dissertaion repseed mustured rakesh

8/7/2019 Dissertaion Repseed Mustured Rakesh

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Micro Nutrient status, Analysis of Fatty acid

Composition and

Effect of Salinity on plant by Indian mustard

DISSERTATION

SUBMITTED TO

LORDS INTERNATIONAL COLLEGE CHIKANI (ALWAR)RAJASTHAN UNIVERSITY (JAIPUR)

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

MASTER OF SCIENCE IN BIOTECHNOLOGY

2009 - 10

SUBMITTED BY UNDER THE SUPERVISION OF

RAKESH KUMAR Dr. N.S.BHOGALEnrollment No. 05/121558 Senior Scientist (soil science)

Work done at
http://images.google.co.in/imgres?imgurl=http://www.uniraj.ernet.in/logo2.gif&imgrefurl=http://www.uniraj.ernet.in/conferences/sociosemi.html&usg=__zlkS7XJ4jSmSeSF2dRKeMw07Jyw=&h=800&w=800&sz=237&hl=en&start=1&tbnid=DeJrBvebdVLlLM:&tbnh=143&tbnw=143&prev=/images%3Fq%3Drajasthan%2Buniversity%2Bjaipur%26gbv%3D2%26hl%3Den


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Fax: 05644- 260565

Email: [email protected]

[email protected]

Website: http://drmr.ernet.in

Dr. N.S.Bhogal

Senior Scientist (Soil Science) DATE: 1st Dec. 2009

Certificate

It is to certify that the thesis entitled Micronutrient status, Analysis Of Fatty

acid composition and effect of salinity on Indian mustard is an original piece of

done by Mr. RAKESH KUMAR S/o Sh. Girraj Prasad, Enrollment No.

RU/05/121558under my supervision for partial fulfillment of the requirement for the

degree ofMasterof Science in Biotechnology (Biotech), to the Lords International

College, Chikani Alwar, Rajasthan University Jaipur (Raj).I further certify that:

It is up to the required standard both the respect of its contents and literary

presentation for being referred to the examiners.

The candidate has worked under my supervision for the required period at

Directorate of Rapeseed-Mustard Research Sewer, Bharatpur (Raj.), India.

The assistance and help received during the course of this investigation and

sources of literature have been duly acknowledged.

(N.S.BHOGAL)
mailto:[email protected]:[email protected]


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Certificate

This is to certify that thesis entitled Micronutrient Status, fatty acid

composition and Effect of Salinity on Indian mustard. has been carried out at

Directorate of Rapeseed-Mustard Research,Sewar, Bharatpur (Raj.) under

the supervision of Dr.N.S.Bhogal, Senior Scientist (Soil Science), by Mr.

Rakesh Kumar Enrollment No. RU/05/121558 student of the Lords

International College, Chikani Alwar, Rajasthan University Jaipur (Raj).

And Submitted in partial fulfillment of the requirements for the degree of

Master of Science in Biotechnology (Biotech). It is further certified that the

candidate has put in the necessary stay in department during the thesis

period as per university rules.

. (UMESH SHARMA)

DIRECTOR

(External Examiner)


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AKNOWLEDGEMENTS

First of All I Thanks To God, Whose showers His

Kindness and blessings to Maintain me all cheerful

throughout the Course of this thesis work / research

programme.

I acknowledge my deepest sense of debt and

gratitude to my esteemed advisor Dr.N.S.Bhogal Senior

Scientist (Soil science), Directorate of Rapeseed-Mustard

Research Sewer, Bharatpur (Raj.) India, under whose

valuable supervision and guidance this work was

competed. I have no words to express my heartiest

gratitude for the undoubting support, constant

encouragement, painstaking efforts and motivation

provided by him at every stage of my present work and

during the preparation of this manuscript.

Words can hardly acknowledge the help made by

Dr. Arvind Kumar, Director DRMR, Bharatpur, for

providing necessary facilities and benevolent patronage.


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I also express my great pleasure and deep sense of

gratitude to Shri. H.P.Meena (technical Officer) for his

valuable advice and help in analysis.

I feel honored to record my highest regard to my

reverend parents and all the family members for

untiring help, love, affection and encouragement

without I would not have reached up to this level of

stage in life.

Last but not the least, I express my sincere thanks

to all beloved and respected people who helped me but

could not find a separate mention.

PLACE: DRMR, BHARATPUR

DATE: (RAKESH

KUMAR)


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DECLARATION

I Here by declare that this project MICRONUTRIENT STATUS ,

FATTY ACID ANALYSIS AND EFFECT OF SALINITY ON INDIAN

MUSTARD Subitted By me for bachelor Degree of Biotechnology is

an Original work done to the best of my knowledge during the

period JUNE to NOVEMBER 2009. This work has not formed the

basis for the award of my degree similar to any other candidate in

any university.


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PLACE:- ALWAR

DATE:- RAKESH KUMAR

CONTENT

1.About DRMR

2.Micronutrient Status

a. Micronutrient Status by AAS(Atomic

Absorption Specto Photometer)

Inastrument

PrincipleMethod&Procedure

b.Micronutrient Status by

Spectrophotometer

Principle

Method &Procedure


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3.Analysis of fatty acid Composition by

GCMS(Gas Chromatography and Mass

Spectrophotometer

1. InstrumentPrinciple

Method & procedure

3.Effect of salinityNa +k Ratio by flame Photometer

Instrument

Principle Method & Procedure

4.RESULT

5. REFERENCE

CHAPTER 1


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INTRODUCTI

ON


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Mandate of Institute:

National repository for rapeseed-mustard genetic resources.

Basic, strategic and applied research to improve the productivity,

quality of oil and seed meal.

Development of ecologically sound and economically viable agro-

production and protection technologies for different situations.

Generation of location specific, interdisciplinary information based on

multi-location testing and coordination.

Establishment of linkages and promotion of cooperation with national

and international agencies objectives envisaged.

To extent technical expertise and consultancies.


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This species is the source of many cultivated forms of Indian and

Chinese mustard. It resembles Broccoli or Collards in appearance, but these

cultivated plants are apparently different forms of Brassica olearacea (Wild

Cabbage). Indian mustard differs from Wild Cabbage by the absence of

leaves that clasp the central stem. It differs from other Brassica spp.

(Mustards) in the wild by the lack of hairs on the foliage, seedpods, or stems.

While some cultivated forms of Indian mustard have leaves that are

incredibly hot and spicy, the wild form of this plant has leaves with a mild

flavor. They are edible and can be used as a potherb.

This plant typically grows in full sun under mesic to dry conditions. It is

not fussy about the characteristics of the soil, and can often be found in clay-loam or gravelly sites. However, fertile soil will produce larger plants.

Disease rarely bothers this plant in the wild, although various insects often

chomp holes in the foliage.


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ECONOMIC IMPORTANCE :-

The leaves, seed, oil and oil cake are economically useful in one way or other.

Rapeseed and mustard yield the most important edible oil. The oil content seeds of

different ranges from 10 to 46 percent. The leaves of young plants are used as green and

plants are used as green fodder for cattle.

Oil :-

(A) Edible oil: The Rapeseed and mustard oil is the most important edible oil in the

northern and eastern India. Oils are used for cooking and for the preparation of

various products. The seed and oil are used as condiment in the preparation of pickles

and for flavoring curies and vegetable. The oil is also used as medicine.

(B) Industrial uses: The mustard oil is used in the preparation of hair oil, soap and

in the manufacture of mineral oil for lubrication. It is also used in bakery, tea industry

and in the preparation of vanaspati ghee. Rapeseed oil is used in the manufacture ofgrease, tanning industry and mustard oil is used for softening leather. But the use of

mustard oil for industrial purpose is rather limited on account of its high cost.

Oil cake:-


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Micronutrient availability to plants can be measured in direct uptake experiments, or

estimated with techniques that correlate quantities of micronutrients extracted chemically

from soils to plant uptake and response to micronutrient fertilization. Rational

management of micronutrient fertility and toxicity requires an understanding of how total

and plant-available soil micronutrients vary across the land.

Digestion of plant sample for estimation of nutrients

It is a method in which the harvested plant material is converted into solution form, so

that the estimation of nutrients could be done.

1- The harvested plant material is first surface cleaned using with a mild

acidic detergent like 3N HCl.

2- Then it is rinsed with distilled water.

3- This washed material is air dried.

4- Once the plant material is dried completely it is ready for grinding.

5- After the material is grinded it is placed in a suitable air tight container.

By above methods the husk, straw, seed and root are digested and the

procedure is as follows;

Digestion

Take 0.5g of sample in a conical flask of 150 ml.

Add 10ml of conc. Nitric acid.

Leave overnight for pre-digestion.

After pre-digestion add 20ml in ratio of 9:3 of Nitric acid and perchloric

acid.

Then heat the conical flask using hot plate in the presence of fume

extraction hood.


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After some time the plant material would appear in a form of a light

colored paste.

Now allow the conical flask to cool down.

Then make up the volume up to 50 ml using volumetric flask.

Precautions:

Gloves should always be used during working with acid.


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Borosil flask should not be used.

Sulphuric acid could be used if we are not interested in estimating sulphur.

Estimation of Micronutrients by

Atomic Absorption System

About AAS instrument

The Atomic Absorption Spectrophotometer (AAS) used in the study was ofThermo make model M5 having Graphite and hydride unit. AAS is based on the

principle that when a radiation from an external light source, emitting the spectral

line(s) that correspond to the energy required for an electronic transition from the

ground state to an excited state, is passed through the flame. The flame gases are

treated as a medium containing free, unexcited atoms capable of absorbing

radiation from an external source when the radiation corresponds exactly to the

energy required for a transition of the test element from the ground electronic

state to an upper excited level. Unabsorbed radiation then passes through a

monochromator that isolates the exciting spectral line of the light source and intoa detector. The absorption of radiation from the light source depends on the

population of the ground state, which is proportional to the solution concentration

sprayed into the flame. Absorption is measured by the difference in transmitted

signal in the presence and absence of the test element.

The layout of a basic flame atomic absorption spectrometer


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Fig. Atomic Absorption Spectrophotometer

Light from a line source of characteristic wavelength for the element being

determined passes through the flame into which it has been sprayed as a fine mist

of the sample solution. The region of spectrum to be measured is selected by a

monochromator. The isolated spectral line falls on the photomultiplier, the

detector and the output is amplified and to a readout device meter, digital or

analogue, to a chart recorder or through a computer data processing system,

printer or digital display unit. The intensity of the resonance line is measured

twice, once with the sample in the flame and once without. The ratio of the two

readings is a measure of the amount of absorption, hence the sample.

Hollow Cathode Lamps


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The hallow cathode lamp is stable, reliable, has a long operating

life and is the standard source in Atomic Absorption Spectrometry.

Lamps may be expected to run in excess of 5000mA hours without

failure and many have been known to run twice as long. The hollowcathode discharge lamp is known as a fine line sources capable of

producing spectra where fine structure could be studied. It consist of a

glass tube with the electrode sealed inside with an optical window at

one end made of glass or silica depending on the wavelength and

attached with a thermosetting resin or vacuum wax. The construction of

a typical lamp in use today is

Fig. Hallow cathode Lamp


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The two electrodes are sealed in the glass envelope and the

window located at the opposite end to the cathode. Electrical

connections are made through a standard octal plug at the base of the

lamp. The cathode is shaped in the form of a hallow cup inside which

the discharge takes place. This cup is constructed of or contains theelement of interest of interest whose spectra is required. A mica shield

holds the structure rigid and helps to contain the discharge inside the

cathode cup. The cup usually has an internal diameter of about 2mm, to

concentrate the discharge into a small area and produce a high intensity

line. The available energy appears to dissipate in the metal resonance

line rather than in the filler gas in this type of construction. The lamp is

usually filled to pressure of 4-10 Torr with an inert gas such as helium,

argon or neon. Highly purified inert gases are used and the glass is out-

gassed at high temperature to remove impurities absorbed onto the

surface of the glass. The emission line of the inert filler gas must not

coincide with the resonance line of the emission line of the inert filler

gas and the line of the metal of interest must not coincide with the

resonance Neon has a higher ionization potential because it improves

the intensity of the discharge.

When a voltage of between 300-400 is applied between the anode and

cathode the discharge is set up and argon is ionized by the and becomes

positive argon by the mechanism of:

Ar +e- = Ar+ + 2e-

The positive Ar+ ion is attracted and accelerated toward the

cathode where it dislodges or sputters excited metal atoms into themetal cathode knocking metallic atoms into the discharge, which

improves the sensitivity of the discharge. The excited atoms emit

energy of their own characteristic wavelength before returning to the

ground state and the emitted light is used as the light sources for the

AAS system. After the atoms return to the ground state they form a

cloud of free atoms which return either to the walls of the glass lamp or

to the metal cathode.


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Lamp as uses to as

Atom Atomic

no

Atomic

mass

Primary

wavelength(nm

)

Fuel flow

rate(L/min)

Flame Type

Mn 25 54.9380 279.5 0.9-.12 Air/acetylene

Fe 26 55.847 248.3 0.8-1.0 Air/acetylene

Cu 29 63.546 324.8 0.8-1.1 Air/acetylene

Zn 30 65.5 213.9 0.9-1.2 Air/acetylene

Double Beam Instruments

The light from the sources is split into two beams by means of a

rotating half-silvered mirror, or by a beam splitter which is a 50%

transmitting mirror. This directs beam alternatively through the flameand along a path which by-passes the flame at a frequency usually at

50Hz or higher. Once past the flame, the beams are recombined with a

half a half-silvered mirror as shown below:


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Fig. Double Beam Instruments

At the detector end, the output signals which correspond to each are

divided, amplified separately and compared in a bridge circuit. The out-

of-balance signal is than compensated electronically and converted toabsorbance.

Estimation of micronutrients

Analyses of different trace element from soils are considered as a

diagnostic tool for identifying nutrient deficiency or excess in soils. The soil test

method in practical/realistic till the nutrient question extracted by the chemical

extractants from the soil shown a high degree of correlation with crop yield. Plant

analysis of as compared to soil analysis can evaluate better considerable from

nutrient deficiency. Mostly deficiency of micronutrient like Zn, Cu, Fe, Mn, B,

Mo etc. appears in the early growing stage and by usual symptom could identify

the micronutrient deficiency the time the usual symptom appear the deficiency of

nutrient have all ready done the damage to the crop. It in hear that soil analysis

has the advantage over that of plant analysis as well as symptom in indicating the

extent of tract element deficiency of their requirement for crops to seeding.

However, despite number of demerits soil analysis has covered itself as a

diagnostic tool in monitoring nutrient status of soil. For the estimation available

micronutrient are in use still most acceptable method as 0.005 m DTPA

extractable method of at pH 7.3 (Lindsay and Norvell 1978).

Apparatus


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Atomic Absorption Spectrophotometer

Reagents

(i) Weigh 1.965 of DTPA.

(ii)Add 600 ml in 1 liter distilled water dissolve in the DTPA.

(iii) Weigh 1.470 gm CaCl2 and then dissolve in the DTPA solution.

(iv) Then add 13.29 ml of Triethanolamine (TEA) to this solution.

(v) Add 300 ml distilled water and then the solution was adjusted at pH 7.3

by adding dilute. HCl or NH4OH

(vi) After pH was adjusted at pH 7.3 the volume was made up

to 1000 ml.

Procedure

(i) 10 gm of soil is taken in 250 gm plastic shaking bottle.

(ii) Add 20 ml of DTPA solution Adjusted at pH 7.3 in the

plastic shaking bottle.

(iii) The plastic bottle is placed on mechanical shaker for 2

hours.

(iv) After 2 hour of shaking the soil solution is filtered with

Whatman no of 42.

(v) The filtrate in used for the analysis of micronutrient and

pollutant element.

(vi) Atomic Absorption Spectrophotometer is use to analysis the

micronutrient and pollutant element of different optimal


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wavelength, current and slit width after it is calibrated for

the specific element.

Calculation for soil

10 gm + 20 ml extracted solution = x 2 dilution factor.

Sample reading by AAS concn mg / kg = M

g / g or mg / kg of Zn / Cu / Fe / Mn = M x 2

Calculation for plant tissue:-

0.5 g plant tissue digested and volume made up 50 ml = X 100

Dilution factor

Sample reading by AAS conc. In mg \ kg = P (ppm)

g \ g or mg\kg of Zn\Cu\ Fe\ Mn = P X

Dilution factor (100)

Reading:-

1. Soil Sample

Sample Zn(ppm) Cu(ppm) Fe(ppm) Mn(ppm)

1 1.2089 1.0712 1.7176 1.81662 1.2785 1.0676 1.7411 1.24413 1.2082 1.0693 2.0504 2.0762

4 1.2012 1.0813 1.4002 1.34055 0.8814 1.088 2.1152 1.52666 0.5973 1.0555 1.7631 1.84957 0.7228 1.0577 1.9081 1.24568 1.3261 1.0725 1.4352 1.00999 0.6257 1.0008 1.9527 0.8847

10 0.8206 1.0666 1.6584 1.2363


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Max-min 1.2785-0.5973 1.0888-1.0008 2.1152-1.4002 2.0762-0.8847

Mean 0.98707 1.06305 1.7742 1.423

Table.1 Soil sample

2.Plant sample

Sample Zn(ppm) Cu(ppm) Fe(ppm) Mn(ppm)

12.344 299.19 395.14 55.51

22.313 152.09 323.89 52.6

31.333 133.58 508.82 27.55

4 2.281 281.97 294.6 25.555

2.507 260.65 321.54 29.33

62.411 253.15 320.87 31.81

72.165 257.25 555.39 27.85

81.097 236.58 530.73 30.97

92.46 254.14 628.6 23.64

102.452 217.13 199.96 55.51

Max-min 2 .507 -1.09 7 2 99.1 9-1 33.58 628.6 -199.96 55 .5 1-2 3.64

Mean2.1363 234.573 407.954

36.032

Result


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1. Critical limit in soil for of Zn, Cu, Fe, Mn are - 0.71, 0.60,7.0 and

3.0 respectively by Component the data in Cu table 1 all the soils are

deficient in Fe and Mn Where, they were sufficient in copper in Zn in

two soils table no 6 and 9 are deficient in zinc

Determination of available Boron in soil

Introduction:-

Boron (B) is unique in soils by its narrow range between deficiency for

(plant growth) and toxicity. Less than 1ppm of B may lead to a deficiency,

yet 3ppm may be toxic. It is associated with calcium uptake by plants, and,

in fertility studies, it is, often, useful to measure boron-calcium ratio. The

most useful measure of available B is the water-soluble form. The range of

water soluble B, in mineral soils of the humid region, is generally, from 0.2

to 1.5ppm onto 2ppm or more in muck soils, and down to 0.2ppm in fairlyfertile sandy soils. The content of water-soluble B in soils is influenced by

pH, organic matter and amount of colloids.

Importance:-

Knowledge of water-soluble B in soils is of considerable agricultural

significance in the context of its narrow limits between deficiency and

sufficiency, its interaction with calcium and its precipitation as calcium

metaborate in extreme cases.

Extraction of Boron from soil:-

Take a 250 ml of low boron conical flask and weigh 20 g of air dried soil.


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of the colour developed. Nitrates are removed by treatment with

hyposulphite in the presence of hydrochloric acid.

Instrument and Apparatus:-

Photoelectric colorimeter, boron-free glassware, viz., conical flasks,

volumetric flasks, pipette, a dispenser or a fine pipette, heater or water bath.

Reagent:-

Copper sulphate, CuSO4.5H2O, hydrogen peroxide, 30%, conc.

H2SO4, NaOH, 0.5N

Quinalizarine in H2SO4 0.01%: Dissolve 0.01g of quinalizarine in a

mixture of 90ml of H2SO4 and 10ml of water.

Standard boric acid solution, 100ppm of B:

Dissolve 0.5716g of boric acid (H3BO3) in distilled water, and dilute to

one litre. One ml of this solution contains 0.1mg of boron. Dilute 50ml of

this solution to 500 ml with distilled water, which will give a B

concentration of 10mg per ml (10ppm).

Procedure:-

* Place 10 g of soil (or 5 g peat) in a 100ml conical flask, and treat with

30 ml hot distilled water.


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*Add 0.5ml of CuSO4. 5H2O solution, and boil it for 5 minutes. Then,

agitate and filter.

*Take 10ml of the filtrate in a 50ml volumetric flask, and add 0.5ml of

30% H2O2 Boil the solution gently for about 1 minute until the solutionloses its colour.

*Add 1ml of 0.5N NaOH solution, and heat the solution gently until

evaporation. Then, add to it 0.5ml of 30% H2O2, and very slowly

evaporate until dry, to avoid loss of boron.

*Treat the residue with 4.5ml of conc. H2SO4 until the salts have

completely dissolved. If the solution is brown in colour, another heating

is required.

*Add to the solution 0.4ml of potassium hyposulphite (a reducing

agent) in HCl system for elimination of nitrates.

*Following the disappearance of gas bubbles, add 0.5ml of

quinalizarine solution in H2SO4, agitate the contents, and make up the

volume to the mark with distilled water.

*Stopper the flask and allow it to stand for 25 minutes.

*Measure the blue color intensity on the colorimeter, using 620m light

maximum, read the % transmittance and record the concentration of B

in the test solution from the calibration curve, prepared from the

working B standards (using 10ppm stock solution) over a range of 0-10

ppm as per the procedure outlined above.


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Observation:-

a. Weight of the soil = W g

b. Volume of the extract (hot water) made up to = V ml

c. Volume of aliquot taken (test solution) = 10 ml

d. Final Volume made up to = 50 ml

e. Transmittance (%) as read from the colorimeter = T (say)

f. Concentration (ppm) of B in the solution = C (say)

Calculations:-

g. First dilution: = (v/w) times

h. Second dilution: (50/10) = 5 times

i. Total dilution = (v/w) x 5 times

Now, available B in the soil (ppm) = Cx [(v/w) x 5]

Thus, available B in the soil (Kg/ha) = Cx [(v/w) x5] x2.24

Where Cx is the conc. of the unknown sample in O.D.


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GC-MSGAS

CHROMATOGRAPHY AND

MASS SPECTROSCOPY


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FATTY ACIDS ANALYSIS

INTRODUCTION:

Fatty acids comprises of wide range of oils and fats which are commercially

mixture of lipids. As reported the production of oils and fats is over 100 million tonnes from 17

sources which include 4 animal fats (21%) and 13 vegetable oils (79%).

The major consumption of vegetable oils is because of its use in human food. Dietary

fat has number of nutritional functions. It serves as an energy source for infants. Also, it is an

important constituent of cell membranes and precursor of Eiconsanoids like Prostaglandins,

Thromboxanes, Leukotriens. It also serves as a carrier for fat soluble vitamins. Although its

further implications are in cardiovascular diseases, obesity and hypertension. So the

recommended intake of fat should be reduced to 30% while that of saturated fat is 10% of total

energy.

Natural fats comprise mainly Triglycerols (or triglycerides), Diacylglycerols (or

Triglycerides), Monoacylglycerols (or Monoglycerides) and free fatty acids which on

hydrolysis yield Sterols, Triterpene, Alcohols, Tocols and fat soluble vitamins.

There are 1000 natural fatty acids which have been identified which vary in chain

length, degree of unsaturation and presence or absence of functional groups. The fatty acids are

divided into four major categories namely.

SATURATED ACIDS

e.g. Lauric acid, Myristic acid, Palmitic acid, Stearic acid

MONOSATURATED ACIDSe.g. Oleic acid, Erucic acid

POLYUNSATURATED ACIDS(n-6)

e.g. Linoleic acid, -Linolenic ( GLA), Arachidonic acid

POLYUNSATURATED ACIDS(n-3)

e.g. Linolenic acid, EPA, DHA


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( n-3 and n-6 relate to the position of the first double bond according to methyl end of the

chain).

COMPONENTS OF NATURAL FATS

1. TRIGYCEROLS:

A crude oil or fat contains 95% Triglycerols. These are esters of trihydric alcohol glycerol and

contain three acyl chains in each molecule, usually from two or three type of acids. In addition

Diacylglycerols, Monoacylglycerols are also present.

2. PHOSPHOLIPIDS:

Crude oil also contain phospholipids. The major components being phosphatidylcholines,

phospha-tiadylethnolmines and phosphatidylinositides. Soybean oil contain 3.2%, rapeseed oilcontain 2.5% and sunflower oil 1.5% of total phospholipids.

3.STEROLS:

Vegetable oils contain 1000-5000 ppm of sterols. Rapeseed oil has 5-11g/kg of sterol.

Sitosterol is the main phytosterol. Soybean contains 0.18%- 0.40% while sunflower contains

0.25%-0.45%.

4. TOCOLS:

Tocols are also present in vegetable oils. They are four in number with saturated C-16

branched tocopherols while tocotrienols are also present. Tocols are known to have vitamin E

activity and antioxidant property.

ESTIMATION OF FATTY ACID :-

The instrument used for quantitative and qualitative analysis of fatty acid is based on two

principles namely of Gas Chromatography and Mass Spectrometer and hence named as GCMS.


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GAS CHROMATOGRAPHY MASS SPECTROPHOTOMETER

Mass spectrometry (MS) was developed at beginning of the 20 th century by J.J.

Thomson (1910), Dempster (1918) and F.W.Aston (1919-1920) to separate and identify

isotopes. In the thirties, Symth (1931) and Tate (1935) recognized that ionization of organicmolecules by electron impact leads to characteristic fragment ions which can be measured by

mass spectrometry and which conclusions on the structure of the molecules. In the forties it

was first commercially used in the petroleum and rubber industry of U.S.A.

In sixties it was introduced to estimate the accurate molecular masses in organicchemistry and thereby for the calculation of elemental composition. Additionally it was used

for unknown compound.

In the seventies mass spectrometry was directly coupled with chromatographic methods

especially with gas which revolutionized the analysis of complex composition of organic

compound because of its high sensitivity and specificity. The great disadvantage of this

technique was the high cost and the complexity of the handling.

In eighties both these disadvantages were abolished by the development of low priced

mass selective detectors or iron trap detectors which can easily be handled. Both mass

analyzers allowed coupled with modern high performance capillary gas chromatography

columns. As a consequence GCMS has become the most powerful method for the highly

specific and sensitive identification and quantification of relatively non-polar organic

molecules especially in complex matrices like biological samples. Therefore GCMS is widely

used for the detection of drugs, poison, pesticides, pollutants and there metabolites in clinical,


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forensic, food technology and in doping control as well as in environmental and occupational

toxicology.

GCMS is the method of choice for screening and confirmation of toxicants which are

volatile in gas chromatography. In GCMS we can use Head - Spacer for the analysis of

volatile compounds.

A gas chromatographic procedure with packed column, Flame Ionization Detection

(FID) and Nitrogen Sensitive Flame (N-FID) as well as with capillary column and mass

selective detection was set up to determine retention indices.

In case of GCMS this is not an essential. In GCMS it unable the differentiation of

isomeric metabolites which yield the same spectra which have now been included in the

computer library search now been included in the computer library search like NIST, Pfleger/

Maurer/ Weber drug library, EPA(Environmental protection act), NTH and Pesticide database.There are different modes of analysis in GCMS mainly EI (Electron Impact Ionization)

and CI (Chemical Ionization). Mostly EI spectra are more useful for the identification of

unknown compound then CI spectra. Even spectra containing no molecular ion can be

identified in almost all cases by comparison with the EI reference spectra. It is the only

collection which also contains data covering the metabolites which were normally present

when using the analytical procedure.

MATERIALS USED:

Syringe (5l capacity)

Capped test tube (5ml)

EQUIPMENTS USED:

GCMS

Vortex shaker

Oven

CHEMICALS USED:

0.5ml Petroleum ether vortex

0.5ml Sodium methoxide (80mg NaOH in 100 ml methanol)

PROCEDURE:

1. Ten oven dried seeds (450C for 6 hours) of Hyola, Varuna and 15 other

germplasms were crushed in capped test tube in duplicate.

2. Add 0.5ml of petroleum ether, vortex for 1 minute and leave for two hours.


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3. Centrifuge at 5000 rpm for 5 minutes to have a clear supernatant.

4. The supernatant was transferred to another capped test tube and 0.5ml sodium

methoxide

solution was added to it.. Vortex it for 30 seconds.

5. After 45 minutes, 0.75ml of 8% NaClsolution was added followed by Vortexing.

6. Layers were separated.

7. One micro litre of upper layer was injected for fatty acid analysis in GCMS.

Fatty Acid Composition:

As we compare the contents of fatty acid in two check varieties i.e. Hyola and Varunaand germplasm (EC-49 and YST-151) using the international dictionary Pfleger/Maurer/Weber

we observed that the prominent peaks are marked as A for palmitic acid, B of Stearic acid, C of

Oleic acid, D of Linoleic acid, E of Linolenic acid and F for Erucic acid that can be identified

using the following characters:

Fatty Acid IUPAC name Empirical

formula

Peaks obtained Molecular

mass

Palmitic

acid

Hexadeconic acid C17H34O2 84, 87, 143, 227,

270

270.25588

Stearic

acid

Octadecanoic acid C19H38O2 74, 87, 143, 255,

298

298.28718

Oleic acid 9-Octadecenoic acid C19H36O2 56, 97, 222, 264,

296

296.27153

Linoleic

acid

9,12octadecadienoic acid C19H34O2 67, 81, 95, 263,

294

294.25588

Linolenic

acid

9,12,15Octadecatrienoic

acid

C19H32O2 79, 95, 121, 191,

292

292.24023

Erucic acid 13-Docosenoic acid C23H44O2 55, 69, 97, 320,

352

352.33413


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lative index of different Fatty acids

Undesirable characters Desirable characters

Germplasm Palmitic Stearic Erucic Oleic Linoleic Linolenic

Hyola 2.30 0.70 Absent 6.70 5.20 2.70

Varuna 1.50 0.49 5.05 3.20 4.05 3.40

EC-49 0.62 0.30 0.42 4.68 3.90 1.60

YST-151 0.51 0.51 5.42 4.25 4.27 2.90

We have taken two check varieties (Hyola and Varuna) and two varieties (EC-49 and

YST-151) for choosing the better germplasm having desirable characters. The undesirable

characters like Erucic acid had the least relative index in Hyola but higher palmitic (5.42) and

Stearic acid (0.70) as compared to others. Whereas, in germplasm EC-49, the palmitic acid and

Stearic acid had least (0.62 and 0.30, respectively) as compared with others. Moreover, the

YST-151 contains similar amount of fatty acids as compared to Varuna.

So it clearly indicates that if we are interested to choose a better germplasm for

increasing desirable characters and decreasing undesirable ones for quality breeding, we could

choose EC-49 which is an Australian germplasm.


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FIG: GC-MS INSTRUMENT

GC-MS INSTRUMENT SETTING:-


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THE FLAME PHOTOMETER FOR THE MEASUREMENT OFSODIUM AND POTASSIUM IN BIOLOGICAL MATERIALSBY PAULINE M. HALD(From the Department of Internal Medicine, Yale University School of Medicine,New Haven)(Received for publication, October 30, 1946)

The flame photometer was devised by Barnes, Richardson, Berry, andHood (1) to measure low concentrations of sodium and potassium in solution.The present paper describes an investigation of its application tothe analysis of biological materials. Measurements of sodium and potassiumin such materials by means of the flame photometer were comparedwith measurements made by chemical procedures of tested accuracywhich have been previously described (2).The InstrumentThe principle of the instrument has been described in detail by Barneset al. (1). The solution to be analyzed is discharged through an atomizerin a fine mist into a chamber, whence it is drawn into a flame. By anoptical system the light produced by the combustion of the elements inthe vaporized solution is conducted through appropriate filters to impingeupon a photoelectric cell which activates a galvanometer. Under properoperative conditions the concentration of sodium or potassium in the

solution can be estimated from the reading of the galvanometer. Theinstrument is equipped with an amplifier which permits the analysis ofsolutions with greatly varying concentrations of sodium and potassium.The measurements reported below were obtained with one of the modelapparatus (model G, series 11) designed and made by the AmericanCyanamid Company for the inventors and similar to the instrument describedby them (1). A small number of these instruments were constructedand distributed by the American Cyanamid Company for experimentalpurposes. The model has not been made generally available.An instrument patterned after the original model and designed for thesame purposes has, however, been placed on the market, and through thekindness of the manufacturer2 we have been allowed to study it. Unhappily,certain features of this apparatus render it unsuitable for the analysis

of biological materials. For the gravity feed of the original model1 The author wishes to thank Dr. Barnes and his associates for making this instrumentavailable to the department.2 Perkin-Elmer Corporation, Glenbrook, Connecticut.499Downloaded from www.jbc.org by guest, on November 24, 2009

500 FLAME PHOTOMETERa suction feed was substituted. This does not yield a steady flow of mistfrom the atomizer and requires more solution for analysis. This is aserious disadvantage, especially in the measurement of serum potassiumin which the amount of solution available is extremely limited. Whencommercial production was undertaken, the emergency of the war made itimpossible to obtain the most desirable types of optical and recording instruments.It is hoped that with the release of restricted materials aphotometer suitable for the analysis of biological media will be commerciallyavailable. Meanwhile it is believed that the studies of the behaviorand operation of the original models have a general significance that warrantstheir publication.For the general description of the instrument and its use the reader isreferred to the original article by Barnes and his associates (1). Thepresent discussion will be confined to the control of the variable featuresof the apparatus upon which its proper operation depends. These are(1) the atomizer, (2) the flame, (3) the air pressure, (4) the preparation ofmaterial, (5) the dilution of materials, and (6) interfering substances.Details of ApparatusThe Atomizer-This is the most critical feature of the instrument.Unless the mist produced is constant, reproducible results cannot be attained.


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All-glassatomizers, permanently fixed, and thereforenot adjustable,have been employed. While such a fixed position is highly desirable,other features of the glass atomizer detracted from its practicability. Itis both expensive and fragile. If it is broken, it must be replaced or repairedby a proficient glass blower.A metal atomizer, similar to that described by the designers of the instrument,has been found more satisfactory. This type of atomizer can

be fashioned from two inexpensive hypodermic needles by the analyst if areplacement is necessary (Fig. 1). The straight needle through which theliquid is fed may be probed with a stylet to dislodge particles or air bubbleswith no fear of breakage. Larger needles than were previously used withthe instrument have proved advantageous because the opportunity forobstruction by minute particles or protein precipitated at the tip is minimized.The needle for the delivery of the sample is a No. 20 gage lumbarpuncture needle which has been cut off to make an over-all length of 30mm. The lumbar puncture needle was chosen because it offered an openinglarge enough to admit a stylet from above. By means of a file a highbevel of approximately 3 mm. is made. This high bevel serves a dualpurpose: (1) it obviates the formation of a droplet of solution which interruptsthe steady delivery of t,he mist; (2) it offers a relatively largesurface to the compressed air in the atomizing flask. This consequentlyDownloaded from www.jbc.org by guest, on November 24, 2009

P. M. HALD 501retards the flow of solution and thus permits the use of a larger needlewhich is less easily obstructed without increasing the volume of solutionnecessary for a satisfactory reading. The needle through which the airis admitted, a No. 19 needle, is curued to meet the lower tip of the bevelin such a way as to send the spray of mist in a direction at right angles tothat of the straight needle, the beveled face of which is directed awayfrom the source of air. While with this type of atomizer the relative positionsof the needle tips are not readily reproducible, it is possible to adjusttheir positions so that a satisfactory mist is produced. A steady, strongspray which is somewhat diffuse near the source is usually satisfactory.A steady, moderately rapid deflection of the galvanometer over the desired

range indicates satisfactory performance of the needles.The flame must necessarily be fed by gas of constant composition.When city gas was tried it was impossible to obtain satisfactory results.This was occasioned by the variability in composition of the gas ratherthan by changes in pressure. A bottled gas (Pyrofax) has since been employedto insure against this variability. A reducing valve was interposedat the tank so that the pressure can be cut down partially at this point.The final pressure is controlled by means of the pressure regulator in theinstrument.The air pressure is supplied from a central source and is led into the photometerthrough a connection made of copper tubing. The pressure,which is 60 to 90 pounds at the source, is cut down to the desired point bythe regulator in the instrument. The final pressure, which is usually between

10 and 25 pounds, is selected experimentally to suit the needs ofthe occasion. It has not been found necessary to filter the air in thesestudies.The preparation of material (see Table I) depends upon several factors.Serum needs only to be diluted for the determination of either element.Water is used as a diluent for sodium analysis because the dilution is sogreat that precipitated globulin offers no obstacle. For potassium analyses,in which the dilution is far lower, 0.9 per cent NaCl is used. Wholeblood must be ashed and iron removed before sodium is determined. Thehigh concentration of iron in cells interferes with the flame reaction.Equal volumes of blood and 4 N sulfuric acid are placed on a steam bathin a suitable porcelain or platinum dish until water has been evaporatedand a charred mass remains. The dish is then transferred to a cold


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muffle furnace and the temperature advanced to 500-550. After 5 to 8hours ashing should be complete. An aqueous extract of the ash is dilutedto a volume of 100 or 200 cc. per cc. of serum represented. Theinsoluble iron salts are filtered out and the filtrate is used for the measurementof sodium. Potassium may be determined on an aqueous solutionDownloaded from www.jbc.org by guest, on November 24, 2009

502 FLAME PHOTOMETERof whole blood without interference from the iron present. Urine needsonly to be diluted with water. If gross particles are present, the urineshould be filtered before the dilution. Food and feces must of course beashed and the ash extracted before it can be introduced into the apparatus.1 or 2 gm. of a weighed homogeneous mixture of diet are placed in a weigheddish of Vycor glass. The weight is noted and approximately 1 cc. of 4N H&SO4 is added per gm. of food. The water is evaporated from thesolution, after which the dish containing the charred material is placed ina muffle furnace, where it is brought to a temperature of 500-550. After

TABLE I

Preparation of Materials for Flame PhotometryMaterialPotassium

Serum. .......Urine .........Food. ........Feces. ........Whole bloodNone Normal saline WaterAsh HCl and water None Waterl:lO-1:25 1.251:20 2.501 gm.-50 cc. 2.501 (6 -50 (( 2.501:lOO 1.25nq. per 1. lbs. lbs.1 20-251 20-25

1 20-251 20-251 20-25SodiumSerum. None Water 1:250-1:500 1.00 14-2 15-20Urine.. . _. 1:40 2.50 13-2 15-20Food . Ash HCl and water 1 gm.-25 cc. 2.50 1+-2 15-20Feces.. I 1 (6 -10 (( 2.50 la-2 E-20Whole blood.. Water 1:200 1.00 la-2 1520

approximately 8 hours a white ash remains. To this white ash is added 1cc. of concentrated HCl for each gm. of food represented. The dish iswarmed to facilitate solution of the ash. With the aid of water, the mixtureis transferred quantitatively to a volumetric flask and diluted to theappropriate volume. This diluted extract is filtered, if insoluble materialremains, before it is admitted to the photometer. Feces are similarly

treated.The dilution of material (see Table I) must necessarily be varied tobring the concentrations of sodium and potassium into ranges which areoptimum for accurate analysis. In serum and whole blood the bases arepresent in concentrations which are limited and which vary over a relativelysmall range. Consequently, significant variations can be determinedDownloaded from www.jbc.org by guest, on November 24, 2009

P. M. HALD 503only by a very sensitive instrument. The control rheostats are set, therefore,so that in the case of sodium determinations 1 milliequivalent perliter is represented by 100 scale divisions. Since it is rarely possible toread the galvanometer closer than the nearest 0.5 division, this sensitiverange is essential. If, therefore, serum is diluted with distilled water


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1: 250 or 1: 500, the resulting readings fall in the neighborhood of 30 and60 scale divisions respectively. When both dilutions are used, obscureerrors are sometimes revealed which are not evident when the concentrationsof the duplicate dilutions are identical. 0.2 cc. of serum, diluted to50 and 100 cc., is an economical amount which gives sufficient materialfor several readings. For sodium in whole blood in which ashing is required,it is advisable to use 1 cc. aliquots. The ash, extracted with water,

may be diluted to 100 or 200 cc. for convenient readings.Because potassium emits a less intense light than sodium when it isburned, it is necessary to use a 1.25 milliequivalent solution for the formerto attain 100 full scale divisions. Serum potassium occurs in concentrationswhich are so low that this analysis offers unusual difficulty if materialis limited to small amounts. The most economical procedure is to dilutethe serum 1:25 with physiological saline. At this concentration thereadings attain a maximum deflection of approximately 12 scale divisionsfor a normal serum potassium. It is therefore possible to reach this relativelylow maximum and maintain it sufficiently long for the operator totake an accurate reading. If an abundance of material is at hand, additionaldilutions or dilutions of varying concentrations may be made.Whole blood, in which the concentration of potassium is approximately 10times as great, offers a wider choice of dilution ranges. Dilutions of 1: 100have proved to be practical.The concentrations of sodium and potassium in urine, food, and fecesoffer no such limitation in either range or quantity. It is often impossibleto predict the concentration even approximately. However, since thematerial is usually readily obtained and the concentration range is soextensive, it is neither necessary nor practical to use the sensitive rangesdescribed above. When 2.5 milliequivalents are represented by 100 scaledivisions, the dilutions tabulated in Table I serve as a general guide. Itis unwise to start with a high dilution unless there is some assurance thatit is indicated. If material is limited, as in the case of an ash, a seconddilution may be resorted to if the first one gives too high a reading.Interfering substances have been found in the course of this study. Itwas found that iron in the concentration in which it exists in whole blood

interferes with the reading of whole blood sodium. While an increaseddeflection is also found under some conditions in the potassium analyses,the effect of the interference in whole blood potassium determinations isDownloaded from www.jbc.org by guest, on November 24, 2009

504 FLAME PHOTOMETERnegligible. The fact that the flame photometer measures sodium andpotassium accurately in the presence of such complex materials as arefound in serum and urine indicates that its operation is not influenced bymost organic compounds in the concentrations encountered in biologicalmaterials. It was found, however, that when 5 per cent glucose wasadded to dilute serum low values for both sodium and potassium resulted.When 5 per cent glucose alone is burned, it imparts a definite color to theflame. This color appears to cause no positive deflection of the galvanometer

with the potassium filter, but an appreciable deflection is observedwhen the sodium filter is in place.It is necessary to determine the effect of any ion added in non-physiologicalconcentrations before it can be assumed that accurate results canbe obtained.General Technical ProcedureThe procedure of using the flame photometer requires first of all the establishmentof a stable calibration curve. This curve is prepared to covera given concentration range, selected to suit the conditions of the analysisunder consideration. This standard curve is not always constant andreproducible but depends on the variables previously discussed. Onlywhen the flow of solution through the atomizer, the air pressure, and thegas pressure are properly related will a stable curve result. Only if all


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three factors are duplicated will it be reproducible. It is necessary,therefore, to construct a curve for each set of analyses. Even when itappears to be constant it must be continually rechecked, or even reconstructed,in the course of a set of analyses. Unless this procedure isthoroughly understood and rigidly adhered to, results within the limits oferror reported cannot be expected.The curve is constructed from readings obtained with a series of stock

solutions of sodium or potassium chloride of the desired concentrations.Four or more points are used to est.ablish the slope of the curve. In TableI suitable concentration ranges for each type of determination are suggested.It has been found convenient to adjust the instrument so thatthe upper standard chosen reads full scale or 100 divisions. Solutions ofone-fourth, one-half, and three-fourths of this concentration are alsorequired.A set of stock solutions is prepared and stored in glass bottles withtightly fitting stoppers. No-Sol-Vit bottles have not been found necessary,but the concentrations of the solutions have been checked from timeto time against freshly prepared solutions to prove that no reaction withthe glass had taken place. Some of the solutions had been stored morethan a year. This stability might not be maintained with all types ofglass and must be established if No-Sol-Vit bottles are not used.Downloaded from www.jbc.org by guest, on November 24, 2009

P. M. HALD 505In order to prepare a calibration curve which is stable or comparativelystable the rate of atomization relative to the size of the gas flame must becarefully adjusted. With a given setting of the atomizer two possiblevariables may be controlled, air pressure and gas pressure. It has provednecessary to use a low pressure of gas (about 1 pound) and a high pressureof air (20 to 25 pounds) in determining potassium. For sodium analysesa higher gas pressure (13 to 2 pounds) and a lower air pressure (15 to 20pounds) have been more satisfactory with the instrument used in thesestudies.With the flame burning properly and the correct filter in place the zeroreading on the galvanometer is set. Distilled water introduced into theinstrument should not alter this reading. Next the standard solution

bevelFIG. 1. Metal atomizer, stainless steel hypodermic needles used as tips. 1, airfeed, 19 gage needle; %, liquid feed, 20 gage lumbar puncture needle.which has been selected to be represented by the full scale is poured intothe funnel and by means of the amplifiers is adjusted to the 100 position.Distilled water is again introduced. If it does not read 0, this point isagain adjusted and the 100 mark reset. This mark is then reestablishedseveral times. Unless the 0 and 100 points are maintained on successivereadings the gas pressure, air pressure, or both, must be adjusted to bringabout a stable reaction. If the upper reading drifts above the 100 mark,the gas pressure should be decreased or the air pressure increased. Whenthe reading tends to drift below the 100 mark, the gas pressure should beincreased or air pressure decreased. When the range has been successfullyestablished, the other points on the curve are read and the 0 and 100

points rechecked. The relation between the galvanometer readings andthe concentration is curvilinear. Readings of approximately 27, 52.5,and 77 are as close to the straight line relationship as can be expected fromDownloaded from www.jbc.org by guest, on November 24, 2009

506 FLAME PHOTOMETERsolutions of 25, 50, and 75 per cent of the concentration of the solutionused to represent 100 divisions on the galvanometer scale. Readings muchfurther removed from proportionality indicate less than optimum conditions.It is often rather time-consuming to establish a satisfactorycurve. However, it is important that a stable curve be attained beforereadings on unknown solutions are taken. A deviation of more than ahalf division may invalidate the reading. Merely resetting the galvanometerdoes not correct such an error. A drifting curve indicates changing


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conditions under which reliable measurements cannot be obtained.When the curve has been defined and repeated satisfactorily, the unknownsolution is introduced. After the reading has been taken, the twopoints on the curve, between which the reading falls, are again checked.Only if these two points have remained stable can the reading be consideredvalid. If these two points have varied, the slope of the curve has changedand it is impossible to say at what time the change in the reaction occurred.

This procedure must be rigidly adhered to when serum is analyzed. Anytendency to change in the rate of flow through the atomizer, due to particlesor air bubbles in or protein precipitated from the solution, is reflectedin a failure of the readings to attain the points originally established.When this situation arises, the delivery needle may be probed with a styletor washed with a protein solvent. Haemo-Sol, a commercial cleaner,3has been found satisfactory. Since it contains both sodium and potassium,the system must. be thoroughly washed with distilled water immediatelyafter its use. When the sodium filter is in place, it is advisable toswitch off the cell to avoid a violent deflection of the galvanometer. Urineand other protein-free solutions rarely necessitate this treatment. Inreading the galvanometer scale, deviations of more than 0.5 division arenot considered acceptable.Whenever possible, it is good practice to read a given unknown at twodifferent dilutions. In this way small discrepancies in the concentrationof the standard solutions, the presence of contaminants, or errors in dilutionare made evident. Duplicate measurements with different concentrationsof a given element constitute the most rigid check on the varioussources of error. In determining serum sodium, dilutions of 1:200and 1:400 or 1:250 and 1:500 will yield results which agree within 1 or1.5 per cent when the conditions are optimum.Serum potassium offers the greatest challenge to the performance ofthe instrument, especially when material is limited. When a dilution of1:25 is used, the reading falls in the lower portion of the scale. Becausethe reaction is slow, more than 25 cc. would be needed to reach a higher3 Obtained from Meinecke and Company, Inc., 225 Varick Street, New York.Downloaded from www.jbc.org by guest, on November 24, 2009

P. M. HALD 507point. However, although the reaction is slow, it is quite stable and satisfactoryreadings result.Blank determinations are important and are as necessary to detect contaminatingor interfering substances as they are in using chemical procedures.In the determination of sodium, the distilled water used to dilutethe sample must be tested as well as the saline solution used as a diluentin the potassium procedure. When material is ashed all of the solutions,sulfuric acid, hydrochloric acid, and distilled water, are possible sourcesof interference or contamination. A blank determination carried outsimultaneously with the analysis of the unknown will indicate whether ornot these solutions are pure. Any deflection of the galvanometer fromthe position of the zero point, established with a current of air, indicates a

positive blank.When serum is analyzed it is advisable to run a control base solution.This solution should contain the usual inorganic elements present in serum,in the approximate concentrations in which they occur. Sodium, potassium,calcium, magnesium, chloride, sulfate, and phosphate should beincluded. Each set of determinations should be controlled by dilutingthis solution in exactly the same manner as the unknown serum. Anycontamination of the diluent or apparatus will then be evident by failureto recover theoretical amounts. Furthermore, a similar procedure isessential in analyzing any other materials than those described in whichinterfering ions might prevent accurate recovery.ResultsIn Table II the results of the comparative analyses are tabulated. It


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is notable that, as is true in the chemical method, the greatest per centerror is likely to be found in serum potassium. This is due largely to thefact that this element is present in such a low concentration. Since materialis usually limited, higher concentrations or multiple analyses are oftenimpossible. In the case of analysis on the flame photometer, the relativelysmall amount of light produced by the potassium flame contributesto the difficulty. Unless more than 1 cc. of serum is used, not more than

two readings can be made. In whole blood, in which the potassium concentrationis much higher, the percentage recovery is much better. Inaddition to the comparative studies presented in Table II many additionalanalyses have been made. They have not differed in general from thosetabulated.Differences in the values for sodium in both whole blood and serum obtainedby the two methods lie within the errors of the methods.Urine values for both potassium and sodium fall within a maximum of5 per cent, with one exception. The average would be much closer.Downloaded from www.jbc.org by guest, on November 24, 2009TABLE II

Comparison ofChemical and Photometric Analyses PISerum Urine FoodPotassium Sodium Sodium Potassium Sodiumm&J.

4.4.3.:4.e4.'hotometricn.eq.

4.:4.14.:4.!4.4T&T I- I rm.eq.

50.1 644.1 352.1 372.f 1

63.: 341.j 356.1 3-hot

dissertaion repseed mustured rakesh

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