1.006 T + 0.506 G = –0.969 T + 0.487 T = T A = 0.976 T + 0.004

0.95 0.97

1 0.88

C = –0.940 A + 0.481 G = –0.860 A+ 0.452 T = 0.800 A + 0.067 A = A

0.98 0.95 0.88

1

The numbers in parentheses represent the sample number examined. R represents the regression coefficient.

Table 2. Regression lines based on plant mitochondria.

Sample Vs. pyrimidine R Vs. purine R C = C G = 0.854 C + 0.037 T = –0.906 C + 0.481 A = –0.947 C + 0.482

1 0.90 0.95 0.84

C = 0.938 G – 0.003 G = G T = –0.806 G + 0.476 A = –1.132 G + 0.527

0.90 1

0.80 0.96

Plant Mitochondria (49)

C = –0.988 T + 0.492 G = –0.799 T + 0.443 T = T A = 0.708 T + 0.065

0.95 0.80

1 0.67

C = –0.755 A + 0.409 G = –0.821 A + 0.445 T = 0.576 A + 0.146 A = A

0.85 0.96 0.67

1





522

(invertebrates I and II) are located below and above line C: this suggests that they diverged from this crossing point. Regression lines representing nucleo-tide content relationships in vertebrates, invertebrate I and II are shown in Tables 3-5. Vertebrate mitochon-dria belonged to the same group as invertebrate I mi-tochondria, and the C content of vertebrate mitochon-dria was relatively high.

Nucleotide contents in vertebrate mitochondria were plotted against C content. T/C contents were correlated, while G and A (purines) were not correlated against C content (Figure 4). This finding may be due to the short range of vertebrate distribution and their variations. Line characteristics representing regression lines are shown in Table 3. Even invertebrate mitochondria, when nucleo-tide contents were plotted against G or A (purine) con-tents, G/A contents were correlated, while C and T (pyrimidines) were not correlated against G or A (purine) content (Tables 4 and 5).

Group I invertebrate mitochondria were examined

and are plotted in Figure 5 (upper panel). Various nu-cleotide content relationships are shown, plotted against C content. The regression coefficients for the equations expressing other nucleotide contents against C content were 0.7-0.8 (Table 4). Extended lines representing G and C content converged at 0.06, forming a clear cunei-form. Similarly, A and T lines converged at around 0.05. These results indicate that separations of G from C started at around 0.05 C content, and around 0.45 for T and A content. Regression values are shown in Table 4.

Group II invertebrate mitochondria were examined using the same procedure as above. When G, A and T content was plotted against C content, there was a corre-lation between G and C content (Figure 4, middle panel). A and T lines also converged when C content was 0.10, although the extended C and G lines crossed when C content was 0.02. When C content was plotted against G content, C and G lines converged when G content was 0.16. Regression lines are shown in Table 5.

Table 3. Regression lines based on vertebrate mitochondria.

Sample Vs. pyrimidine R Vs. purine R

C = C G = 0.192 C + 0.093 T = –0.772 C + 0.479 A = –0.420 C + 0.429

1 0.25 0.78 0.37

C = 0.340 G + 0.223 G = G T = –0.119 G + 0.286 A = –1.221 G + 0.491

0.08 1

0.09 0.82 Vertebrate

Mitochondria (39) C = –0.782 T + 0.482

G = –0.068 T + 0.163 T = T A = –0.150 T + 0.355

0.78 0.09

1 0.67

C = –0.333 A + 0.377 G = –0.549 A + 0.317 T = –0.118 A + 0.306 A = A

0.37 0.82 0.13

1


Table 4. Regression lines based on invertebrate I mitochondria.


C = C G = 0.386 C + 0.039 T = –0.782 C + 0.476 A = –0.604 C + 0.485

1 0.83 0.84 0.72

C = 1.804 G – 0.012 G = G T = –1.383 G + 0.482 A = –1.422 G + 0.553

0.83 1

0.68 0.78

Invertebrate I Mitochondria

(30) C = –0.897 T + 0.485 G = –0.339 T + 0.224 T = T A = 0.236 T + 0.292

0.84 0.68

1 0.26

C = –0.860 A + 0.511 G = –0.433 A + 0.273 T = 0.293 A + 0.216 A = A

0.72 0.78 0.26

1


Table 5. Regression lines based on invertebrate II mitochondria


C = C G = 1.488 C + 0.009 T = –0.291 C + 0.402 A = –2.197 C + 0.607

1 0.71 0.22 0.75

C = 0.342 G + 0.066 G = G T = –0.102 G + 0.383 A = –1.239 G + 0.551

0.71 1

0.16 0.88

Invertebrate II Mitochondria

(24) C = –0.160 T + 0.186 G = –0.244 T + 0.270 T = T A = –0.596 T + 0.544

0.22 0.16

1 0.27

C = –0.253 A + 0.211 G = –0.622 A + 0.384 T = –0.125 A + 0.406 A = A

0.75 0.88 0.27

1




523523


Figure 4. Nucleotide relationships in vertebrate mito-chondria. Nucleotide contents were normalized, and nu-cleotide contents were plotted against C content. The horizontal axis represents C content, and the vertical axis represents four nucleotide contents. Pink square, C; blue diamond, G; green triangle, T; and red triangle, A.

Figure 5. Regression lines representing nucleotide al-ternations in various organelles. Upper panel, inverte-brate I mitochondria; middle panel, invertebrate II mi-tochondria; and lower panel, invertebrate I plus verte-brate mitochondria. The vertical axis represents four nu-cleotide contents and the horizontal axis represents C content. Blue diamond, G; pink square、C; green dia-mond, T; red triangle, A; dark red squares, chloroplasts; and large black square, vertebrates.

3.4. Origin of Life

When G/C contents were plotted for various organelles and nuclei, all extended regression lines converged when C content was 0.03 0.02 (mean value s. d.) (Figure 6). Vertebrate mitochondria (a relatively re-cent group) are located towards the right of the slope. This confirms the evolutionary direction (left to right), and confirms that all organisms diverged from the same origin. In fact, Ureaplasma urealyticum, which has the smallest genome size [25], is located towards the left of the slope, though this position is not abso-lute because of reversible nucleotide alternations on the genome.

4. DISCUSSION

This study used recent genomic data and knowledge of Chargaff’s second parity rule to demonstrate common ancestry across species.

Although evolution by natural selection applies to all organelles, animal mitochondrial evolution seems to differ from both nuclei evolution and plant organelle evolution. Brown et al. previously reported the rapid evolution of animal mitochondrial DNA [26]. Animal mitochondria do not follow Chargaff’s second parity rule, but this study revealed that they evolved from a common ancestor. We previously showed that plasmids (not com-partmentalized from the nucleus) have codon frequen-cies that resemble those of the parent organism, although there is no evidence that plasmids pass nuclear genomic material across generations [27]. Thus, the compartmen-talization of cellular organelles strongly influences characteristically organelle evolution.

Although deviations from Chargaff’s second parity rule have been previously discussed [22,23], the results obtained here either demonstrate evolutionary phenom-ena or are caused by other confounding factors. In the

Figure 6. C content (horizontal axis) and G content (ver-tical axis) in nuclei and various organelles. Blue dia-monds, invertebrate I and vertebrate mitochondria; pink diamonds, invertebrate II mitochondria; red squares, plant mitochondria; green triangles, chloroplasts; and black squares, nuclei.



524


present study, deviations from Chargaff’s second parity rule in plant mitochondria depended on the genome size and disappeared in the larger genome size (Figure 2). Thus, differences in gene density between the cyto-sine-rich light and guanine-rich heavy strands affect Chargaff’s second parity rule in the relatively small animal mitochondria, while they were cancelled out in the larger plant mitochondria. In fact, the ratios (C/G and T/A) were extremely close to 1 in the chloroplast DNA where genome sizes were more than 5 × 105 nucleotides; no exceptions were observed in the samples examined (unpublished data). This fact clearly shows that genome size is an important factor in Chargaff’s second parity rule [22]. In the Treponema pallidum genome, although the gene density differs between the forward and reverse strands [28], this organism obeys Chargaff’s second par-ity rule [21]. The nuclear genome of Ureaplasma urea-lyticum, which also obeys Chargaff’s second parity rule, consists of 7.5 × 105 nucleotides [25]. This reflects the fact that plant mitochondrial genome sizes are much smaller than plant nuclear genomes.

Animal mitochondria did not obey Chargaff’s second parity rule, even after classification into vertebrate, in-vertebrate I and II mitochondrial genes. This suggests that nuclear, chloroplast and plant mitochondrial evolu-tion is governed under the same rule, while animal mi-tochondrial evolution is governed under different rules.

The fact that evolution is expressed by linear formulas suggests that it proceeded linearly. The crossing of two regression lines suggests two evolutionary distinct proc-esses, and a crossing point suggests either divergence or convergence at a single origin. The degree of difference in two evolutionary processes is expressed by the dif-ference in linear regression slopes: small and large dif-ferences are expressed by sharp and dull angles, respec-tively. A single evolutionary process is expressed by a single regression line. The appearance of many regres-sion lines which have the same slope but different inter-cept values would indicate multiple evolutionary origins. A previous study found that regression lines representing nucleotide relationships in the coding region were al-most identical in chromosomal DNA among bacteria, archaea and eukaryotes [20]. In our previous study [24], two regression lines representing homonucleotide con-tents in chloroplasts and plant mitochondria converged at the top of the cuneiform in both coding and non-coding regions. This suggests that chloroplasts and plant mitochondria diverged from the same origin. As research suggests that the former are derived from cyanobacteria [29] and the latter are derived from pro-teobacteria [30], both organelles are likely to be derived from the same origin. In addition, the formation of the cuneiform is obtained naturally in the comparison be-tween coding and non-coding regions, because both fragments belong to the same strand [24].

5. CONCLUSIONS

When evolutionary direction is discovered, elucidating whether it occurs by divergence or convergence is not straightforward. In invertebrate mitochondria, as more recently evolved (and more advanced) vertebrates were located on the end of invertebrate I data, results indi-cated that invertebrate I and II evolution diverged from the opposite side of vertebrates. Nuclear, chloroplast and plant mitochondrial evolution is expressed by the same regression line based on Chargaff’s second parity rule (Figure 6). In nuclei, chloroplasts and mitochondria from plants, amino acid compositions deduced from complete genome data were very similar, although they differed from animal mitochondria [24]. In the present study, regression lines based on plant chloroplasts, mi-tochondria and nuclei overlapped, while animal mito-chondrial regression lines converged at the same single point. Finally, all extended regression lines representing chromosomes, chloroplasts, plant mitochondria, verte-brates and invertebrates I and II converged at the same point (Figure 6). Therefore, I conclude that there is one single origin of life from which all organisms derived. This is consistent with the chemical conditions during prebiotic evolution, in which primitive replicators such as ribosomes would have formed [31], and in which primitive life forms would have similar cellular amino acid compositions presumed from those of present or-ganisms [32,33]. Thus all advanced forms of life, as de-duced using genomic data in this study, descended from a single origin.

6. ACKNOWLEDGMENTS

The author would like to thank David Bann of Edanz Writing for edi-torial support.

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