University of Szeged

Pharmaceutical Analysis Practicals

Edited by:

Gyrgy Dombi

Gerda Szakonyi

Authors:

Gyrgy Dombi

va Kalmr

Gerda Szakonyi

Henriett Dina Szcs

Reviewed by:

Krisztina Novk-Takcs

Szeged, 2015

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of "Coordinated, practice-oriented, student-friendly modernization of

biomedical education in three Hungarian universities (Pcs, Debrecen, Szeged), with focus on the strengthening of international competitiveness" TMOP-4.1.1.C-13/1/KONV-2014-0001

project.

The curriculum can not be sold in any form!

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TABLE OF CONTENTS

CONDUCTOMETRY ....................................................................................................................... 3CONDUCTOMETRIC TITRATION OF CARBOXYLIC ACIDS .............................................................. 7ANALYSIS OF ACETYLSALICYLIC ACID ....................................................................................... 8ANALYSIS OF BENZOIC ACID ........................................................................................................ 9POTENTIOMETRIC (pH-METRIC) TITRATIONS ............................................................................ 10HOW TO USE THE GLASS ELECTRODE ....................................................................................... 15POTENTIOMETRIC TITRATION .................................................................................................... 15EVALUATION OF THE MEASUREMENT ....................................................................................... 15QUANTITATIVE ASSAYS BY TITRATION WITH ALKALINE SOLUTIONS ........................................ 16DINATRII PHOSPHAS DIHYDRICUS ............................................................................................. 17NATRII DIHYDROGENOPHOSPHAS DIHYDRICUS ......................................................................... 18CHININI HYDROCHLORIDUM ..................................................................................................... 19UNGUENTUM AD VULNERA ....................................................................................................... 21SPECTROPHOTOMETRY .............................................................................................................. 23PULVIS CHINACISALIS CUM VITAMINO C .................................................................................. 33TABLETTA ASPIRINI 500 (ASPIRIN TABLET 500) ....................................................................... 37SUPPOSITORIUM PARACETAMOLI 500 MG .................................................................................. 39SPARSORIUM ANTISUDORICUM ................................................................................................. 41SOLUTIO METRONIDAZOLI ........................................................................................................ 43PULVIS CHOLAGOGUS ............................................................................................................... 44DETERMINATION OF PROTEIN CONCENTRATION WITH THE BIURET REAGENT .......................... 47ATOMIC ABSORPTION SPECTROMETRY ..................................................................................... 49DETERMINATION OF MAGNESIUM CONTENT OF SPARSORIUM ANTISUDORICUM BY FLAME ATOMIC ABSORPTION ................................................................................................................ 53DETERMINTION OF MAGNESIUM CONTENT OF PULVIS NEUTRACIDUS BY FLAME ATOMIC ABSORPTION ............................................................................................................................. 54DETERMINATION OF ACTIVE INGREDIENTS OF PANADOL EXTRA BY HPLC .............................. 55COMPLEXOMETRIC TITRATIONS ................................................................................................ 58PULVIS NEUTRACIDUS ............................................................................................................... 63SUSPENSIO ZINCI AQUOSA ........................................................................................................ 65ARGENTOMETRIC ANALYSIS ..................................................................................................... 66SPARSORIUM SULFABORICUM ................................................................................................... 67REDOX TITRATIONS .................................................................................................................. 68SUPPOSITORIUM ANTIPYRETICUM PRO INFATE VEL PRO PARVULO ........................................... 74INJECTIO ALGOPYRINI 50% ....................................................................................................... 76ACIDBASE TITRATIONS ........................................................................................................... 77SPIRITUS IODOSALICYLATUS ..................................................................................................... 80TEST YOURSELF SAMPLE TEST QUESTIONS ............................................................................ 82APPENDIX .............................................................................................................................. 92UNICAM UV/VIS SPECTROPHOTOMETER MANUAL .................................................................. 93UV-1601 SHIMADZU SPECTROPHOTOMETER MANUAL ............................................................. 95MARS CEM MICROWAVE DESTRUCTOR MANUAL ..................................................................... 97ATOMIC ABSORPTION SPECTROMETER MANUAL ...................................................................... 98HPLC MANUAL ...................................................................................................................... 100NMR SPECTRA ........................................................................................................................ 109

3

CONDUCTOMETRY

(MEASUREMENT OF SPECIFIC CONDUCTANCE)

Conductometry is based on the measurement of the conductance of electrolyte solutions.

The passage of electric current through a chemical cell is carried out by the ionic species in the solution. It is an additive property, with the participation of all of the ions in the solution. The conductance is specified by the measurement of the resistance of a certain segment of the solution. The conductance (G) is the reciprocal of the resistance (R), its unit is 1/ Siemens; S):

G = R1

The conductance is directly proprotional to the surface area (A) of the electrodes and inversely proportional to the distance (d) between the electrodes:

R1 =

dA

is the specific conductance, where the resistance of the solution is measured between two electrodes of 1 cm2 area 1 cm apart.

The conductance depends on the number of ions in the solution and on the identity of the ions. Some ions move faster than others in an electric field, and their mobility is therefore an important factor too.

Dilution of an electrolyte solution will decrease the specific conductance: the lower number of ions present in a given volume, the lower the current flow is. The molar specific conductance () was introduced to characterize of the conductance of certain ions:

= 1000c

where c is the concentration of the electrolyte solution. The ions in an infinitely dilute solution contribute to the conductance independently

from each other, and the molar specific conductance of an infinite dilute solution can therefore be calculated by summing the conductances of each of the ions in the solution:

and are the conductances of cations and anions, respectively in infinitely dilute solution.

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The electrode Special eletrodes are used during conductometric measurements. The conductance is

determined by measurement of the resistance of the solution in a certain volume between two electrodes made of platinized platinum. The surface area of the electrodes is increased and the polarization resistance is decreased by platinization. The electrodes are fixed tightly in a cylindrical unit. The fixed geometry specifies the distance of the electrodes during both the calibration and the measurement. Alternate current is used for the conductometric analysis so as to avoid disturbing electrode processes. A Wheatstone bridge is used to measure the resistance.

Concentration measurement (direct conductometry) and conductometric titrations (indirect conductometry) are distinguished in conductometry. In direct conductometry, the concentration is determined by the measurement of conductance. This method is used, for example, to check Aqua purificata or Aqua destillata. An electrode is built into the ion-exchange system that continuously monitors the conductance of ion-exchanged water. When the conductance is above a given limit, the system must be regenerated. According to the European Pharmacopoeia 8th Edition, the maximum allowed conductance of Aqua purificata is 4.3 S/cm, while that of Aqua ad iniectabilia is 1.1 S/cm.

Conductometric titrations can be applied when the ion concentration changes during a reaction, or when the ion concentration remains constant, but the mobility of the ions changes.

Types of conductometric titrations Acidbase titrations

It is easy to determine the equivalence point in these titrations because hydrogen ions (H+) are the most mobile of all ions, and hydroxide ions (OH-) are the second most mobile, and the mobilities are well above those of other ions.

1. Titration of a strong acid with a strong base The titration of hydrochloric acid (HCl) with sodium hydroxide (NaOH) may serve as

an example.

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The neutralization of HCl does not change the electrolyte concentration of the solution before the equivalence point because the H+ are replaced by sodium ions (Na+). The lower mobility of the Na+ results in decreased conductivity. There are two reasons why the conductivity increases after the equivalence point. The excess NaOH increases the electrolyte concentration in the solution, and the mobility of the OH- is high.

HCl + NaOHH2O + NaCl

2. Titration of a weak acid with a strong base As an example, acetic acid may be titrated with NaOH.

At the beginning of the titration, the dissociation of acetic acid is blocked by the acetate ions. The H+ concentration is decreased, and the conductance is therefore also decreased, so that a minimum is visible in the titration plot. The concentration of acetate ions increases on the addition of NaOH, and the conductance increases too then slowly. Na+ also contribute to the increase of conductance. The conductance increases sharply after the equivalence point because of the presence of excess Na+ and OH-. The plot becomes steeper than in the previous phase because the OH- are not neutralized and their mobility is higher than that of acetate ions.

CH3COOH + NaOH CH3COO- + Na+ + H2O

CH3COOH CH3COO- + H+

The intersection of the linear sections of the graph is often not clearly visible, and the measurement is therefore not precise. This problem can be avoided by using the method described in Section 3.

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3. Titration of a weak acid with a weak base As an example, oxalic acid may be titrated with N-Methylglucamine (meglumine).

Oxalic acid is a dicarboxylic acid. Its first proton (pKa1 = 1.05) can be titrated as a medium strength acid, while the second proton (pKa2 = 4.28) can be titrated as a weak acid. N-methylglucamine, (C6H11O5NHCH3), a hexosamine, is used as standard solution; it contains a basic secondary amino group (pKa = 9.20) that can accept protons. The conductance of N-Methylglucamine is negligible because its dissociation is very low.

The first proton of oxalic acid influences the conductance in the early stages of the titration, because of its mobility. The mobile protons react with N-Methylglucamine and form N-Methylglucammonium ions which have strongly decreased mobility. That is why the conductance of the solution initially drops. The second proton of oxalic acid reacts according to the following equation:

HOOCCOO- + C6H11O5NHCH3 -OOCCOO- + C6H11O5NH 2 CH3

The number of charged particles increases, and the conductance is therefore also increases. The titration graph shows a decrease at the beginning until it reaches a minimum, after which it increases slowly. After the equivalence point, N-Methylglucamine becomes dominant in the solution. Its dissociation is practically zero and the dilution of the solution is negligible. The conductance reaches a plateau. The intersection of the linear sections of the graph is relatively clear, and the determination of the equivalence point is therefore easy.

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CONDUCTOMETRIC TITRATION OF CARBOXYLIC ACIDS

Background: Conductometric titrations are suitable for the analysis of reactions in which the ion

concentration changes or in which the concentration is kept constant but the mobility of the ions changes.

Acetylsalicylic acid and benzoic acid are weak organic acids. N-Methylglucamine, a weak base, is used as a standard solution for their analysis. The conductance decreases at the beginning of the titration, and then increases moderately. The conductance does not change or only negligibly after the equivalence point is reached. The conductance is plotted as a function of the volume of standard solution. The intersection of the two sections is clearly visible, and the equivalence point can be determined graphically. The conductance increases rapidly after the equivalence point if a strong base (NaOH) is used for the analysis.

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ANALYSIS OF ACETYLSALICYLIC ACID

Definition: Acetylsalicylic acid contains not less than 99.5 per cent and not more than the equivalent of 101.0 per cent of 2-(acetyloxy)benzoic acid, calculated with reference to the dried substance.

Characters: A white, crystalline powder or colourless crystals, slightly soluble in water, freely soluble in ethanol. It melts at about 143C (instantaneous method).

Quantitative analysis: Weigh 0.1500 g acetyl salicylic acid. Prepare two independent samples! Use two 150

ml beakers. Dissolve them in 5 ml of methanol and then add 40 ml of distilled water. Put one of your samples on the magnetic stirrer and place a stir bar in the solution. Immerse the electrode of the conductometer into the solution and add as much water to reach the black mark on the electrode. Turn on the conductometer with the right button under the screen. On the left of the screen S (micro siemens) mS (milli siemens) and C signs are visible. The instrument shows the actual setting that should be changed to S range. Titrate the sample by addition of 0.5 ml portions of 0.1 M N-methyl-glucamine (meglumine) standard solution. Record the conductance after each 0.5 ml. Continue the titration until a total 15.0 ml of titrant is added. Plot the conductance as a function of the volumes of the standard solution. Calculate the percentage of acetyl salicylic acid content of the sample. Enter the result with two-decimal precision.

1 ml of standard 0.1 M N-methyl-glucamine is equivalent to 18.016 mg acetyl salicylic acid.

Repeat the titration with 0.1 M sodium hydroxide standard solution and evaluate the different titration curves.

1 ml of standard 0.1 M NaOH is equivalent to 18.016 mg acetyl salicylic acid.

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ANALYSIS OF BENZOIC ACID

Definition: Benzocaine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of ethyl 4-aminobenzoate, calculated with reference to the dried substance.

Characters: A white, crystalline powder or colourless crystals, very slightly soluble in water, freely soluble in alcohol.

Quantitative analysis: Weigh 0.1200 g benzoic acid. Prepare two independent samples! Use two 150 ml

beakers. Dissolve them in 5 ml of methanol and then add 40 ml of distilled water. Put one of your samples on the magnetic stirrer and place a stir bar in the solution. Immerse the electrode of the conductometer into the solution and add as much water to reach the black mark on the electrode. Turn on the conductometer with the right button under the screen. On the left of the screen S (micro siemens) mS (milli siemens) and C signs are visible. The instrument shows the actual setting that should be changed to S range. Titrate the sample by addition of 0.5 ml portions of 0.1 M meglumine standard solution. Record the conductance after each 0.5 ml. Continue the titration until a total 15.0 ml of titrant is added. Plot the conductance as a function of the volumes of the standard solution. Calculate percentage of benzioc acid content of the sample. Enter the result with two-decimal precision.

1 ml of standard.1 M meglumine is equivalent to 12.21 mg benzoic acid.

Repeat the titration with 0.1 M sodium hydroxide standard solution and evaluate the different titration curves.

1 ml of standard 0.1 M NaOH is equivalent to 12.21 mg benzoic acid. Notes:

The temperature of the sample is not needed to be monitored to correct the result with a temperature factor because the temperature is kept constant during the measurement. The instrument need not to be calibrated with solutions of known conductance as relative changes are being recorded.

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POTENTIOMETRIC (pH-METRIC) TITRATIONS

Visual observation of the end-point by using an acidbase indicator is simple and convenient, but it may cause several problems. Instrumental methods are being used in most of the quantitative analyses in the Pharmacopeia; for acidbase titrations, the measurement of pH can be a possible solution.

Pontentiometry is an analytical method that is based on the measurement of electrode potential. The electrode potential of the indicator electrode immersed in the analyte is used to determine the concentration of the sample.

It is possible to measure only the potential of a cell, which is the potential difference between two electrodes. It is universally agreed that an arbitrary electromotive force (emf) is assigned to one electrode, and the potential of the second electrode can be measured.

Two types of galvanic cells can be distinguished:

a cell without transmission: Ag / AgCl / ZnCl2(c1) / Zn, and a cell with transmission: Ag / AgCl/ KCl(c2) // ZnCl2(c1) / Zn

The major difference between the two cells is that there is an electrodeliquid interface in a cell without transmission, while there is a liquidliquid interface in a cell with transmission, where KCl and ZnCl2 solutions are in contact. The potential difference here is called the liquidliquid interface potential or diffusion potential. The potential of the cells includes the diffusion potential in every liquidliquid interface cell.

The value of the diffusion potential can be decreased by using a salt bridge. A salt bridge consists of a concentrated or saturated solution of a specific salt, where the mobilities of its anions and cations are nearly the same. Potassium chloride (KCl) is most frequently used for this purpose, but when Cl- ions disturb the analysis, potassium or ammonium nitrate (KNO3 or NH4NO3) is used. A salt bridge actually means the insertion of two diffusion potentials.

As the electrolyte concentration of the salt bridge is much higher than the analyte concentration, the two diffusion potentials are influenced by the K+ and Cl- or NH4+ and Cl- ions. The mobilities of these ions are nearly the same, the value of the diffusion potential is low, and the potentials at the two interfaces are also really close to each other.

The value of the diffusion potential will be very low; however, it cannot be eliminated completely, but only decreased to a minimum.

The electrode potentials agreed by convention are determined by comparison with the standard hydrogen electrode. The reference electrode, the standard hydrogen electrode, is set to 0.00 V. The standard hydrogen electrode is a platinized platinum electrode that is immersed in 1.0 mol/dm3 HCl and pure hydrogen gas (H2) is bubbled through it. The pressure of the H2 is 0.1 MPa. Any electrode for which the electrode potential is not yet known, can be coupled with the standard hydrogen electrode to form a galvanic cell, and the potential of the galvanic cell gives the the potential of the unknown electrode.

The table below shows several specific electrode potential values. The electrode potential can be positive or negative. A negative electrode potential means that the electrode is rather reducing relative to H+, while a positive value indicates a stronger oxidizing property than that of hydrogen.

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Types of electrodes in potentiometry:

1. Electrodes working on the basis of equilibrium reactions (e.g. primary electrodes, secondary electrodes and redox electrodes)

2. Ionselective electrodes (membrane electrodes) (e.g. pHselective glass electrodes, metal ion selective glass electrodes and liquid membrane electrodes)

3. Moleculeselective electrodes (e.g. enzyme electrodes and gas moleculeselective electrodes)

The pHsensitive glass electrode is the most important in the pharmaceutical analytical practicals.

The most important part of the glass electrode that is used for pH measurement is a thin bulbform membrane made of hydrogensensitive glass attached to a nonhydrogenselective glass tube. The resistances of both the tube and the membrane are high. The H+ response is given only by this special glass membrane. The depth of immersion does not influence the measurement if the membrane is fully covered by the solution.

Table of standard electrode potentials

Li+(aq) + e- Li(s) -3.04

K+(aq) + e- K(s) -2.92

Ca2+(aq) + 2 e- Ca(s) -2.76

Na+(aq) + e- Na(s) -2.71

Mg2+(aq) + 2 e- Mg(s) -2.38

Al3+(aq) + 3 e- Al(s) -1.66

2H2O(l) + 2 e- H2(g) + 2 OH-(aq) -0.83

Zn2+(aq) + 2 e- Zn(s) -0.76

Cr3+(aq) + 3 e- Cr(s) -0.74

Fe2+(aq) + 2 e- Fe(s) -0.41

Cd2+(aq) + 2 e- Cd(s) -0.40

Ni2+(aq) + 2 e- Ni(s) -0.23

Sn2+(aq) + 2 e- Sn(s) -0.14

Pb2+(aq) + 2 e- Pb(s) -0.13

Fe3+(aq) + 3 e- Fe(s) -0.04

2H+(aq) + 2 e- H2(g) 0.00

Sn4+(aq) + 2 e- Sn2+(aq) 0.15

Cu2+(aq) + e- Cu+(aq) 0.16

ClO4-(aq) + H2O(l) + 2 e- ClO3-(aq) + 2 OH-(aq) 0.17

AgCl(s) + e- Ag(s) + Cl-(aq) 0.22

Cu2+(aq) + 2 e- Cu(s) 0.34

ClO3-(aq) + H2O(l) + 2 e- ClO2-(aq) + 2 OH-(aq) 0.35

IO-(aq) + H2O(l) + 2 e- I-(aq) + 2 OH-(aq) 0.49

Cu+(aq) + e- Cu(s) 0.52

I2(s) + 2 e- 2 I-(aq) 0.54

ClO2-(aq) + H2O(l) + 2 e- ClO-(aq) + 2 OH-(aq) 0.59

Fe3+(aq) + e- Fe2+(aq) 0.77

Hg22+(aq) + 2 e- 2 Hg(l) 0.80

Ag+(aq) + e- Ag(s) 0.80

Hg2+(aq) + 2 e- Hg(l) 0.85

ClO-(aq) + H2O(l) + 2 e- Cl-(aq) + 2 OH-(aq) 0.90

2Hg2+(aq) + 2 e- Hg22+(aq) 0.90

NO3-(aq) + 4 H+(aq) + 3 e- NO(g) + 2 H2O(l) 0.96

Br2(l) + 2 e- 2 Br-(aq) 1.07

O2(g) + 4 H+(aq) + 4 e- 2 H2O(l) 1.23

Cr2O72-(aq) + 14 H+(aq) + 6 e- 2 Cr3+(aq) + 7 H2O(l) 1.33

Cl2(g) + 2 e- 2 Cl-(aq) 1.36

Ce4+(aq) + e- Ce3+(aq) 1.44

MnO4-(aq) + 8 H+(aq) + 5e- Mn2+(aq) + 4 H2O(l) 1.49

H2O2(aq) + 2 H+(aq) + 2 e- 2 H2O(l) 1.78

Co3+(aq) + e- Co2+(aq) 1.82

S2O82-(aq) + 2 e- 2 SO42-(aq) 2.01

O3(g) + 2 H+(aq) + 2 e- O2(g) + H2O(l) 2.07

F2(g) + 2 e- 2 F-(aq) 2.87

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The inner and outer surfaces of the membrane are hydrogensensitive. The electric potential at the outer surface, which depends on the proton concentration of the analyte, is usually measured with a secondary electrode, e.g. Hg-Hg2Cl2 or Ag-AgCl. There is a high buffer capacity reference solution inside the electrode where the reference electrode (usually Ag-AgCl) can be found.

The schematic diagram of the whole electrochemical cell:

Ag-AgCl internal electrode

Internal buffer solution

pH sensitive glass membrane

Analyte solution

Outer reference electrode

(Hg-Hg2Cl2

(Single vertical lines indicate the phase borders, while the double vertical line denotes a salt bridge or diaphragm.)

A combined glass electrode in which the reference electrode is inbuilt is used during the pharmaceutical analytical practicals.

Shematic diagram of a glass electrode

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The practice of potentiometric analysis: The measurement requires the following components:

a solution of the analyte

an indicator electrode (working (half) cell)

a reference electrode (reference (half) cell)

a potentiometer

a closed circuit (salt bridge) Types of potentiometric analysis:

direct potentiometry

indirect potentiometry (potentiometric titration) The concentration of the electrode active material is calculated from the emf or the

value of the electrode potential by using the Nernst-equation:

)ln(0 aFzTREE

where:

a = activity (a = f c; f = activity coefficient; f 1, so a c in the case of dilute solutions)

R = universal gas constant, 8.314 J/(mol K) T = absolute temperature (K) F = Faraday constant (96,487 C/mol)

Introducing the constants:

)log(059.00 czEE

where

z = moles of electrons transferred in the cell reaction c = concentration

The concentration of the electrodeactive sample can be calculated if the electrode potential is determined (known).

Direct potentiometry is fast and easy to automatize, but there are limitations of its use because of the possible errors.

Determination of the endpoint of a titration is also possible with potentiometric titration. The indicator electrode is immersed in the solution of the sample that contains the

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electrodeactive material in this case and the emf is measured as a function of the volume of the standard solution. The titration curve is determined experimentally and its inflexion point indicates the equivalence point of the titration. The accuracy of the measurement depends on the determination of the endpoint of the titration and not on the accuracy of the measurement of the emf, and thus the error will be smaller.

The use of indicator dyes is not necessary during the application of potentiometry, so any indicator error is eliminated. Potentiometric determination of the end-point is more sensitive than visual methods. It can be applied for solutions one order of magnitude more dilute than those where visual end-point determination is used.

This method can be applied for all the titrations where either of the reactants can participate in a reversible electrochemical reaction for which a potentiometric electrode can be built. It is used for neutralization analysis, complexometry, argentometry and oxidimetry.

Determination of the equivalence point graphically and numerically:

The precision of the determination of the endpoint can be increased by the derivation of the titration curve. A local maximum or local minimum is visible in the first derivative of the curve; while the second derivative of the curve is zero.

The electrode potential is directly proportional to the pH of the solution:

]log[0

HFzTREE

pHE 059.0

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HOW TO USE THE GLASS ELECTRODE

Combined glass electrodes are usually used when pH is monitored.

The electrode must never be allowed to dry out.

After use, the electrode must be rinsed with distilled water or, if it has been used in some non-aqueous medium, with other appropriate solution. In special cases, washing with water is not sufficient, and the electrode must be immersed in a cleaning solution for about 30-60 min, after which it must be rinsed and stored in an appropriate storage solution.

The electrode must be detached from the main unit before it is turned off.

The electrode must not be stored or turned upside down.

The electrode is expensive. It must be immersed into the sample solution with extreme care, with the electrode kept about 4 cm beneath the surface. At the same time, it must be kept as far as possible from the magnetic stirrer.

The pH-meter must be calibrated before use by immersing the electrode in different commercially available calibrating solutions. Various calibration points may be chosen for the calibration routine. However, calibration at two different pH values is used most frequently. During the calibration, the electrode is attached to the main unit, and is then rinsed with distilled water, dried and immersed in the chosen calibrating solution. The same measurement range is set and a waiting period is necessary until the pH value on the screen has been stabilized. The value is then recorded and stored in the memory unit of the pH-meter. After calibration, the electrode must be rinsed again with distilled water, and the analysis can then start. The calibration range must be chosen so that the measured pH or potentials fall into this range. The most frequently used calibration solution is pH 7.01 buffer solution.

POTENTIOMETRIC TITRATION

The combined glass electrode is immersed into the sample solution, the magnetic stirrer is set up for slow mixing and small quantities of standard solution are added. It is necessary to wait for a few seconds after the addition of each volume to allow the pH to stabilize. The pH values are recorded and plotted as a function of the volume of standard solution used. In acidbase titrations, the temperature may rise in case of because of the neutralizing heat. The pH may change if the temperature is not constant, and the sample flask must therefore be thermostated.

EVALUATION OF THE MEASUREMENT

The potentiometric titration can be evaluated numerically or graphically. The numerical evaluation may be performed manually or by computer. The pH values are plotted as a function of the volume of standard solution used.

The inflexion points should be determined. The easiest way to find inflexion points is the tangential method.

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For appropriate analysis of the curves, very accurate graphs should be plotted. The basic requirement is multiple recordings of the pH changes around the inflexion points. It is therefore necessary to know the expected equivalence point. This is possible if the dissociation exponents and/or the equivalence ranges are known. The standard solution is usually added in

Tangental method for evaluation of a potentiometric titration curve

to the sample 0.5-ml quantities, but in the range close to equivalence the quantities should be smaller, e.g. 0.1-0.2 ml.

The pH change is monitored continuously, and when it changes by more then 0.1 pH unit the added volume of standard solution should be decreased. The added volume of standard solution can be increased again if there are two equivalence points between the two inflexion points and the pH does not change significantly after the second inflexion point.

In order to determine the equivalence point, parallel straight lines are fitted to the initial and final sections of the titration curve, and a third straight line is fitted to the linear points around the inflexion points of the curve. The mean V (cm3) value of the intersections gives the volume at the equivalence point.

Before use, the pH-meter must be set with the help of the tutor.

QUANTITATIVE ASSAYS BY TITRATION WITH ALKALINE SOLUTIONS

Titration with NaOH or KOH is used for the quantitative assay of acidic substances in around 100 cases, in the Pharmacopoeia. More than 50% of these assays are potentiometric titrations. Organic substances are usually dissolved in alcohol because of their limitied solubility in water. Inorganic substances are tested in hydrophilic solutions.

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DINATRII PHOSPHAS DIHYDRICUS DISODIUM HYDROGENPHOSPHATE DIHYDRATE

Na2HPO42H2O Mr 178.0

Definition: Content: 98.0 per cent to 101.0 per cent (dried substance).

Characters: Appearance: a white or almost white powder or colorless crystals. Solubility: soluble in water, practically insoluble in ethanol (96 per cent).

Background: Potentiometric titration is used in the Pharmacopoeia to assay the hydrated and

dehydrated forms of NaH2PO4 and KH2PO4. The samples must be dried before the analysis and the mass loss on drying must be taken into account. The different compounds can be measured together during the titration, which means when Na2HPO4 is analyzed, NaH2PO4 content can also be determined. Two-step titration curves are obtained in all cases during the analysis of these substances. An analytically accurate quantity (25.0 ml) of 1 M HCl is added at the beginning of the measurement, and the sample is then titrated with standard 1 M NaOH.

The phosphates react with HCl to form orthophosphoric acid (H3PO4). The excess HCl and the H3PO4 are titrated to reach the first inflexion point (V1 ml), and (25 ml - V1 ml) is proportional to the concentration of HPO42-. The first inflexion point can be calculated by using the dissociation exponents: (pKs1+pKs2); its value is ~4.6. Frequent measurement intervals should therefore be applied around the first inflexion point at pH 4.6. The second inflexion point is at pH ~9.7 (V2 ml), (pKs2+pKs3), when all the H2PO4- is converted to HPO42-. Frequent measurement intervals should again be recorded around the inflexion point and the titration should be continued until the pH changes is decreased dramatically (pH ~11). The result is calculated by using the equation below.

Quantitative analysis: Disolve 2.0000 g of sample weighed with analytical accuracy in 50 ml of water R and

add 25.0 ml of 1 M HCl. Titrate the sample potentiometrically to the first inflexion point (V1 ml) by using standard 1 M NaOH solution. Then continue the titration to the second inflexion point (total volume of 1 M NaOH solution required V2 ml).

Calculate the percentage of Na2HPO4 by using the following formula:

)100()25(1420 1

dmfVf NaOHHCl

where

d = percentage loss on drying.

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Calculate the percentage Na2HPO4 contamination of the sample according to the following equation:

NaOHHCl

HClNaOH

fVfffV

1

2

2525

This percentage content should not be greater than 0.025%.

NATRII DIHYDROGENOPHOSPHAS DIHYDRICUS SODIUM DIHYDROGENPHOSPHATE DIHYDRATE

NaH2PO42H2O Mr 156.0

Definition: Content: 98.0 per cent to 100.5 per cent (dried substance).

Characters: Appearance: a white or almost white powder or colorless crystals. Solubility: very soluble in water, very slightly soluble in ethanol (96 per cent).

Quantitative analysis Dissolve 2.5000 g of sample weighed with analytical accuracy in 40 ml water R. Titrate

it with carbonate-free 1 M NaOH, determining the end-point potentiometrically.

1 ml of standard 1 M NaOH is equivalent to 0.120 g of NaH2PO4. Calculate the percentage NaH2PO4 content of the powder in a similar way as for Na2HPO4.

19

CHININI HYDROCHLORIDUM QUININE HYDROCHLORIDE

C20H25ClN2O22H2O Mr 396.9

Definition: Content: 99.0 per cent to 101.0 per cent of alkaloid monohydrochlorides, expressed as (R)-[(2S,4S,5R)-5-ethenyl-l-azabicyclo[2.2.2]oct-2-yl]-(6-methoxy-quinolin-4-yl)methanol]-hydrochloride (dried substance).

Characters: Appearance: white or almost white or colorless, fine, silky needles, often in clusters. Solubility: soluble in water, freely soluble in ethanol (96 per cent).

Background: The method described below is frequently specified for the analysis of organic amine

salts amine hydrochlorides in the Pharmacopoiea. There are 78 such quantitative analyses, including papaverineHCl, quinineHCl, ephedrineHCl and pseudo-ephedrineHCl.

The method is called displacement titration because the amine base is liberated from its hydrochloride form during the titration. Organic amine bases are not very soluble in water, and an alcoholic aqueous medium is therefore used. The acidic natures of the amine hydrochlorides differ, and the sample is therefore dissolved in ethanol and before the measurement 5 ml of 0.1 M HCl is added as an adjuvant solution. This HCl is not a volumetric solution; it must be added to the sample to allow the accurate determination of the first inflexion point, from where the amine hydrochloride is measured. In other words, at the beginning of the titration the excess HCl is measured by the addition of 0.1 M NaOH solution up to the first inflexion point. The volume relating to the second inflexion point depends on the quantity of the amine base. It is recommended to use smaller steps (e.g. 0.1-0.2 ml) around the inflexion points. If the pH jump is more than 0.1 unit, use 0.1 ml NaOH should be used as the amount added. The quantity of the amine base is proportional to the volume of NaOH added, which can be calculated by subtracting the volume added up to the first inflexion point that up to the second one. There are

20

special cases when diamine dihydrochlorides are tested (e.g. histamine2HCl, meclozine2HCl); in these titrations, three inflexion points can be observed.

Standardization of the 0.1 M NaOH solution The procedure for the standardization of 0.1 M NaOH solution is similar to the

displacement titration method:

Accurately measure 0.1000 g of dried benzoic acid and dissolve it in 50 ml of alcohol. Add 5 ml of 0.1 M HCl and titrate the solution potentiometrically with 0.1 M NaOH. 1 ml of 0.1 M NaOH is equivalent to 12.21 mg of benzoic acid. Calculate the theoretical volume by using the equivalent mass of benzoic acid. Calculate the practical volume by subtracting the volume added up to the the first inflexion point from the volume added up to the second inflexion point. Calculate the factor of NaOH by using the following equation:

practical

ltheoretica

VVf

The benzoic acid should be very pure for the standardization. If only inpure benzoic acid is available, it should be purified in an appropriate sublimation apparatus.

Quantitative analysis:

Dissolve 0.2500 g of accurately weighed sample in 50 ml of alcohol R and add 5 ml 0.1 M HCl. Titrate the sample with 0.1 M NaOH, determining the end-point potentiometrically. Read the volume added between the 2 inflexion points.

N.B. standard 0.1 M NaOH solution is made by the dilution of 1 M NaOH stock solution. NaOH solutions should always be standardized because NaOH pellets are hygroscopic and adsorb carbon dioxide (CO2). The standardization of standard NaOH solution is described in detail in the theoretical guidelines.

1 ml of 0.1 M NaOH is equivalent to 36.09 mg of C20H25ClN2O22H2O. Calculate the C20H25ClN2O22H2O percentage of the powder.

21

UNGUENTUM AD VULNERA (UNG. AD VULNER.)

DERMATOLOGICUM. ANTISEPTICUM.

Composition: Acidum salicylicum 0.6 g

Vaselinum acidi borici ad 30.0 g

Background:

At the beginning of the titration, standard 0.1 M NaOH solution is added in 0.2-ml portions to the sample. The pH is recorded after each 0.2 ml. The pH may decrease and then slowly increase during the titration. The change is more dramatic around the equivalence point of the salicylic acid, after which the rate of pH increase slows down. After the equivalence point has been reached, the sample is overtitrated by the addition of at least 5 ml of 0.1 M NaOH solution in 0.5-ml portions and 2.0 g of mannitol is then added. Mannitol forms a complex with boric acid (H3BO3) that can be titrated as a stronger monoprotic acid than H3BO3 itself. The pH of the solution drops by 2-3 units.

B

OH

HO

HO

OHOH

OH HO

HOB

O

O O

O+ H

NaOH+ Na + H2O

boric acid - mannitol complex

The titration is carried oot to reach the equivalence point of H3BO3 and continued with 5-6 ml of standard solution after the potential jump so as to be able to evaluate the result graphically. The pH is plotted as a function of the volume of standard 0.1 M NaOH solution. Two inflexion points are visible in the curve. The first is directly proportional to the amount of salicylic acid. The difference between the second and first equivalence points is directly proportional to the amount of H3BO3. The amount of salicylic acid and H3BO3 should be calculated in 30.0 g of sample.

Quantitative analysis: An HI 9321 type pH-meter and an HI 1331 type combined glass electrode are used

during the potentiometric measurements. The instrument can be connected to the mains through

22

an adaptor. Between measurements, the electrode is stored in a special storage solution that can be found in the cap of the electrode. Before the measurement is started, this cap should be removed and the electrode must be rinsed with water R before use. The lid should be kept in such a way as to keep the storage solution intact. The electrode must be connected to the instrument before the pH meter is turned on with the ON/OFF button. The electrode must be immersed into the sample solution so that a distance of at least 4 cm is kept between the bottom of the electrode and the surface of the solution. The monitor of the pH-meter shows the actual pH (it is necessary to wait a few seconds to let the value stabilize).

Heat 1.4000 g of sample weighed with analytical accuracy with of 50.0 ml water to 100 C, then shake it to dissolve the active ingredients, cool it down to room temperature and filter it to remove the base of the ointment. Put the sample solution (in a 100.0 ml beaker) on a magnetic stirrer and place a clean stir bar into the solution. Set the stirring speed to medium. The sample solution is titrated with 0.1 M NaOH. It is recommended to use smaller quantities at the beginning of the titration, e.g. 0.2 ml. Check and record the pH after the addition of each quantity of NaOH (wait a few seconds after the addition of NaOH to let the pH stabilize). During the titration, the pH first decreases slightly and increases slowly. Around the equivalence point of salicylic acid, the pH rises rapidly, and after the equivalence point it reaches a plateau. Continue the titration with 5-6 ml of additional 0.1 M NaOH after the equivalence point of salicylic acid has been reached. Then add 2.0 g of mannitol to the solution. Mannitol forms a complex with H3BO3 and this complex is a stronger acid than H3BO3 itself. At this point, the pH of the solution drops by 2-3 units. Continue the titration with 0.1 M NaOH to reach the next potential jump at the equivalence point of H3BO3; it is necessary to overtitrate so that graphical determination of the equivalence points is possible.

Plot the pH values as a function of the volume of 0.1 M NaOH added. A curve with two potential jumps gives the volumes of NaOH needed to titrate salicylic acid and H3BO3, respectively. The volume added up to the first potential jump is equivalent to the amount of the salicylic acid, and the difference between the second and first potential jumps is equivalent to the amount of the H3BO3.

Calculate the salicylic acid and H3BO3 contents of a 30.0 g sample. Give the results in grams with four-decimal precision.

1 ml of standard 0.1 M NaOH solution is equivalent to 13.812 mg of salicylic acid (C7H6O3).

1 ml of standard 0.1 M NaOH solution is equivalent to 6.183 mg of H3BO3.

23

SPECTROPHOTOMETRY

Energy is absorbed by all atoms and compounds depending on their chemical structure. The structure of the molecule determines the interaction of the molecule and the electromagnetic radiation. The electromagnetic radiation absorbed is directly proportional to the concentration of the sample, and this phenomenon can therefore be used for analytical purposes. The method is simple, fast, sensitive, and specific, and is frequently used in analytical chemistry for quantitative determinations.

The interpretation of absorption phenomena that occur in ultraviolet (UV) and visible (VIS) light is at the main focus of the pharmaceutical analysis practicals. The excitation of single -bonds in a molecule is very difficult; it may be achieved when far-UV light is used. Nonbonding electrons (n-electrons) in the outer shell (that are not involved in chemical bond formation) can be excited by UV light. -electrons (double or triple bonds) can be excited by both UV and VIS light.

The chromophore group of a molecule is responsible for its light absorption. Most chromophore groups contain one or more unsaturated bonds. A group of atoms attached to a chromophore which is able to modify how the chromophore absorbs light is called an auxochrome. The absorption maximum of a molecule can be influenced in the following ways:

A bathochromic effect occurs when the absorption maximum shifts to longer wavelengths. The opposite is a hypsochromic shift, when the absorption maximum shifts toward shorter wavelengths. Hyperchromicity is the increase in absorbance of a material, while hypochromicity is the decrease in absorbance of the substance. These phenomena are used in practice when chromophore groups are built into a molecule:

24

Nitrobenzene is a typical chromophore; the nitro group of the aromatic ring intensifies the conjugation. Another similar molecule is trinitrophenol (picric acid), a yellow compound; its salts are called picrates.

The measurement of steroids in the UV-VIS range on the basis of own light absorption at short wavelengths is really difficult. However when steroid derivatives are used on the basis of the following reactions: the analysis can be performed:

When an aldehyde reacts with a primary amine, a Schiff base is formed. In the case of an aromatic amine, the conjugation of the molecule is extended. A bathochromic shift occurs.

25

The determination of protein concentration is possible in the UV range at a wavelength of 280 nm, when the absorption of the aromatic side-chains of phenylalanine, tyrosine and tryptophan is maximal. Complex formation is often used in practice, when the protein concentration is measured in the VIS range:

Most such measurements are based on the fact that the peptide bonds of proteins are able to reduce copper ions (Cu2+) in alkaline media. The extent of the reaction is directly proportional to the amount of protein in the sample. The reduced copper ions (Cu+) form a colored product with a chelating agent, e.g. the BCA assay shown above.

26

The ninhydrin reaction is used to detect peptides/proteins. Ninhydrin can react with primary and secondary (noncyclic) amines. The product of the reaction is a conjugated Schiff base with an absorption maximum in VIS range. Since proline is an imino acid, it does not react with ninhydrin.

O

O

OH

OH

ninhydrin

Ninhydrin reaction

+ RHC

NH2

COOH

O

O

H

OH

NH3 + CO2 +RCHO

O

O

OH

OH

+ NH3

O

O

H

OH

+

OH

O

N

O

O

Determination of the amino groups in amino acids, peptides and proteins

blue complex, max = 570 nm

The blood glucose level can normally vary between certain values. It is very important to monitor it regularly to obtain acurate information about the carbohydrate metabolism of the body, and particularly to identify diabetes.

Two basic techniques are used to determine the blood glucose level. The first is chemical method, in which the nonspecific reducing property of glucose is used. The concentration of glucose is revealed by the color and its intensity of an indicator.

27

1. Glucose + alkaline copper(II)tartrate copper(I)-oxide

2. Cu2+ + phosphomolybdic acid Mo2O5 phosphomolybdic oxide (blue-colored end-product)

The colored end-product of the reaction can be measured by spectrophotometry. The measurement may be influenced by other reducing substances present in the blood, e.g. higher values may be detected in uremic patients.

These problems can be eliminated by using newly developed enzymatic techniques. The most frequently used enzymes are (a) glucose oxidase and (b) hexokinase.

(a) D-glucose + O2 glucose oxidase D-glucono--lacton + H2O2

Reducing agents, e.g ascorbic acid, bilirubin, gluthathione and certain drugs may interfere with the determination. The method is not suitable for the determination of the blood glucose level in urine.

(b) glucose + ATP hexokinase glucose-6-P + ADP

glucose-6-P + NADP glucose-6-P-dehydrogenase 6-phosphogluconate + NADPH + H+

The reduced NADP coenzyme is determined at 340 nm.

Besides photometry, electrochemical methods, wih the use of biosensors, may be applied to determine blood glucose levels.

Amperometric glucometers measure the current on the surface of a working electrode due to the chemical reaction, as a function of the working electrode potential. Several factors like temperature, hematocrit, drugs, etc., may interfere with the measurement.

The amount of substance involved in the electrode reaction can be determined by means of Faradays law: the amount of substance released is proportional to the total electric charge passed through the cell in coulometric glucometers:

28

FnQMm

m = the mass of substance liberated at an electrode (g) M = the molar mass of the substance (g/mol) n = is the valency number of the ions of the substance (electrons transferred per ion) F = 96,500 (C/mol), the Faraday constant Q = the total electric charge passed through the cell (C)

The measurement is precise only if the system detects the electrons released during the redox reaction of the analyte. The interfering reactions must therefore be blocked. Coulometric analysis is slightly influenced by environmental factors.

The reactions on the biosensors:

D-glucose + O2 glucose oxidase D-glkono--lactone + H2O2

H2O2 platinum anode 2 H+ + O2 + 2 e-

The Beer-Lambert law describes the relationship between the concentration (c) and the absorbance (A) of the sample; it is valid only for dilute solutions:

clTI

IA o 1loglog

Transmittance is the ratio of the transmitted light and the total incident light, usually expressed as a percentage. It is not often used in practice because its graph is hyperbolic.

oIIT

A = absorbance Io = intensity of the incident light I = intensity of the transmitted light T = transmittance = molar absorbance (absorbance of a 1 mol/dm3 concentration solution if l=1 cm ) l = path length c = concentration of the sample (mol/dm3)

29

The analysis of a single compound in a sample is most often required. The absorption maximum of the molecule should first be determined, after which the absorbances of the calibration solutions are measured. The absorbance is plotted as a function of the concentration and the concentration of the sample can then be calculated on the basis of the fitted straight line.

Multiple components in the same sample can also be analyzed. The choice of the appropriate wavelength is critical in this case. The best choice is a wavelength where only one of the molecules has an absorption maximum and the absorbances of the other molecules are close to zero.

The absorbances of all the components in a sample are added:

A = Acell + Asolvent + Areagents + Aunknown sample A = Acell + Asolvent + Areagents

A = blank solution Aunknown sample = A A

The absorbance of the blank solution should be used to set the scale of the spectrophotometer to zero because there are colored reagents such as yellow iron(III) chloride (FeCl3) solution.

Double-beam spectrophotometers are used during the practicals of pharmaceutical analysis. A schematic diagram of the instrument is as follows:

Double-beam spectrophotometer I.

A tungsten lamp is used as a light source in the VIS range. The lamp contains a straight filament. The emitted electromagnetic waves cross the wall of the lamp perpendicularly. Light sources in the UV range are the mercury-vapor lamp, the hydrogen lamp and the deuterium lamp. The deuterium lamps used in modern spectrophotometers cover the UV range completely. The mercury-vapor lamp is official in Ph.Hg.VIII. The disadvantage of the mercury-vapor lamp relative to the deuterium lamp is that it has an energy minimum at ~200 nm and it cannot be used in this wavelength region. The lamps used in spectrophotometry are filled with special gas. Because of the high pressure inside them, the glass can cause serious injuries if they are broken so they cannot be disposed of as communal waste. Spectrophotometers should be turned on 10-15 minutes before use.

Prisms, half-prisms or diffraction gratings are used as monochromators in spectrophotometers.

Light source

Mono- chromator

Sample cell

Reference cell

Detector

30

Double-beam spectrophotometer II.

Photocells that are especially sensitive for the electromagnetic radiation in the UV, VIS and near IR range can be used as detectors in spectrophotometers. Incident photons excite electrons and these free electrons fly from the cathode toward the anode when electromagnetic waves reach the surface of the photocell. The photocurrent depends on the intensity and wavelength of the exciting electromagnetic radiation. Semiconductors are more effective than photocells and they operate in a broad wavelength region. CCD (charge-coupled device) detectors are combined with photodiodes that transform light into an electronic signal. CCDs comprise major technology in digital imaging such as in photography. In photodiode array detectors, hundreds or thousands (e.g. 256, 512, 1024 or 2048) of photodiodes are used. Photodiode array detectors can be used in nanometer resolution.

Light source

Sample cell

V-shaped mirror

Beam splitter

Detector

Reference cell

Aperture

31

Spectroscopic methods can be distinguished by the energy of the electromagnetic radiation:

Name Wavelength Effect Practical use

Gamma-rays 0.5-10 pm Excitation of nuclei Material sciences, synchrotrons.

X-rays 0.01-10 nm Excitation of inner shell electrons

Structure determination: X-ray diffraction

Diagnostics

UV light 10-380 nm Excitation of outer shell electrons Analytical chemistry: spectrophotometry.

VIS light 380-780 nm Excitation of outer shell electrons Analytical chemistry: spectrophotometry.

Near-IR 780-2500 nm Excitation of vibrational and rotational states of molecules

Near-IR spectroscopy:

Quality control, identification of products based on pigment dyes

IR radiation 2.5-300 m Excitation of vibrational and rotational states of molecules IR spectroscopy: identification tests

Microwaves 0.3 mm - 1 m Electron spin excitation of spin transition, excitation of rotational states of molecules

ESR = Electron spin resonance spectroscopy

Radiowaves 1-300 m Excitation of nuclear spins NMR = Nuclear magnetic resonance spectroscopy: structure and qualitative analysis

32

33

PULVIS CHINACISALIS CUM VITAMINO C (PULV. CHINACISAL. C. VIT. C)

ANTIPYRETICUM. ANALGETICUM.

Components: Chinini sulfas 0.15 g

Acidum ascorbicum 1.50 g

Acidum acetylsalicylicum 6.00 g

For 10 doses of divided powder

Background:

The acetyl group in acetylsalicylic acid can be removed by alkaline hydrolysis. The hydrolysis is faster at higher temperature.

34

In acidic media, salicylic acid forms a violet complex with Fe3+ that can be analyzed in the visible range.

I. Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of acetylsalicylic acid:

Dissolve 0.1000 g of acetylsalicylic acid R (pure reference material) weighed with analytical accuracy in 10 ml alcohol and then add 1.25 ml of freshly prepared 10% aqueous KOH solution. Five min later, add 0.25 ml 25% HCl to the solution and then dilute it to 100.0 ml with water in a volumetric flask. The acetylsalicylic acid concentration of this stock solution is 1 mg/ml or 1000 g/ml. Use this stock solution to make a 10-fold diluted solution A: mix 10.0 ml of the stock solution with 5.0 ml of 1% FeCl3 solution made with 0.1 M HCl (solution I) and dilute it to 100.0 ml with water in a volumetric flask. The concentration of solution A is 100 g/ml. Use solution A to prepare the solutions needed for the calibration curve. Make 20, 40, 60 and 80 g/ml solutions in 25-ml volumetric flasks. Measure the appropriate amounts from solution A into 25-ml flasks and make up to volume with the reference FeCl3 solution (solution II: 5.00 ml of 1% FeCl3 made with 0.1 M HCl and diluted to 100.0 ml with water) to keep the FeCl3 concentration constant (0.05%). The 100 g/ml solution is solution A itself. (If the weight of salicylic acid is different from 0.1000 g, the concentrations will be different, and that must be taken into account during the calculation.)

Amount of solution A to prepare: Absorbance

20 g/ml solution ml

40 g/ml solution ml

60 g/ml solution ml

80 g/ml solution ml

Determine the absorption maximum of acetylsalicylic acid according to the manual of the spectrophotometer between wavelengths of 500 and 600 nm. (The 60 g/ml calibration solution should be used.) Measure the absorbances of the calibration solutions at the absorption maximum of the acetylsalicylic acid. The spectrophotometer draws the calibration curve; check and record the r2 value. Preparation of the sample for determination of its acetylsalicylic acid concentration:

Dissolve 80 mg of substance in 10.0 ml of alcohol then add 1.25 ml of freshly prepared 10% KOH solution. Five min later, add 0.25 ml of 25% HCl and then dilute it up to 100.0 ml with water. Mix 10.0 ml of this solution with 5.0 ml of 1% FeCl3 solution made with 0.1 M HCl (solution I) and dilute to 100.0 ml with water.

Calculate the acetylsalicylic acid content of the powder, using the concentration calculated by the spectrophotometer on the basis of the calibration curve.

Determine the specific absorbance of the acetylsalicylic acid too.

35

Background: The concentrations of multiple components in a sample can be analyzed by spectro-

photometry. Measurements can be made when a wavelength can be found where one compound has an absorption maximum and the absorbances of the other compounds are zero. Appropriate wavelengths can be found for two-component samples, but the probability decreases when three- or multiple-component samples are to be analyzed. The amounts of both acetylsalicylic acid and quinine sulfate can be determined by spectrophotometry in Pulivis chinacisalis. Acetylsalicylic acid absorbs UV light, but its absorption maximum is shifted toward the VIS range (bathochromic effect) as a result of complex formation, and therefore quinine sulfate does not influence the measurement.

The absorbance changes rapidly if a peak is sharp, whereas the change is not so dramatic

if peak is broad. The shape of the absorption maximum peak should be considered during the measurement. Choosing a broad peak is more appropriate, as indicated in the figure above. The measurement wavelength chosen for the analysis is closer to the absorption maximum in the case of a broad peak.

II. Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of quinine sulfate:

Dissolve 50.0 mg of accurately pure quinine sulfate R weighed with analytical accuracy with gentle heating in a mixture of 1.0 ml 0.05 M H2SO4 and 10.0 ml of alcohol. Cool the solution to room temperature and dilute it to 50.0 ml with water. The quinine sulfate concentration of this solution is 1.0 mg/ml. Prepare a 10-fold dilution in a 50.00-ml volumeric flask with the solution that contains 2.50 ml of 0.05 M H2SO4 and 25.0 ml of alcohol per 250.00 ml. The concentration of the 10-fold diluted solution is 100 g/ml. (If the weight of quinine sulfate is different, the concentration will be different and that must be taken into account.) Use

36

the 100 g/ml solution to make the following calibration solutions in 25.0-ml volumeric flasks: 10.0, 20.0, 30.0, 40.0 and 50.0 g/ml. Use the above-mentioned acidic alcoholic solution to make the dilutions.

Amount of 100 g/ml solution to prepare: Absorbance

10 g/ml solution ml

20 g/ml solution ml

30 g/ml solution ml

40 g/ml solution ml

50 g/ml solution ml

Use the 30 g/ml calibration solution to determine the absorption maximum of quinine sulfate between wavelengths of 300 and 400 nm according to the manual of the spectrophotometer. When the absorption maxima are known, set the spectrophotometer to the longer wavelength and determine the absorbances of the calibration solutions. The reference solution is the acidic alcoholic solution used for the dilutions.

The spectrophotometer draws the calibration curve; check and record the r2 value.

Preparation of the sample for determination of its quinine sulfate concentration: Dissolve 0.1500 g of sample weighed with analytical accuracy with gentle heating in

the mixture of 1.0 ml of 0.05 M of H2SO4 and 10.0 ml of alcohol. Cool the solution to room temperature and dilute it to 100.0 ml with water. Measure the absorbance of the sample.

Calculate the quinine sulfate content of the powder, using the concentration calculated by the spectrophotometer on the basis of the calibration curve.

Determine the specific absorbance of the quinine sulfate too.

N.B. The powder is made only for the pharmaceutical analysis practicals. The acetylsalicylic acid and quinine sulfate contents may be different from those in the original formulation.

37

TABLETTA ASPIRINI 500 (ASPIRIN TABLET 500)

ANTIPYRETICUM. ANALGETICUM.

Composition: Acidum acetilsalycilicum 500 mg

Cellulosum (pulvis) qu. s.

Amylum maydis qu. s.

for each tablet

Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of acetylsalicylic acid:

Dissolve 0.1000 g of acetylsalicylic acid R weighed with analytical accuracy in 10 ml of alcohol and add 1.25 ml of freshly prepared 10% KOH. Five min later, add 0.25 ml 25% HCl to the solution and then dilute it to 100.0 ml with water in a volumetric flask. The acetylsalicylic acid concentration of this stock solution is 1 mg/ml or 1000 g/ml. Use this stock solution to make a 10-fold diluted solution A: mix 10.0 ml of the stock solution with 5.0 ml of 1% FeCl3 made with 0.1 M HCl (solution I) and dilute it to 100.0 ml with water in a volumetric flask. The concentration of solution A is 100 g/ml. Use solution A to prepare the solutions of the calibration curve. Make 20, 40, 60 and 80 g/ml solutions in 25-ml volumetric flasks. Measure the appropriate amounts from solution A into 25-ml flasks and make them up to volume with the reference FeCl3 solution (solution II: 5.0 ml of 1% FeCl3 made with 0.1 M HCl and diluted to 100.0 ml with water) to keep the FeCl3 concentration constant (0.05%). The 100 g/ml solution will be solution A itself. (If the weight of the salicylic acid differs from 0.1000 g, the concentrations will differ and this must be taken into account during the calculation.)

Amount of solution A to prepare: Absorbance

20 g/ml solution ml

40 g/ml solution ml

60 g/ml solution ml

80 g/ml solution ml

Determine the absorption maximum of acetylsalicylic acid between 500 and 600 nm according to the manual of the spectrophotometer. (The 60 g/ml calibration solution should be used.) Measure the absorbances of the calibration solutions at the absorption maximum of the acetylsalicylic acid.

The spectrophotometer draws the calibration curve; check and record the r2 value.

Preparation of the sample for determination of its acetylsalicylic acid concentration: Weigh an intact pill with analytical accuracy into a 100-150-ml beaker and add 20.0 ml

of alcohol, and then add 10.0 ml freshly prepared 10% KOH solution. Cover the beaker with a watch glass and boil the sample for 5 min. Cool the sample to room temperature, add 1.0 ml of 25% HCl and dilute make the volume up to a 100.0 ml with water in a volumetric flask. Mix

38

1.00 ml of this solution with 5.0 ml of 1% FeCl3 made with 0.1 M HCl (solution I) and dilute it to 100.0 ml with water.

Calculate the acetylsalicylic acid content of the powder, using the concentration calculated by the spectrophotometer on the basis of the calibration curve.

Determine the specific absorbance of acetylsalicylic acid too.

39

SUPPOSITORIUM PARACETAMOLI 500 MG (SUPP. PARACET. 500 MG)

ANTIPYRETICUM. ANALGETICUM.

Composition: Paracetamolum 3.00 g

Adeps solidus 50 : Butyrum cacao 7 : 3 qu. s.

for 6 suppositories

Background: The following factss should be borne in mind when measurements are made in the UV

range.

Solvents that absorb UV light cannot be used, e.g. benzene or toluene.

Some solvents can absorb UV light at shorter wavelengths (e.g. ~180 nm), which disturbs the analysis. In measurements at 200-210 nm, the absorption is independent of the structure of the the molecule. The absorption is slightly dependent on the structure at ~250 nm, and therefore it is very important to note

the absorption maxima. The absorption peak is definitely not specific for the analyte when the absorption maximum is at ~200 nm and the absorbance is higher than 1 (A>1). The measurement should be performed by setting the spectrophotometer to the absorption maximum of the second peak, e.g. at 280 nm in the figure above.

40

Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of paracetamol:

Weigh 0.0500 g of paracetamol R with analytical accuracy, dissolve it in 5 ml of chloroform and add methanol to make it up to volume in a 50-ml volumetric flask (solution A). Dilute 1 ml of solution A with methanol to 50 ml (solution B) and use this solution to make 2.0, 4.0, 6.0, 8.0 and 10.0 g/ml calibration solutions in 25-ml volumeric flasks.

Amount of solution B to prepare Absorbance

2 g/ml solution ml

4 g/ml solution ml

6 g/ml solution ml

8 g/ml solution ml

10 g/ml solution ml

Determine the absorption maximum of paracetamol by using the 6.0 g/ml calibration solution between 200 and 300 nm according to the manual of the spectrophotometer. When the absorption maxima are known, set the spectrophotometer to the longer wavelength and determine the absorbances of the calibration solutions. The reference solution is the acidic alcoholic solution used for the dilutions.

The spectrophotometer draws the calibration curve; check and record the r2 value.

Preparation of the sample for determination of its paracetamol concentration: Weigh 0.3600 g of suppository, which contains ~60 mg of paracetamol, melt it on a hot

plate and mix it with 10.0 ml of chloroform. Add methanol to make it up to volume in a 100-ml volumetric flask. Wait 20-30 min and then dilute 0.5 ml of the clear supernatant up to 50.0 ml with methanol. Measure the sample solution at the same wavelength and determine its concentration by using the calibration curve.

Calculate the paracetamol content of a suppository of average weight.

Calculate the specific absorbance of paracetamol.

41

SPARSORIUM ANTISUDORICUM (SPARS. ANTISUDOR.)

DERMATOLOGICUM. ANTISUDORICUM. ADSTRINGENS. DESODORANS.

Composition: Hexachlorophenum 0.60 g

Acidum salicylicum 1.80 g

Alumen 6.00 g

Magnesii subcarbonas 20.00 g

Zinci oxydum 20.00 g

Talcum 20.00 g

Background:

Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of salicylic acid:

Weigh 0.0500 g of salicylic acid R with analytical accuracy into a small beaker and dissolve it in a few ml of methanol and then wash the solution into a 50.0-ml volumetric flask with methanol and make it up to volume with the same solvent. Dilute 5 ml of the previous solution with solution II (5.00 ml of FeCl3 solution made with 1% 0.1 M HCl and come up to volume 100.0 ml with water) in another 50.0-ml volumeric flask. The salicylic acid concentration of this solution is 100 g/ml. (If the weight of the salicylic acid is different, the concentration will be different.) Use this solution to prepare 10, 20, 30, 40 and 50 g/ml calibration solutions in 25.0-ml volumetric flasks, using the same solution II for the dilutions.

42

Amount of 100 g/ml solution to prepare Absorbance

10 g/ml solution ml

20 g/ml solution ml

30 g/ml solution ml

40 g/ml solution ml

50 g/ml solution ml

During the spectrophotometric measurements, use solution II as reference solution. Use the 30 g/ml calibration solution to record the absorption curve according to the manual of the spectrophotometer and determine the absorption maximum of the curve. Measure the absorbances of the calibration solutions

The sSpectrophotometer draws the calibration curve; check and record the r2 value.

Preparation of the sample for determination of its salicylic acid concentration: Weigh 0.60 g of material, mix it vigorously with methanol and make the final volume

up to 50.0 ml with the same solvent. Filter the suspension, throw away the first 10 ml and continue the filtration through the same filter paper into a clean beaker. Take 5 ml of the second filtrate to make a 10-fold diluted solution, using solution II (5.00 ml FeCl3 made with 1% 0.1 M HCl per and come up to volume 100.0 ml with water). Measure the sample solution an the same wavelength and determine its concentration by using the calibration curve.

Calculate the salicylic acid content of the sample.

Determine the specific absorbance of salicylic acid.

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SOLUTIO METRONIDAZOLI (SOL. METRONIDAZ.)

ANTIAPHTOSUM.

Composition: Metronidazoleum 0.30 g

Lidocainum 0.05 g

Glycerinum 20.00 g

Ethanolum 70% ad 30.00 g

Preparation of the solutions of the calibration curve, determination of the absorption maximum of metronidazole:

Weigh 0.0500 g of metronidazole R into a small beaker, dissolve it in methanol and wash the solution into a 50.0-ml volumeric flask (solution A). Prepare a 10-fold diluted solution: transfer 5.0 ml of solution A into a 50-ml volumetric flask and make it up to volume with methanol (solution B). The concentration of solution B is 0.100 mg/ml (100 g/ml). (If the weight of the metronidazole differs from 0.0500 g, the concentration of the solution will differ and this should be taken into account.) Use solution B to make 2.0, 5.0, 10.0, 15.0 and 20.0 g/ml calibrating solutions in 25-ml volumeric flasks.

Amount of solution B to prepare Absorbance

2 g/ml solution ml

5 g/ml solution ml

10 g/ml solution ml

15 g/ml solution ml

20 g/ml solution ml

Determine the absorption maximum of metronidazole using the 10.0 g/ml calibration solution, scanning the region between 300 and 400 nm according to the manual of the spectrophotometer. Measure the absorbances of the calibration solutions at the absorption maximum of metronidazole.

The spectrophotometer draws the calibration curve; check and record the r2 value.

Preparation of the sample for determination of its metronidazole concentration: Weigh 0.5000 g of sample and dilute it to 50.0 ml with methanol. Transfer 5.00 ml of

the solution to a 50.0-ml volumetric flask and make it up to volume with methanol. Measure the absorbance of the sample solution at the same wavelength and determine its concentration by using the calibration curve.

Calculate the metronidazole content of the sample.

Determine the specific absorbance of metronidazole.

44

PULVIS CHOLAGOGUS (PULV. CHOLAGOG.)

CHOLAGOGUM. SPASMOLYTICUM.

Composition: Homatropini methylbromidum 0.03 g

Phenolphthaleinum 0.50 g

Papaverini hydrochloridum 0.60 g

Acidum dehydrocholicum 2.50 g

Natrii salicylas 2.50 g

Natrii bensoas 2.50 g

For 10 doses of powder

Background:

Phenolphthalein turns colorless in acidic solutions and is pink in basic solutions. If the concentration of the indicator is particularly strong, it can appear purple. In very strongly basic solutions, the pink color of phenolphthalein fades and it becomes colorless again. Phenolphthalein is often used as an indicator in acidbase titrations. Its earlier use as a as laxative was stopped recently because its long-term application can cause malignancy. Phenolphthalein absorbs UV light in both its protonated and its deprotonated forms, due to its three aromatic rings. In the deprotonated form (slightly alkaline pH), the delocalization extends to the entire molecule. The excitation energy is decreased by the more extensive delocalization, and longer wavelengths (VIS) can therefore be used for its quantitative analysis. Phenolphthalein absorbs green light and transmits the complementary color pink (the one we see).

The pH of the sample is shifted into the alkaline range by Na2CO3 during the analysis of phenolphthalein.

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Barbiturates exhibit a similar phenomenon. The heterocyclic system of barbituric acid can be is stabilized in the oxo form at acidic pH, while the enol form is stable at alkaline pH. Barbituric

acid shows both enol-oxo and lactam-lactim tautomerism. Barbiturates (5,5-disubstituted derivatives) shows only lactam-lactim tautomerism.

Buffering of the sample solutions is therefore very important. However, possible decomposition reactions, e.g. acidic or alkalic hydrolysis must be avoided.

The absorption spectrum changes as a function of pH. The molecule has different absorption maxima and minima at different pH values. The wavelength at which the absorbance is independent of the pH is called the isobestic point. This fact should be remembered during measurements in clinical chemistry. If the pH is set wrongly, the result will be false. Isobestic points of reference materials can be used to calibrate the spectrophotometer.

Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of phenolphthalein:

Weigh 0.0060 g of phenolphthalein R and dissolve it in methanol using a 100-ml volumetric flask (solution A). Take 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml portions of solution A and dilute them with 0.1 M Na2CO3 to 50.0 ml.

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Concentration of the calibration solution

Amount of solution A Absorbance

g/ml solution 1 ml

g/ml solution 2 ml

g/ml solution 3 ml

g/ml solution 4 ml

g/ml solution 5 ml

The reference solution is 0.1 M Na2CO3. Determine the absorption maximum of phenolphthalein by using the 3rd calibration solution, scanning the region between 500 and 700 nm according to the manual of the spectrophotometer. Measure the absorbances of the calibration solutions at the absorption maximum of phenolphthalein.

The spectrophotometer draws the calibration curve; check and record the r2 value.

Preparation of the sample for determination of its phenolphthalein concentration: Weigh 0.0300 g of sample and dissolve it in methanol in a 100.0-ml volumetric flask.

Prepare two dilutions: take 4.0 ml and 8.0 ml in different 50.0-ml volumetric flasks and dilute them with 0.1 M Na2CO3. Measure the absorbances of the sample solutions at the same wavelength and determine their concentrations by using the calibration curve.

Calculate the mass percent phenolphthalein content of the sample with four-decimal accuracy.

Determine the specific absorbance of phenolphthalein.

47

DETERMINATION OF PROTEIN CONCENTRATION WITH THE BIURET REAGENT

The proteins are macromolecules that have a large variety of functions and are essential for human life. They are built up from 20 amino acids, which are connected through peptide (amide) bonds. 50% of the dry material of a cell is protein. The total protein concentration in the human body is ~60-80 g/l.

Background:

Several methods are known for the determination of protein concentration. Some methods make use of the properties of peptide bonds or an amino acid side chains that react with certain reagents or dyes forming colored complexes. One well-known method is called the biuret reaction. The reagent, which contains Cu2+ ions in alkaline medium, forms bluish-violet complexes with peptide bonds. At least two peptide bonds are necessary for the reaction. Proteins fulfil this requirement in all cases. The following complex is formed:

The biuret reagent contains CuSO4, NaOH, K,Na-tartrate and KI. (The K,Na-tartrate is necessary to keep Cu2+ in solution in the alkaline medium. KI stabilizes the reagent, so that the shelf life becomes longer.)

Preparation of the solutions required to produce the calibration curve, and determination of the absorption maximum of BSA:

20 mg/ml of protein stock solution made from BSA (bovine serum albumin) is used for the calibration solutions. Pipette 700 l of biuret reagent into each of the test tubes and then add the following components according to the table:

48

Number of test tube

Dist. water (ml)

BSA stock solution

(ml)

Concentration (mg/ml) Absorbance

1 0.3 0

2 0.3 0

3 0.275 0.025

4 0.250 0.05

5 0.225 0.075

6 0.200 0.1

7 0.175 0.125

Incubate the reaction mixture tubes in a 35-40 C water bath for 10 min.

Calculate the concentrations of the calibration solutions (column 4). 10 min later, use tube 5 to determine the absorption maximum in the range 500-700 nm. The reference solutions are in tube 1 and tube 2, which do not contain BSA. Measure the absorbances of the calibration solutions at the absorption maximum of BSA.

The spectrophotometer draws the calibration curve; check and record the r2 value.

Preparation of the sample and determination of its protein concentration: Take aliquots of 75 l-t and 125 l from the unknown sample into separate test tubes.

Dilute them up to 300 l with distilled water and then add 700 l of biuret reagent. The measurement is more precise if the unknown sample is washed into a mixture of distilled water and the biuret reagent. It is recommended to pipette the reagent first, then add distilled water and finally wash the required amount of sample into the reaction mixture.

Measure the absorbances of the samples.

Calculate the protein concentration and give the final result in g/l with two-decimal accuray. Average the results on the individual samples.

49

ATOMIC ABSORPTION SPECTROMETRY

Methods of sample preparation The following methods are used in the pharmaceutical industry to prepare samples for

atomic absorption spectrometric measurements:

dry ashing

wet decomposition o in open vessels at atmospheric pressure o in closed Teflon beakers with steel covers, which allows the use of high

pressure

o with microwave radiation in closed plastic vessels: microwave-assisted sample preparation

Microwave-assisted sample preparation will be described in detail here as this technique will be used during the practicals.

The microwave-assisted wet decomposition of samples is becoming widely used in the pharmaceutical industry. This technique is suitable for the preparation of both solid and liquid samples. The procedure is carried out in a microwave oven. The inner wall of the oven is made of acid-proof steel covered with Teflon. The samples are fixed on a rotating plate in the microwave oven. These instruments have built-in pressure control units; the latest ones can also monitor the temperature. The sample vessels are made of acid-, temperature- and pressure-proof plastic.

The samples are weighed directly into the plastic vessels. 23 ml of liquid or 0.20.5 g solid sample is usually weighed for decomposition and concentrated H2SO4, HCl, HNO3, and/or H2O2 is then added. The membrane in the pressure valve should be checked. The vessels are then thightly sealed with the appropriate tool (torque wrench) and placed into a Kevlar jacket. The pressure valve opens only if overpressure develops. If the membranes are damaged, acid vapor is released and the samples are not suitable for pharmaceutical analysis, but the vessels remain unaffected. After the microwave treatment, the samples should be cooled to room temperature. During the process the carbon and hydrogen present in the organic components of the samples are turned into water and CO2 by the end of the wet decomposition; the inorganic compounds are dissolved in water or in the applied acids. The cooled samples are transferred into volumetric flasks for the analytical measurements, the vessels must also be rinsed out. Water molecules and other dipolar solvents are affected by the microwave treatment, making them oscillate with high frequency. This phenomenon heats the samples rapidly. The advantages of the microwave-assisted wet decomposition are:

only small amount of reagents are necessary

the closed system keeps the corrosive vapors inside the vessel

only a short time is required for sample preparation

Atomic spectroscopy Each element has a unique atomic electron configuration, and therefore a characteristic

atomic spectrum. As each spectral line is sharp, the spectral lines of different elements do not or only rarely overlap.

50

Most elements vaporize at elevated temperatures and the molecules then dissociate into free atoms. The first step in atomic spectroscopy is the conversion of molecules into free atoms in the gaseous phase. If the atoms are then converted into an excited phase, they emit characteristic radiation when they relax. Analysis of the emitted radiation provides analytical information. These are the basic principles of the atomic spectroscopy. In other cases, the vaporization and atomization are not accompanied by excitation of the atoms, and the basis of the analytical measurements is the absorption of electromagnetic radiation by specific light sources. This latter technique is atomic absorption spectroscopy (AAS).

Both methods are very effective, with detection limits in the ppm (parts per million) range, and the inductively coupled plasma (ICP) method (see below) is even suitable for the analysis of ppb (parts per billion) concentrations. As these are sensitive methods, problems arise from the aspect of accuracy. The methods are less accurate than, for instance, spectrophotometry.

Atomic absorption spectroscopy AAS is useful for the analysis of more than 70 metals and semimetallic elements. Only

a small amount of sample is usually used for the analysis.

In the atomization unit of the instrument, the electrons of the atoms are promoted to higher orbitals by absorbing definite quantities of energy (e.g. in the cases of certain electromagnetic waves). The energy required for the excitation is specific for the electron transition of the element; a unique wavelength is specific for a certain element. The method is therefore selective.

The sample is transformed into the gaseous phase and atomized during the analysis. Light passed through the atomized sample and is absorbed by the sample. The rate of absorption is directly proportional to the concentration of the analyte. Before the analysis, calibration solutions with known concentration must be prepared and the instrument must be calibrated.

The major units of the instrument are the light source, the sample introduction unit, the atomization unit, the monochromator and the detector. The various spectrometers may differ in the atomization unit and the sample introduction unit. The methods available for sample analysis are flame, electrothermal and cold vapor-hydride atomization.

Hollow cathode lamps are most commonly used as light sources in AAS. High voltage (100-400 V) is applied across the anode and cathode, resulting in ionization of the gas inside the sealed lamp. The emitted radiation is characteristic of this fill gas and the material of the cathode. The fill gas is usually Ar or Ne, depending on the analyte element. The spectral lines of the fill gas should not overlap with the analyte element. The cathode is made of the analyte element or covered with it. The electrons are accelerated between the cathode and the anode and ionize the fill gas, and the gas ions are therefore accelerated toward the cathode, causing the sputtering of atoms from the cathode. The atoms of the cathode become excited upon collisions with the fill gas and emit light as they fall back to the ground state. The emitted light is characteristic of the element. The intensity of the hollow cathode lamp can be influenced by the current.

The atomization unit produces free atoms capable of the absorption of electromagnetic radiation. The atomization is either chemical or thermal; the latter is used for all elements except Hg. Three methods are available for thermal atomization: flame, electrothermal furnace (graphite tube) or radiofrequency-induced plasma. The sample is sprayed through the nebulizer into the flame, where the solvent is first evaporated rapidly. The molecules are atomized due

51

to the high temperature. A large amount of sample is usually required to obtain a continuos signal during the measurement.

Premixed laminal gases are used in flame atomization. This type of flame is stable, the background is low and the transformations of the sample are separated throughout the height of the flame. Two types of gas mixes are used in most cases: acetyleneair and acetyleneN2O. Their temperatures differ; one is ~2000 K and is capable of atomizing most elements. The analytes should be volatile, e.g. Cl-, F- or H- salts. Other elements may form stable oxides at high temperatures, (e.g. Al, Ti, V, and W) and higher temperatures are necessary for their atomization; this is provided by acetyleneN2O (~3000 K). The atomization may be blocked by oxygen either in the flame or in the sample, resulting in the formation of oxides that dissociate only at very high temperatures. A reducing flame is therefore used, when the amount of fuel gas is higher than the oxidant gas.

Flame atomization requires a large amount of sample, whereas in the case of a graphite furnace several l of sample is sufficient and solid samples can also be analysed. The graphite furnace is heated up gradually to the appropriate temperature. The sample is first dried at ~400 K, and is next heated to 1000-1500 K to ash all the organic molecules. Finally, at 3000 K, the sample evaporates and atomizes. The gradual heating is necessary to avoid smoke in the light path. The graphite furnace serves as a cell. Graphite is an extremely good reducing agent at 2800-3200 K and therefore blocks the formation of metal oxides. The efficiency of the atomization is improved too. The sensitivity of this technique is 23 orders of magnitude higher than that of flame atomization. The method is sutable for the analysis of volatile samples.

The emission spectra of hollow cathode lamps consist of multiple spectral lines due to the elements present: the material of the cathode, the fill gas (He or Ne) and the contaminants, if any. A monochromator system is therefore necessary for the selection of