u · 3.6 Signal of 2 /xg citrate at various wavelengths 56 3.7 Data for the calibration of citrate 58 3.8 Precision test of the proposed method 60 3.9 Description of pharmaceutical

u “

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l H I k J ^ "

%、"停i^: / .

•'• J (：,.： z

DETERMINATION OF CITRATE, CAMPHOR AND MENTHOL BY

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

By

TSOI Yeung-pang

( 蔡仰鵬）

A thesis submitted in partial fulfilment of

the requirement for the degree of

Master of Philosophy in

The Chinese University of Hong Kong

1994

Thesis Committee : Dr. H. F. CHOW

DR. Glen K. C. HUI, Chairman

Dr. O. W. LAU

Prof. T. S. West, External Examiner

ACKNOWLEDGEMENTS

工 wish to express my deepest gratitude to my supervisor, Dr. 0. W. Lau, for her support, guidance and encouragement throughout the project, and for her invaluable advice given in the preparation of this thesis.

I am also indebted to Dr. C. S. Mok for his helpful discussion and technical advice throughout this project.

My thanks are also due to Mr. K.C. Cheng, Ms W.N. Ho, Ms. Y.T. Leung, Mr. K.L. So, Mr. M.C. Wong, Ms. Y.O. Lam, Mr. C.M. Chan, and other staffs of the Chemistry Department for their support and prompt assistance.

Department of Chemistry The Chinese University of Hong Kong June 1994

TSOI Yeung-pang

f 1

ABSTRACT

High Performance Liquid Chromatographic ( HPLC )

methods for the determination of organic compounds having

no special functional characteristic for detection by

ultra-violet ( UV ) or fluorescence have been developed and

illustrated by two examples. The first example is the

determination of citrate in pharmaceutical preparations by

indirect photometric detection. The second example

involves indirect conductometric detection for the

determination of camphor and menthol.

The first method applied ion-interaction

chromatography, employing an indirect method of detection,

where salicylate is used as a co-anion to an ion-

interaction reagent ( IIR ) , tetrabutylammonium ion, and

produces a constant background of UV absorption.

The citrate in the sample was separated and determined

quantitatively by isocratic reversed phase HPLC on a column

(25 cm X 4.6 mm ) of Adsorbosphere C18 ( 5 /Lim ) with an

aqueous solution containing tetrabutylammonium iodide (

0.12 mM ) and sodium salicylate ( 0.15 mM ) as the mobile

phase ( 1.8 mL / min. )• Detection at 242 nm vs. air with

the polarity of the integrator reversed. The calibration

graph was linear from 20 to 200 /ig / ml ) . Recoveries

ranged from 95 to 106% for 8 samples.

• • 11

The second method employed an indirect conductometric

detection in the quantitative determination of camphor and

menthol in pharmaceutical products using a Adsorbosphere

cyano column ( 25 cm X 4.6丽，5/m ) with 4 mM sodium

citrate in 30 % aqueous acetonitrile as the mobile phase (

1 ml / min. ) with the polarity of the integrator reversed.

The calibration graph was linear from 0.05 to 1 mg / ml for

both compounds. Recoveries ranged from 95 to 105% for

camphor and 97 to 100% for menthol from 10 samples.

• • « 111

TABLE OF CONTENTS

Page

• I. Acknowledgements i II. Abstract ii III. Table of contents iv IV. List of Tables and Figures v

Chapter 1. Introduction 1 1.1 Modes of chromatography 1.2 Objective of the present study

References

Chapter 2. Instrumentation and theory 8 2.1 Instrumentation of HPLC 2.2 Theory of liquid chromatography

References

Chapter 3. Determination of citrate in pharmaceutical 21 preparations by HPLC using indirect photometric detection

3.1 Introduction 3.2 Review of the analytical methods 3.3 Theory of detection 3.4 Experimental 3.5 Results and discussion 3.6 Conclusion

References

Chapter 4. Determination of camphor and menthol 74 by HPLC using indirect conductometric detection

4.1 Introduction 4.2 Review of the analytical methods 4.3 Theory of detection

(

4.4 Experimental 4.5 Results and discussion 4.6 Conclusion

References iv

LIST OF TABLES AND FIGURES

1. List of Tables : Page

3.1 Intensity of the light source at different

wavelength 43

3.2 Peak area of 2ng citrate at different

background settings 45

3.3 Effect of co-anions in the mobile phase on the

sensitivity and retention of citrate 48

3.4 The absorbance of TBAI and co-anions 49

3.5 Signal and retention at different TBAI and NaSa

concentrations in the mobile phase 54

3.6 Signal of 2 /xg citrate at various wavelengths 56

3.7 Data for the calibration of citrate 58

3.8 Precision test of the proposed method 60

3.9 Description of pharmaceutical preparation 61

3.10 Assay of content of citrate in drug sample 63

3.11 Recovery test for the proposed method 67

4.1 Effect of conducting species in the mobile phase

on the sensitivity and retention on camphor and

menthol 88

4.2 Concentration effect of background conducting

species on peak intensity and retention time 90

4.3 Concentration effect of acetonitrile on

retention time 92

4.4 Data for the calibration of camphor 93 4.5 Data for the calibration of menthol 95

V

4.6 Precision test of the proposed method 97

4.7 Description of pharmaceutical preparation 99

4.8 Assay of the contents of camphor and menthol in

drug sample 100

4.9 Results of recovery test for the proposed

method 103

2. List of Figures :

2.1 General instrumentation for a high performance

liquid chromatograph 8

2.2 Six-port sample injection valve 12

3.1 The paired-ion model 31

3.2 The dynamic ion-exchange model 31

3.3 The ion-interaction model 33

3.4 Retention of an oppositely charged sample 33

3.5 Intensity of the detector light source vs

wavelength 44

3.6 Signal to noise ratio of 2 ng citrate at

different background settings 47

3.7 Absorption spectrum of 0.12 mM tetrabutyl-

ammonium iodide 50

3.8 Absorption spectrum of mobile phase containing ,

0.12 mM tetrabutylammonium iodide and 0.15 luM

sodium salicylate 50

3.9 Absorption spectrum of mobile phase containing

0.12 itiM tetrabutylammonium iodide and 0.15 mM

vi

r.

sodium benzoate 51

3.10 Absorption spectrum of mobile phase containing

0.12 mM tetrabutylammonium iodide and 0.15 mM

potassium hydrogen phthalate 51

3.11 Calibration graph for the determination

of citrate 59

3.12a Chromatograms of sample No. 2 ( left ) and

sample No. 3 ( right ) 64 3.12b Chromatograms of sample No. 4 ( left ) and

sample No. 5 ( right ) 65

3.12c Chromatograms of sample No. 7 ( left ). and sample No. 8 ( right ) 66


of camphor 94


of menthol 96

4.3a Chromatograms of sample No. 4 ( left ) and


4.3b Chromatograms of sample No. 6 ( left ) and


•) J,

vii

CHAPTER 1

INTRODUCTION

Liquid chromatography is the generic name used to

describe any chromatographic procedure in which the mobile

phase is a liquid. High performance liquid chromatography

( H P L C ) is probably the single most used analytical

technique today, surpassing even gas chromatography in use

for the separation and analysis of mixtures. This is due

mainly to the extensive versatility of the technique which

results from the fact that both stationary phase and mobile

phase interactions may be utilised to alter the selectivity

of the system. Modern liquid chromatography has the

advantages that the columns are reusable, sample

introduction can be automated and detection and

quantitation can be achieved by the use of continuous flow

detectors. These features lead to improved accuracy and

precision of analysis.

1.1 MODES OF CHROMATOGRAPHY

The exact mode of chromatography operating in a

given application is determined principally by the nature

of the column stationary phase and the mobile phase. It

must be stressed that , while there may be one dominant

mechanism, the modes are not mutually exclusive. HPLC

separation methods can be classified as :

1

1. Adsorption chromatography；

2. Partition chromatography；

3. Ion-exchange chromatography；

4. Exclusion chromatography.

1.1.1 Adsorption chromatography

Adsorption chromatography was introduced by Tswett^ and

Day The separation is based on the selective adsorption

of solutes on the active sites on the surface of the

adsorbent such as silica and alumina. The active sites on

silica are hydroxy1 groups. The eluent systems are usually

non-polar solvents containing a small amount of polar

additive called polar modifier. When the sample is applied

to the column packing, polar molecules with polar

functional groups will be attracted towards the active

sites and will subsequently be displaced by the polar

modifier molecules and will pass down the column to be

readsorbed on new sites. More polar molecules will be

retained more strongly and hence eluted more slowly by the

column. This kind of chromatography is commonly applied in

the separation of isomer like propellants, vitamins and

alkaloids .3-5

1.1.2 Partition chromatography

Partition chromatography can be divided into liquid-

liquid partition chromatography and organo-bonded partition

2

chromatography. This method was developed by Martin and

Synge^ and in both modes of partition chromatography,

separation is achieved by partition of an organic solute

between a liquid mobile phase and an organic liquid

adsorbed on , or chemically bonded onto, a solid support.

Liquid-liquid partition chromatography is seldom used today

because of the problems of solvent stripping and limited

hydrolytic stability. This technique is displaced largely

by organo-bonded partition chromatography, which is, in

fact, a modified mode of the liquid-liquid partition

chromatography, where the liquid stationary phase is

chemically bonded or organo-bonded to an insoluble matrix.

This method is further divided into normal phase where the

stationary phase is more polar than the mobile phase and }

vice versa in the case of reverse phase partition

chromatography.

The selectivity in reversed phase chromatography is

controlled mainly by eluent effects and it is therefore

unnecessary to have an extensive range of packing

materials. With different mobile phases used, different

form of retention mechanisms will be resulted. The

technique found its use in the separation of plasticizer,

saccharide and fatty acids.^ -

Ion interaction chromatography belongs to this class

of chromatography which is a modified mode of reversed bond

phase chromatography having the ability of ion-exchange

3

chromatography.

1.1.3 Ion-exchange chromatography

Ion exchange chromatography is performed with packing

materials which contain ionic functional groups. These are

either acid groups such as sulphonic or carboxylic acids

for the separation of cations and amine or quaternary

ammonium basic groups for the separation of anions.

The original definition of ion chromatography

referred to an ion exchange separation with conductometric

detection making use of a suppressor column to remove the

background conductance of the eluent. The latest trend

however, moves away from the use of suppressor columns and

the definition has been considerably broadened to include

any high performance ion exchange separation which uses a

low capacity resin and eluents of low concentration. Ion

chromatography has been one of the fastest growing areas of

HPLC in recent years. Again, silica based materials have

proved popular but recently several manufacturers have

produced polymeric ion exchangers with low exchange

capacity specifically for ion chromatography. The main

advantage of these materials is their pH stability which

allows weakly acidic or basic species to be chromatographed

in their ionic form. They also show different selectivity

from the silica ion exchangers.^

4

1.1.4 Exclusion chromatography

Exclusion chromatography, also called gel permeation

chromatography ( GPC ) , utilizes the selective diffusion of

solute molecules within the solvent filled pores of a

three-dimensional lattice. Small molecules will permeate

the pores while large bulky molecules will be excluded.

Thus separation is achieved principally on the basis of

molecular weight and size, with larger molecules being

eluted from the column more quickly.^ It is commonly used in

the analysis of biological materials and polymer.

1.2 OBJECTIVE OF THE PRESENT STUDY

The objective of this work is to develop HPLC methods

for the determination of organic compounds, which do not

require the analytes to have specific structural

properties.

The work is divided into two parts. The first part of

this work is to develop an HPLC method for the

determination of citrate, which is an organic anion, by

using ion-interaction chromatography, which is usually used

for the determination of inorganic ions. Also, the

detection method used in this project is by UV detection, J

which is uncommon for citrate because it is UV transparent.

The detection method is based on the decrease in UV

absorption of the eluent when the analyte is eluted out and

5

the peak is recorded as a negative peak. This method is

usually called indirect photometric detection. Details of

this work will be shown in Chapter 3 of this thesis.

The second part of this work is to develop an HPLC

method for the determination of camphor and menthol, which

are neutral compounds without UV chromophores, and hence

are non-conducting and UV inactive, by using indirect

conductometric detection. Details of this work will be

shown in Chapter 4.

6

REFERENCES

1. M. Tswett, Trav, Soc• Nat, Warsowie, 1903, 14, 6

2. D.T. Day, Science, 1903, 14, 1007

3. “ J.O. Doali and A. A. Juhasz, Anal. Chem., 1976, 48,

1859

4. M.E. Evenson and B.L. Warren, Clin. Chem., 1976, 22,

851

5. M.G.M. De Ruyter and A.P. Deheenheer, Clin. Chem.,

1976, 22, 1593

6. A.J.P. Martin and R.L.M. Synge, Biochem. J., 1941,

35, 1358-1368

7. N.E. Hoffmann and J.C. Liao, Anal• Chem., 1976, 48,

1104

8. M.T. Gilbert, High Performance Liquid Chromatography,

lOP Publishing Limited, Bristol, 1987, p.1818

9. A. Braithwaite and F.J. Smith, Chromatographic

Methods, Chapman and Hall, New York, 1985, p.414 I

7

CHAPTER 2

INSTRUMENTATION AND THEORY

2.1 OVERVIEW OF HPLC INSTRUMENTATION

The high performance liquid chromatograph comprises of

the following components' ( Figure 2.1 ) : (1) a solvent

reservoir for the mobile phase; (2) a solvent pump,

equipped with a damping unit if a pulasating action

results, to force the mobile phase through the

chromatographic system; (3) a sampling or injection devices

to introduce the sample into the column; (4) the saparation

column; and (5) a detector with recorder readout or other

data handling device.

GRADIENT DEVICE

I Thtrmojtated

r INJECTOR CHR0MATCX5RAM

B L r ° [ f i f^ESEfWOm PUMP / / RECORDER

# @ COLUMN DETECTOR

Figure 2.1 General instrumentation for a high performance liquid chromatograph

8

2.1.1 Mobile phase

The mobile phase employed for HPLC separations may

comprise water, aqueous buffer solutions, organic solvents

such as methanol and acetonitrile or a mixture of the

above. All solvents should be of high spectroscopic

purity, dust free, and should be degassed before use.

They should also, if UV detection is being employed, be

transparent to the wavelength for detection.

2.1.2 Solvent delivery system̂ ''*

‘Several features of the solvent delivery system must

be considered: precise delivery of solvent over a

relatively broad range； maximum pressure attainable; ,

compatibility with other components in the HPLC system;

compatibility with the choice of solvents; and noise level

in the detector resulting from any pulsations. The final

choice of pump will be interwoven with the type of

separation column, the detector employed, whether isocratic

or gradient elution is to be performed, the minimum

detectability limit desired, precision in quantitation, and

the cost of the packaged chromatograph.

Three main types of pumps are used in HPLC to propel

the liquid mobile phase through the system: (1) the

constant pressure type； (2) the syringe design, which

delivers a constant non-pulsating flow; and (3) the

9

reciprocating piston pump, which may be of the single, dual

or triple head design.

2.1.2.1 Constant pressure pumps

Constant pressure pumps deliver solvents via a small

head piston which is driven by a pneumatic amplifier. The

main advantages of these pumps are low cost, ability to

deliver high pressure and stability of flow during the

delivery stroke of the pump. Although pulseless, the

flowrate and, hence, the elution volume, can vary with the

changes in permeability of the column or the- viscosity of

the solvent leading to poor precision and accuracy of

analysis.

2.1.2.2 Syringe type pumps

Syringe type pumps work on the principle of positive

solvent displacement by a piston mechanically driven at a

constant rate in a piston chamber (250-500 ml capacity)

with the generation of a pulseless flow and with high-

pressure capabilities (200-475 atm). This overcomes the

major disadvantage of pneumatic amplifier type pumps, and

makes syringe pumps ideal to yeild reproducible retention

times. However, the major problem encountered is the design

of a suitable refill mechanism.

2.1.2.3 Reciprocatincf piston pumps

Reciprocating piston pumps are most commonly used in

HPLC since they permit delivery of a wide range of flow

10

rates and are relatively inexpensive. Reciprocating pumps

of the single piston design function by having a slow

solvent delivery cycle compared to rapid refilling of the

piston chamber. However, there is some pulsing of the flow

because of the finite time taken to fill the piston

reservoir, and also the fact that the initial part of the

delivery stroke is concerned with compression of the

solvent prior to pumping. Improved precision and smoothing

of flow is provided by twin-piston reciprocating pumps,

where the pistons are driven approximately 180° out of I

phase. i.

2.1.3 Sample introduction

The ideal sample introduction method should be able to

insert reproducibly and conveniently a wide range of sample

volumes into the pressurized column as a sharp plug with

little loss in efficiency. The injection system should

possess zero dead volume to prevent loss of resolution. }

Sample application techniques can be broadly classified as

either syringe or valve injections.

2.1.3.1 Syringe injection''

In the syringe injection method, a small ( 10 pi )

sample is introduced into the pressurized column with a

high-pressure syringe through a self-sealing elastomer

septum and directly on top of the column packing. The

method is limited to low pressure unless a double septa

11

design is employed. At higher pressures there is sample

leakage around the syringe plunger, and it is difficult to

maintain the septum leak proof and to insert the needle

into the pressurized system. Advantages include: low

initial cost, variable and small sample volumes and low

band spreading.

2.1.3.2 Valve injection'*

Valve injection of the sample is now the preferred and

accepted technique in modern HPLC, particularly for routine

and quantitative analysis. In this method a fixed-volume

loop is filled with the sample using a syringe, then the

eluent flow is directed through the loop to flush the

sample onto the column. For most analytical separations a

six-port valve is used with a 10 or 20 external loop, as

illustrated in Figure 2.2/

^ Meedle port ； ^ ^

Vents

Loop ^ ^ LOAD INJECT

I Figure 2.2 Six-port sample injection valve

12

2.1.4 Separation column^

Columns for analytical HPLC are typically 10-25 cm

long and with 4-6 mm internal diameter. The columns are

made of stainless steel to cope with the high back pressure

and are glass lined to prevent metal catalysis of solvent-

solute reactions at high column pressures. The tubing must

have a smooth, precision-bore internal diameter to ensure

that a well-packed column will not channel near the

wall/packing interface because of wall irregularities.

This would result in broader peaks and lower efficiency.

Straight columns are preferred. They are operated in the

vertical position with the flow being directed either up or

down through the packing. Connections to the columns are

made with low dead-volume fittings designed to eliminate

stagnant pockets of mobile phase.

Current practice utilizes column packing that lies in

the range from 3 to 7 /Ltm in diameter; occasionally up to 10

jLtm or higher, especially for exclusion chromatography. The

type of packing material used depends on the type of

separation mode utilised.

2.1.5 Detector

Detectors used in liquid chromatography should ideally

function with high precision, high sensitivity, high

stability and fast response to record rapid eluting peaks.

13

They should also have a wide linear dynamic range to ensure

good quantitative analysis and be easy to operate and

maintain. Unfortunately there is no universal detector

that meets all these criteria. Thus, it is necessary to

select a detector on the basis of the problem at hand so

that in doing a variety of separations, more than one

detector will be needed.

There are three basic types of detectors :

1. Detectors which monitor a specific property of the

solute not shared with the solvent e.g. UV absorbance and

fluorescence. Possession of such a property by the solute

affords its detection in the effluent.

2. Detectors which monitor a bulk property of the eluent e.g. refractive index； in this case the solute

modifies the base value of the property associated with the

solvent.

3. Desolvation/transport detectors which function by

separating the solvent from the eluent, thus allowing

subsequent detection by techniques such as flame

ionization?’^ or mass spectrometry.^

2.1.5.1 Selective property detectors

UV-visible photometers and spectrophotometers

An ultraviolet photometer operating at fixed

wavelengths of 254 or 280nin is one of the most widely used

detectors for HPLC. The advantages of this detector are

14

its relatively low cost, high sensitivity ( nanogram level

)achieved for many compounds of chemical and biological

interest provided that they absorb UV light, and its

insensitivity to changes in temperature, flowrate, and

mobile phase composition.

The variable wavelength detectors are an order of

magnitude less sensitive than the fixed wavelength

detectors but are considerably more versatile, since any

wavelength within the range of the detector may be

selected.

Detectors are now commercially available which allow

programmable wavelength switching during analysis, thus

optimizing sensitivity and selectivity. As many as 12

wavelengths can be selected. The systems use reversed-

optics geometry, i.e., the light from the source is

dispersed after passing through the sample by a holographic

grating on to an array of photodiode detectors. The

photodiode array is a row of detectors ( up to 400 )

mounted on a 1 cm silicon chip, each diode receiving a

different wavelength. The full spectra obtained from diode

array spectrophotometer are a useful aid to component

identification. The examination of the many spectra taken

during the elution of a peak gives information on the peak

homogeneity thus aiding accurate quantitation.

15

Conductivity detectors

The conductivity detector which measures the

electrical resistance of a conducting medium between two

electrodes in solution is usually used in ion

chromatography. The sensitivity is about 10 jug/ml using an

eluent of relatively high conductance. Reducing the eluent

conductance can increase the sensitivity by about two

orders of magnitude. The detection of ionic species in

HPLC eluents using conductivity detector is very difficult

because of high background eluent conductance. The

development of suppressed ion chromatographic systems^®

obviated this problem.

Electrochemical detectors

Electrochemical detectors can be used for the

detection of compounds which are electroactive. The

detection depends on the fact that electroactive species

undergo electrolysis at an electrode when a voltage is

applied and that such solutes may be detected by monitoring

the resulting current. The applicability of

electrochemical detectors is limited by the requirement

that the mobile phase must be electrically conductive.

This may often be achieved by adding buffer to the eluent.

There are still other selective property detectors

such as fluorescence detector, which is sensitive and

selective; infrared detector; atomic absorption detector

and radioactivity detector.

16

2.1.5.2 Bulk property detectors

Refractive index (RI) detectors

Refractive index detector is the commonest bulk

property LC detector and under properly controlled 1

conditions this is virtually a universal detector, although

it is less sensitive than the selective property detectors.

The detector response depends on the difference between the

refractive index of the pure mobile phase and that of the

mobile phase together with the solute eluting from the

column. Therefore, this type of detection is- sensitive to

fluctuations in pressure, temperature and composition.

However, it is an ideal universal detector because most

compounds modified the RI to some extent and both positive

and negative changes in the solvent RI can be detected.

2.1.5.3 Desolvation/transport detectors

. The detection using transport detectors is based on

the concept of physically separating the solvent, which is

necessarily volatile, from the non-volatile solute. It is

ideal for most gradient elution applications, the major

limitation being not applicable to systems with non-

volatile buffers. I

Examples of such detectors include the flame

ionization detector, electron capture detector and mass

spectrometric detector.

17

2.2 THEORY OF LIQUID CHROMATOGRAPHY

Chromatography is a dynamic separation system

consisting of two media, a stationary phase and a mobile

phase. When the solute molecules in the mobile phase come 會

into contact with the stationary phase, there is now

competition between the two phases for the solute

molecules, which depends on their physical properties and

affinity for the stationary phase. This process is termed

partition with each component distributed between the two

phases as they pass through the system. Since different

component molecules have different affinities, they will

proceed through the system at different speeds and hence

separation is achieved.

The molecular interactions leading to the distribution

of a component between the mobile phase and stationary

phase are attributed to a combination of polar forces

arising from induced and permanent electric fields and i.

London's dispersion forces which are influenced by the

relative molar masses of the solute and solvent.u

工ntermolecular forces predominate in chromatography with

polar and dispersion forces having a major contribution to

the overall interactions.

18

Solute retention is normally measured by the capacity

factor, k', which is defined by the expression :

= (Vr - Vo)/Vo = (tR - to) /to

where Vr, V。/ t^ , t。are respectively the retention volume

/ time of solute peak and unretained peak of the system at

constant flowrate of the mobile phase. The larger the value

of , the better the separation but longer the analysis

time and broader the peak/

The efficiency of a column is obtained from the number

of theoretical plates, N :

N = 16 ( tR/Wb )2 J (

where W^ is the base width of the solute peak.

The larger the value of N, the better the resolution

of the column and the value of N is between 2,000 to 20,000

for a good column.

19

*

REFERENCES

1. N. Hadden, F. Baumann, F.MacDonald, M. Munk, R. Stevenson, D. Gere, F. Zamaroni and R. Majors, Basic Liquid Chromatography, Varian Aerograph, 1971

2. L. Berry, and B.L. Karger, Anal. Chem., 1973, 45, 819

3. A. Braithwaite, and F.J. Smith, Chromatographic

Methods, Chapman and Hall, 1985, p.414

4 . M.T. Gilbert, High Performance Liquid Chromatography,

lOP Publishing Limited, Bristol, 1987, p.481

5. H.H. Willard, J.A. Dean, L.L. Merritt and F.A. Settle, Instrumental Methods of analysis, Litton Education Publishing Inc., p.1030

6. Rheodyne Inc. Bull. 106 Cotati, Calif.

7. M. Kreji, et al, J. Chromatogr., 1981, 218, 167

8. V.L. McGuffin and M. Novotny, Anal. Chem., 1981, 53, 946

9. T.E. Young and R.J. Maggs, Anal. Chim, Acta, 1967, 38, 105

10. H. Small, T.S. Steven and W.C. Bauman, Anal• Chem.,

1975, 47, 1801

11 A. B. Littlewood, Gas Chromatography Academic Press, 1970, p.71

20

CHAPTER 3

DETERMINATION OF CITRATE IN PHARMACEUTICALS

BY HPLC USING INDIRECT PHOTOMETRIC DETECTION

3.1 INTRODUCTION «

citric acid is a well known food ingredient, both to

consumers and food processors. Consumers know it as the

• predominant acid in oranges, lemons, and. limes, but

processors use it for its many functions in many foods.

Citric acid has complete acceptance by consumers as a

"natural" ingredient.丨

Citric acid is used as acidulant in beverages,

confectionary, effervescent salts, in pharmaceutical

syrups, elixirs, in effervescent powders and tablets, to

adjust the pH of foods. It is used in beverages, jellies,

jams, preserves and candy to provide tartness； in the

manufacturing of citric acid salts； as mordant to brighten

colours; in electroplating； in special inks； in analytical

chemistry for determining citrate-soluble P2O5； as reagent

for albumin, mucin, glucose, bile pigments

Citric acid is widely used as a metal deactivator or

chelating agent,3 and in combination with phenolic

antioxidants, it is used as synergistic antioxidant in

21

*

processing cheese. Also, detection of added acidulants in

juice drink would be indicative of adulteration provided

they were not declared on the label. The citric acid

content of these foods, therefore, determines their

acceptability and keeping quality. The accurate

quantification of citric acid in such foods is therefore

essential for quality control requirements and meeting

legal regulations.

\

22

3.2 BRIEF REVIEW OF THE ANALYTICAL METHODS FOR

THE DETERMINATION OF CITRIC ACID

3.2.1 Spectrophotometric methods

3.2.1.1 Enzymatic method Citric acid ( citrate ) is converted by citric

lyase to oxaloacetate and acetate. In the presence of the

enzymes malate and lactate dehydrogenase, oxaloacetate and

its decarboxylation product pyruvate are reduced to L-

malate and L-lactate, respectively, by reduced

nicotinamide-adenine dinucleotide ( NADH ) and

stoichiometric amount of NAD+ is produced. The NADH

consumed is measured at 340 nm with a spectrophotometer.^

This method is modified'' where a redox reaction

in which 2-( 4-iodophenyl )-3-( 4-nitrophenyl )-5-phenyl-

2H-tetrazoliuin chloride ( INT ) is reduced to NADH and then

to a red formazan, the concentration of which is

proportional to the original citrate concentration. The

method has good sensitivity and a short analysis time ( 10-

25 min ).

3.2.1.2 Derivatisation

Citric acid is derivatised to form a chromophore,

and the absorbance at the absorption maximum is measured.

23

An example is the modified method^ based on the

chromophore formed between pyridine, acetic anhydride and

citrate. The absorbance was measured at 428nm. Another

derivatisation agent is the acid alizarin black SN? and the

absorbance was measured at 603 nm vs. a blank prepared

similarly. The derivatisation technique gives a faster

analysis time than the enzymatic method while the enzymatic

method is more specific.

3.2.2 Titrimetric methods

Titrimetric method for the determination of

citric acid and citrate is based on complex formation

between copper ion and citrate ion in buffered solutions

(boric acid/borate or sodium hydrogencarbonate). The

equivalence point has been established with an indicator or

from potentiometric titration curves obtained with a

copper or a silicone rubber-based copper(II)-selective^

indicator electrode. The best reported precision was

about 0.9%. 01in9 employing a copper-selective electrode

titrimetric method where equal increments of copper(II)

solution were added stepwise and the sample solution was

kept at constant pH by simultaneously running a pH-

stat addition. Titration curves obtained at constant pH

can be linearized as described by Gran'o, so that the

precision of the results is increased considerably.

A dropping copper-amalgam electrode^^ was used

24

in conjunction with a SCE for the potentionmetric

titration of citrate with aq. 0.5M-CUSO4 in 0.IM-NaHCOj

medium (pH 8.3). The range of the method was 0. ImM to

0. IM-citrate and was used in the analysis of the citrate

content in orange and lemon juices.

3.2.3 Atomic Absorption Spectrometry (AAS)

A new indirect AAS method for determining citric

acid in soda water is investigated by Che et al. After

adding the CU3(P04)2 reagent to react with citric acid in the

sample solution, the unreacted reagent is isolated by .

centr if ligation, and Cu is then determined by AAS after

dissolving the reagent and the content of citric acid is

then calculated. The result is not affected by pH in the

range of 1 to 11. The only drawback for this method is

that it needs 20 hours to obtain a stable and maximum

signal of Cu.

3.2.4 High Performance Liquid Chromatography (HPLC) Methods

High performance liquid chromatography (HPLC)

affords a rapid and simple technique for analyzing

certain mixtures of organic acids. Many analytical

methods have been reported for the separation and

determination of organic a c i d s .歸 Three main methods are

commonly u s e d , 28 namely, ion-exchange chromatography,

solvophobic chromatography and reverse-phase chromatography

25

of the derivatized products.

3.2.4.1 Ion~exchanqe Chromatography

Ashoori6 applied this method to determine citric acid

in a wide variety of food products. The citric acid was

detected as a single peak in all samples analyzed with no

interference from other compounds. These results

indicated that the method is specific for citric acid.

Zhu27 used this method to determine saccharine and citric

acid in beverage.

3.2.4.2 Solvophobic Chromatography

The addition of acids or acidic buffers to the

mobile phase lowers the pH and suppresses the ionization of

the acidic functional groups of the solutes. Ionization

suppression-aided separations are therefore based on the

hydrophobicities of the solutes. The retention is the

result of hydrophobic interactions of the hydrocarbonaceous

moiety of the solute with the octadecyl chains of the

stationary phase. Relatively polar substances can be

separated on Cig-silica gel columns with neat aqueous

eluents of the appropriate pH. This technique is termed

hydrophobic chromatography or ion-supression

chromatography. ‘

A reversed-phase HPLC method described by

Coppola29 uses 2% aqueous potassium dihydrogen phosphate

solution adjusted to pH 2.4 with phosphoric acid for the

26

determination of organic acids (including citric acid) in

cranberry juice. Distler^® used sulphuric acid to adjust

the pH of the pure aqueous mobile phase for the separation

of short-chain carboxylic acids on Cig-silica gel. Marce^^

employed a direct method for the simultaneous determination

of organic acids (including citric acid) in fruit juices

and wines by isocratic reversed phase HPLC. The selection

of the experimental conditions (pH, ionic strength, flow

and temperature) has been carried out by optimizing the

resolution and time of analysis using a modified sequential

simplex method.

3 , 2 .4» 3 Reversed-phase chromatocfraphy of derivatized

products

Chemical derivatization has been required for the

selective and sensitive detection of analytes such as

biological or bioactive substances and metals in tissues,

body fluids, and pollutants. It also affords selectivity

in the separation of the analytes. Hence, growing numbers

of papers have been published recently reporting methods

and applications for derivatization in liquid

chromatography, especially high-performance liquid

chromatography. Review papers and monographs that have

appeared in the past several years give information on this

subject. 31 •

Recent progress in the chemistry of derivatization and

techniques of liquid chromatography has made possible trace

27

analysis of organic functional groups of low-molecular-

weight compounds and, with the aid of enzymes,

differentiation of certain functional group(s) from other

functional groups of the same type in large molecules such

as proteins.

Most chemical reagents reported so far for carboxylic

acids are not appropriate in terms of being undetectable

themselves by UV, fluorescence, and other detectors.

Nevertheless, some of them are useful in practice.

Carboxylic acids in beverages such as wines and other

commercial drinks or natural fruit juices were derivatized

with p-bromophenacyl bromide in 50% acetone-water (pH 7 to

8) containing 8.5 mM 18-crown-6 in boiling water for 75

min, separated on RP-18, and detected at 254 nm.^^

3.2.5 A brief comparison of the method

The AAS is an indirect method while the titrimetric

method and spectrophotometric methods for the determination

of carboxylic acids are lengthy. Hence, chromatographic

methods must be regarded as an attractive alternative.

However, as citric acid (or citrate) is UV transparent

making detection difficult. Nevertheless, this problem' can

be overcome by using ion-interaction chromatography. The

theory of detection of this technique will be described in

the next section.

28

3.3 THEORY OF DETECTION

3.3.1 Indirect photometric detection

Analyte which is ultraviolet transparent cannot be detected in the usual way and the method of indirect detection has been developed.32-35

In this kind of detection method, an anion ( in this

case, salicylate ion which is ultraviolet, UV, active ) is

used as co-anion to an ion-interaction reagent ( tetra-

butylammonium ion ) • At equilibrium, a constant background

of UV absorption is maintained when the co-anions

dynamically occupy all the sites in the primary layer of

the stationary phase.

When an analyte ion of the same charge as the co-anion

first enters the column, it will compete with the coanion

and displace it out of the primary layer to the mobile

phase and hence a positive peak is developed. After some

time when the analyte leaves the column, it will be

released from the primary layer to the mobile phase, and

there will be an excess of charge in the primary layer and

the co-anion in the mobile phase will enter the primary

layer in order to maintain the charge balance. Therefore,

the concentration of the co-anion in the mobile phase will

be decreased. • Thus, the UV detector responds to the

29 T

presence of the analytes by producing a negative peak in the baseline absorbance plot. The magnitude of this peak is directly related to the difference in the concentration and the molar absorptivity between the eluent and analyte species .34

3.3.2 Models for ion-interaction chromatography

Most applications of reversed-phase ion-pair

chromatography involve the addition of a long-chain alkyl

sulfonate ions to the mobile phase to give enhanced

separation of oppositely charged sample ions. The exact

mechanism to describe the ion-pair phenomenon is still

uncertain. There are three popular hypotheses. Two

models propose extreme situations and each covers a

substantial amount of chromatographic data. These two

proposals are the ion-pair model and the dynamic ion-

exchange model. A third view, which is broader in scope

than the previous two concepts, accommodates both extreme

views without combining the two models. This proposal is

the ion-interaction model.

3.3.2.1 The ion-pair model

The ion-pair postulate stipulates that the formation

of an ion pair̂ '̂ ( Figure 3.1 ) occurs in aqueous mobile

phase prior to its adsorption onto the bonded, hydrophobic

stationary p h a s e R e t e n t i o n is governed by the amount of

non-polarity of the "ion-pair", which determines the

30 *

o參 O眷

Figure 3.1 The paired-ion model

• : ion of +ve charge ^ : ion of -ve charge

^^^^^^^ : ion-pair reagent /^M^hT : sample molecule

/ ° ； j

Figure 3.2 The dynamic ion-exchange model

書 :ion of +ve charge O ： ion of -ve charge

/vvWV^ : ion-pair reagent /SA/wn/^ : sample molecule

31

affinity to the stationary phase. A longer alkyl chain on

the pairing agent simply makes a less polar ion pair and

the retention of the pair increases as a result of its

greater affinity for the stationary phase.

3.3.2.2 Ion-exchange model

A second view stipulates an ion-exchange mechanism

In this hypothesis, it is the unpaired lipophilic alkyl

ions that adsorb onto the nonpolar surface and cause the

column to behave as an ion exchanger. This concept^^ is

depicted in Figure 3.2. The longer the chain length of the

ion-pairing reagent the more surface coverage of "ion-

exchanger" will occur and the longer will be the retention

of the ionic sample.

3.3.2.3 The ion-interaction model

Most recently, an ion-interaction mechanism has been

proposed by Bidlingmeyer, et a l , which is less restrictive

than the two previous models. This model is based upon

conductance measurements, which show that upon a series of

experiments involving neutral and charged samples injected

into systems containing positively and negatively charged

lipophilic compounds added to the mobile phase, ion-pairs

do not form in the mobile phase. Neither the ion-pairing

nor the ion-exchange model can explain the data in a

consistent way. Instead, the results suggest a retention

32

BULK ELUANT

SECONDARY\ ， O LAYER \

\ P R I M A R Y / SURFACE LAYER

Figure 3.3 The ion-interaction model

• : ion of +ve charge

o : ion of -ve charge

/VSAAT : ion-interaction reagent

/

/ o \ o O . .

- c:> ；' \ ：

Figure 3.4 Retention of an oppositely charged sample

molecule

/SAA/V^ ： analyte ion

Dotted figure denotes that it has moved from that

position

33 r

*

mechanism that is broader in scope and is best described as

one of ion interaction. The ion-interaction mechanism does

not require ion-pair formation in either phase and is not

based on classical ion-exchange chromatography. The ion-

interaction mechanism assumes dynamic equilibrium of the

lipophilic ion resulting in an electrical double layer

forming on the surface. The retention of the sample

results from an electrostatic force due to the surface

charge density provided by the reagent ion and from an

additional "sorption" effect onto the nonpolar surface.

This has been considered by same workers as

"pseudoexchange". .1

In the ion一interaction model depicted in Figures 3.3

and 3.4, a layer of lipophilic ions from the ion-pair

reagent ( or ion-interaction reagent, IIR ) is absorbed

onto the nonpolar surface ( see Figure 3.3 ). Because these

lipophilic ions have the same charge, they are well spaced

from one another. Most of the surface area on the nonpolar

packing surface is unaffected with only a small fraction of

the surface area being coated with the ion-pair reagent.

A primary ion layer and an oppositely charged counter-ion

layer are formed on the top of the non-polar surface. This

is an electrical double-layer model. The adsorbed ions are

in dynamic equilibrium between the bonded phase and mobile

phase so that if the reagent concentration is increased in

the mobile phase, the amount of reagent ion adsorbed is

also increased, thus increasing the amount of charge on

34

the surface. Transfer of samples through this double layer

is affected by electrostatic and van der Waals forces. For

instance, an ionic organic sample of opposite charge to the

reagent ion is attracted to the charged surface.

Chromatographic retention results from this coulombic

attraction and from an additional "sorption" of the

lipophilic portion of the sample molecule ( Figure 3.4 )

onto the nonpolar surface. The net result is that a pair

of ions ( not necessarily an ion pair ) has been adsorbed

onto the stationary phase.

In ion-pair chromatography many parameters can be

varied in order to effect a separation. In addition to the

stationary and mobile phases and the type, size and

concentration of the counter ion, the pH is a very

important parameter as it determines the concentration of

the ionic form of the solutes.

I. .

Ion interaction chromatography has several

advantages over ion exchange chromatography in some

applications .45 A number of applications rely on the

ability to modulate the "capacity" of the

"pseudoexchanger" simply by altering one or more of the

following:

1. the concentration of the IIR in the mobile phase. 2. The lipophilicity of the IIR-in a homologous series

of IIRs: the longer the nonpolar chain the more the

35

IIR is adsorbed by the substrate.

3. The solvent polarity of the mobile phase - the

addition of water-miscible solvents such as

alcohols or acetonitrile will diminish the

interaction between the lipophile and the nonpolar

stationary phase.

This ability to raise and lower the capacity can be an

advantage in dealing with samples that contain ions with a

wide range of affinities. Furthermore, since large pore

substrates are used and the functionality of the

pseudoexchanger is readily accessible on the pore walls,

IIR methods are especially suitable for the chromatography

of large ions/^

i,

36

3.4 EXPERIMENTAL

3.4.1 Apparatus

3.4.1.1 The liquid chromatoqraph

The high performance liquid chromatographic system was

composed of a solvent delivery module ( Beckman llOB ) with

a controller ( Beckman 4 21A ) ； a system organizer ( Beckman

)；an injection port ( Beckman Altex 210A Value ) ； an

adsorbosphere C18 column of length 250 mm, internal

diameter of 4.6 mm and particle size of 5 jtzm ( Alltech

Associates ), protected by a lOmm x 4. 6inm guard column

packed with the same material as the column; a detector

(Waters Lambda Max Model 481 LC Spectrophotometer ) and an

integrator ( Hitachi 833A Data Processor ).

3.4.1.1.1 Parameter setting of the detector

The following settings for the UV

detector were kept unchanged during the

experiment.

Parameter Settings

Response time 1

Coarse zero -0.3 AU

3•4•1•2 Spectrophotometer

All absorption spectra and absorption measurement

(for the counter check spectrophotometric method ) were

recorded by using a Spectrophotometer ( Hitachi Model

37

U-2000 ) with matched 1 cm quartz cells.

3.4.1.2.1 Operational conditions

Wavelength scanning for the compounds

were taken with the following settings :

Parameters Settings .

Data mode ABS

Start WL 340 nm

Stop WL 2 00 nm

Scan speed 200 nm/min

Response Medium

Baseline User

Lamp change 340 nm

VIS On

UV On

List interval 0.1 nm

Threshold 0.001

Sens 1

3 . 4 . 1 . 3 P H meter

A digital pH ( Jenway 3 02 0 ) meter was used for all

pH measurements. The meter was calibrated by using standard

buffer solutions of pH=4 and pH=7 solution before used.

3.4.1.4 Glassware

All volumetric flask and pipettes were of grade A and

grade B and were calibrated to relevant BS Standard before

use.

38 r

3.4.2 Reagents and materials All reagents used were of analytical reagent grade and

used without any further purification.

3.4.2.1 Water ( Ultra pure water )

All water used during the experiment was distilled

water purified by the Millipore Milli-Q50 ultra pure water

system ( Millipore, France ). The water was deionized and

filtered through the 0.1 /xm filter from the system. The

resistivity of water produced was 18 Mfl/cm.

I. 3.4.2.2 Aqueous stock solutions

3.4.2.2.1 Stock solution of 50 mM tetrabutylammonium

iodide ( TBAI )

50 mM of the aqueous stock solution of TBAI was

prepared by dissolving 9.2346 g of TBAI in 500 ml of ultra

pure water in a calibrated volumetric flask. The solution

was then filtered through a Millipore HA type filter of

0. 45 /Ltm pore size.

3.4.2.2.2 Stock solution of 200 mM sodium salicylate (NaSa) 200 mM of aqueous stock solution of NaSa was prepared

f -by dissolving 8.0063 g of NaSa in 250 ml of ultra pure

water in a calibrated volumetric flask. The solution was then filtered through a Millipore HA type filter of 0.45 fim

pore size.

39 f

3.4.2.2.3 Stock lOmg/ml sodium citrate solution lOmg/ml aqueous stock solution of sodium citrate was

prepared by dissolving 3.9268 g of tri-sodium citrate

dihydrate in 250 ml of ultra pure water in a calibrated

volumetric flask. The solution was then filtered through

a Millipore HA type filter of 0.45 jLtm pore size.

3.4.3 Aqueous mobile phase

Various mixtures of the mobile phase were prepared by

diluting appropriate volumes of the stock IIR and co-anion

solutions in suitable volume of ultra pure water. The

mobile phases so prepared were then filtered through a

Millipore HA type filter of 0.45 jum pore size to remove any

trace of solid particles. The filtered mobile phase was d

then degassed by shaking in an ultrasonic bath ( Branson

1200 ) for at least half an hour before use.

3.4.4 Column conditioning

The column was conditioned each time when a new mobile

phase was used. This normally took two hours to do so.

Whenever the new mobile phase was changed from one

system to another, the column had to be cleaned thoroughly

by 2 0 column volume of water ( this usually took two hour

at a flowrate of 1 ml / min ) followed by another 20 column

volume of 1 : 1 mixture of methanol and water so as to

40 t

ensure that the IIR and co-anion adsorbed on the column bed

were completely removed. 1

3.4.5 Sample treatment

Generally the liquid samples were diluted to

appropriate concentrations with the mobile phase. The

solid samples were first ground and then dissolved in the

mobile phase with shaking in an ultrasonic bath for fifteen

minutes. Any undissolved particles were filtered and the

solution was transferred to a calibrated volumetric flask

and made up to the mark with the mobile phase.


Samples were introduced into the column through the

external loop injection valve by using a 1 ml luerlock

syringe ( Becton Dickson ) fitted with a 0.2 /m disposable

filter.

41

3.5 RESULTS AND DISCUSSION

The objective of this work was to find out the optimum

conditions for the determination of citrate using ion-

interaction chromatography. A calibration graph was then

obtained. A series of samples^Xxere then taken for

analysis to assess the practicability of the proposed

method. Finally the analytical results obtained were

compared with those obtained by an established method in

order to check the accuracy of the proposed method.

3.5.1 Checking of the detector light source

The intensity of the UV detector light source was

first checked to see whether the detector was functioning

properly. Pure methanol was used as the mobile phase at

a flowrate of 1 ml/min. The data and graph were shown in

Table 3.1 and Figure 3.5 respectively.

Figure 3.5 shows a spectral response curve similar to

common spectrophotometer light sources and we can conclude

that the detector is reliable.

{

\

42

Table 3.1 Intensity of the light source at different wavelength

Wavelength (nm) Intensity

26

12

^ ^

^

230 193

201 •

198

192

270 ^

^

290 2J_9

115

^ 171

^

330

^

3£0 ^ ^ ^

138

43

220.00 ]

170.00 一 / X

： \ . >N -

CD -

o 120.00 — c -L J -

70.00 -u 20.00 I I I I I I I I I I I I I I I I I I I I I I I I I I

160.00 260.00 360.00

Wavelength / nm

Figure 3.5 Intensity of the detector light source vs wavelength

44

3.5.2 Choice of background setting The mobile phase used was 0.60 mM TBAI together with

0.75 mM sodium salicylate at a flow rate of 1.8 ml/min. The

wavelength of detection was 248 nm.

An aliquot of standard citrate ( 2 jitg ) was injected at different background settings ( i.e. different absorption intensities of the mobile phase ) of the detector. The peak area so obtained together with the noise level and the signal to noise ratio ( S / N ) are shown in Table 3.2.

Table 3.2 Peak area of 2jLtg citrate at different background settings

BG SET Peak area Noise S/N

( X 103 counts) ( X 103 counts)

-0.10 130.181 48 2.712

-0.05 139.201 50 2.784

0.00 139.262 49 2.842

0.05 85.104 36 2.364

0.10 14.489 8 1.811

0.15 7.936 4 1.984

0.20 4.329 2 2.165

BG SET : background setting

S/N : signal to noise ratio

45

From Table 3.2, it was shown that the peak area

obtained varies greatly with the background setting chosen.

The setting that gives the highest S/N value is the one

that set at zero value. Therefore, the peak area

measurement should be done at zero background, or at least

near it, in order to have a higher sensitivity and hence

more accurate results.

Figure 3.6 shows graphically the relationship between

signal to noise ratio with different background settings.

46

3.00 q

2 . 8 0 二

？ \

0 2.60 二 \

1 ： \ .

一 2.40 - \

I 丨 \

� I \ z 2.00 - \ /

I : V y 1.80 £ ^

1 .60 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

-0.10 -0.00 0.10 0.20

Background setting ‘

Figure 3.6 Signal to noise ratio of 2fMg citrate at different background settings

47

3.5.3 Retention and peak area due to different co-anions

Three kinds of co-anions ( of 0.15 mM each ) were

mixed with tetrabutylammonium iodide of concentration of

0.12 mM to produce three mobile phases. They were then

passed in turn to the column for conditioning, and 2 /ig of

citrate was injected after equilibrium had been reached.

The peak area were recorded in each case and the results

are summarized in Table 3.3. Besides, the UV spectra from

340 to 200 nm of the IIR ( TBAI ) and different mobile

phase containing different co-anions were taken and shown

in Figures 3.7 to 3.10.

Table 3.3 Effect of co-anions in the mobile phase on the sensitivity and retention of citrate

Co-anion pH Peak area k'

(X 10^ counts)

Sa" 6.08 482.41 9.00

Bz- 6.15 210.66 15.03

HP" 4.68 155.76 10.58

Sa" , : salicylate

Bz" : benzoate

HP" : hydrogen phthalate

pH : pH of the mobile phase

： capacity factor of the eluted peak

Detection wavelength at 240 nm

Flow rate : 1.8 ml/min

48

The absorbance of 0.12 mM TBAI and mobile phase

containing different co-anions were measured at 240 nra and

the results are shown in Table 3.4.

Table 3.4 The absorbance of TBAI and co-anions

Absorbance

•12 mM TBAI 0.477

•12 mM TBAI + .15 mM NaSa 1.081

.12 mM TBAI + .15 mM NaBz 0.868

.12 mM TBAI + .15 mM KHP 1.308

.15 mM NaSa O . 604

.15 mM NaBz 0.391

.15 mM KHP 0.831

49

1.500 ,

ABS \ j \

0. 000 r - ^ ^ , ：_, rim 200 240 280 320

Figure 3.7 Absorption spectrum of 0.12 mM tetrabutyl-ammonium iodide

1+500 j

ABS \

I 八 _ 0. 000 , _ — _ , \

nm 200 240 280 320

Figure 3.8 Absorption spectrum of mobile phase containing 0.12 mM tetrabutylammonium iodide and 0.15 mM sodium salicylate

50

1.500 ^

ABS \

0. 000 , • nm 200 240 280 320

Figure 3.9 Absorption spectrum of mobile phase containing 0.12 mM tetrabutylammonium iodide and 0.15 mM sodium benzoate

1；500 ^

ABS \

0. 000 , ^ _ _ ^ ^ •

nm 200 240 280 320

Figure 3•10 Absorption spectrum of mobile phase containing 0.12 mM tetrabutylammonium iodide and 0.15 mM potassium hydrogen phthalate

51 t

From Table 3.3, we can conclude that salicylate ion

gives the highest signal with the smallest retention.

The signal obtained in ion interaction chromatography

relies very much on the absorptivity and the amount of co-

anions present in the mobile phase. The reason why

salicylate gives the largest signal may probably be that it

is a monovalent ion. When the analyte ( citrate ion )

leaves the column, salicylate will replace the vacancy left

by the analyte in the primary layer in a one to one mole I

ratio. Since hydrogen phthalate will exist in both the

monovalent and divalent form, the replacement of the

analyte ions will be less than a one to one mole ratio.

Therefore, hydrogen phthalate gave a smaller signal than

the salycilate despite that hydrogen phthalate has a higher

molar absorptivity than that of salycilate.

For the co-anion benzoate, it gave a larger signal

than the hydrogen phthalate but a smaller signal than the

salicylate ion although it is also a monovalent ion. The

reason is possibly that benzoate possesses the lowest

absorptivity at 240 nm ( refer to Figures 3.7 to 3.10 or

Table 3.4 ) and therefore it gives the smaller signal than

that of salicylate.

52

t

The elution order for the citrate anions by the co-

anions was as follows: »

salicylate < hydrogen phthalate < benzoate

This is probably due to the reason that benzoate

possesses only one polar functional group ( -COOH ) whereas

hydrogen phthalate has two identical polar functional

groups ( -COOH ) , so that benzoate is less polar and is

attracted less strongly by the primary layer. As ion

interaction chromatography is a competition between the

analyte ions and the co-anions for sites in the primary

layer, the analyte ions are displaced comparatively less

easily by benzoate as compared with hydrogen phthalate.

Similar reasoning applies to the comparison between

benzoate and salicylate, which possesses two functioal

groups, namely, the -COOH group and the -OH group. When

compared with the hydrogen phthalate, salicylate is more

polar because the hydroxy 1 group is more polar than the

carboxylic acid group ( 一COOH ) and therefore held more

strongly by the primary layer and hence the analyte anions

is less strongly retained. ,

It is obvious that sodium salicylate should be chosen

as the CO一anion for the determination of citrate rather

than potassium phthalate or sodium benzoate because it can

produce the highest signal with a resonably short retention

time.

53

3.5.4 Choice of mobile phase concentration

The effect of the concentrations of TBAI and NaSa on

the signal and retention for the analyte was tested. For

simplicity, the same concentration ratio for the TBAI and

NaSa was used and 2 /ig of the analyte was injected for the

different mobile phases. The area counts and the retention

time for the analyte peak were recorded and the capacity

factors were then calculated. The results are shown in

Table 3.5.

Table 3.5 Signal and retention at different TBAI and NaSa concentrations in the mobile phase

Cone, of TBAI and NaSa Peak area Capacity

in the mobile phase (xlO^counts) factor

.12 mM TBAI + .15 mM NaSa 178.46 9.00

.24 mM TBAI + .30 mM NaSa 156.54 8.84

.36 mM TBAI + .45 mM NaSa 155.99 8.47

.48 mM TBAI + .60 mM NaSa 146.21 7.93

•60 mM TBAI + .75 mM NaSa 136.38 7.39 ”

.72 mM TBAI + .90 mM NaSa 112.64 6.88

Detection wavelength : 248 nm

Flow rate : 1.8 ml/min

54

From Table 3.5, it is apparent that the higher signal �

were obtained at lower concentration of TBAI and NaSa. With

increase in concentration of TBAI and NaSa in the mobile

phase, a decrease in retention of the analyte was observed.

It seems contradictary at first since an increase in

concentration of TBAI will cause an increase in the active

sites in the column an hence the analyte will be retained

longer in the column. However, the increase in the

concentration of the co-anion will increase the competition

between the analyte ions and the co-anions towards the

primary layer. In this case, it is apparent that the effect

of CO-anion outweighs the effect of the IIR and therefore

the analyte ions will be eluted out more easily with an

increase in the concentration of the co-anions.

It can be concluded that it is better to employ low

concentrations of both TBAI and NaSa for the analysis of

citrate since a high signal can be obtained within a

reasonable period of time. However, it is no good to test

lower concentrations. If the concentrations of TBAI and

NaSa in the mobile phase are too low, re-establishment of

equilibrium condition of the system may take a very long

time or becomes difficult, which will lead to failure in

the analysis. Hence, the optimun condition is a compromise

between sensitivity and a reasonable analysis time.

55

3.5.5 Choice of detection wavelength

The peak area was recorded and the absorbance was

measured at various wavelengths using a UV-visible

spectrophotometer. The results are shown in Table 3.6.

Table 3.6 Signal of 2 /ig citrate at various wavelengths

Wavelength (nm) Area (xlO^counts) Absorbance

239 468.87 1.249

240 421.83 1.081

242 352.18 0.763

246 244.96 0.333

248 178.46 0.291

254 20.36 0.080

256 4.09 0.066

258 24.13 0.061

260 51.19 0.061

280 387.35 0.312

296 669.79 0.564

310 321.31 0.298

Mobile phase : .12 mM TBAI + .15 mM NaSa

56 *

The best wavelength for analysis is normally at the

peak maximum, which is 296 nm for sodium salicylate, and

the observed signal was highest at this wavelength, as

shown in Table 3.6. However, 296 nm was used, the

correlation coefficient of the calibration graph was

0.9984, which is not satifactory. The wavelength 242 nm is

choosen, which gave a higher sensitivity and precision for

the analysis of citrate. The other reason for choosing 242

nm is that the maximum of the intensity vs wavelength for

the light source ( see Figure 3.5 ) occurs at this

wavelength and hence a better lamp stability, that will

reduce fluctuation of the detected signal.

3.5.6 Preparation of calibration graph

The calibration graph was obtained by plotting the

peak area against the corresponding concentration of

citrate, which was made by the appropiate dilution of the

standard stock solution. The data for the calibration

graph are list in Table 3.7 and the calibration graph is

shown in Figure 3.11.

57

Table 3.7 Data for the calibration of citrate

Concentration ( jug/ml ) Peak area (xlO^counts)

‘ 20.0 72.35

40.0 165.58

60.0 260.72

80.0 362.09

100.0 455.55

120.0 548.92

140.0 637.58

160.0 737.53

180.0 826.84

200.0 922.12

Linear working range : 20-200 jitg / ml

Slope ： 4721 ml / jLtg

Intercept : -20.42

Correlation coefficient : 0.9999

Conditions of the chromatograph

Detection wavelength : 242 nm

Mobile phase : .12 mM TBAI + .15 mM NaSa

Flow - rate : 1.8 ml / min

Operating pressure : 3,000 psi

58 f

1000.00 q

； /

800.00 - /

？丨 /

0 / (A / D 600.00 - /

J ： / O 400.00 - /

；丨 /

2 0 0 . 0 0 一 /

： / , . . .

Q.00 —tn~~I~~II~1n~~I~I~~I~I~II~~II~III~~I~I~I~I 0.00 100.00 200.00

Concentration ( ug / mL )

Figure 3.11 Calibration graph for the determination of citrate

59

3.5.7 Precision test for the proposed method

In order to test the reproducibility of the proposed

method using the optimised system setting and mobile phase,

aliquots of citrate with a known concentration are

injected ten times into the column. The relative standard

deviation ( R.S.D. ) of the peak area was calculated and

was found to be 1.2 % ( Table 3.8 ). This means that the

proposed method is stable and good for analytical purposes.

Table 3.8 Precision test of the proposed method

2 fig citrate injected Peak area (counts)

1 456,250

2 449,717

3 - 448,997

4 456,887

5 459,894

6 455,994

7 456,667

8 447,098

9 453,891

10 441,864

R.S.D. 1.2 %

60

3.5.8 Determination of citrate in pharmaceuticals

The pharmaceutical products for this project were

purchased from Watson's medicine department store and a

brief description of the samples are shown in Table 3.9.

Table 3.9 Description of pharmaceutical preparation

Sample Sample Brief description no.

1 Cooling ENO, Antacid; contains sodium chrysanthemum carbonate, citric acid,

flavoured sodium saccharin, etc.

2 Cooling ENO, Ditto lemon flavour

3 Aspro Clear Anti-pain; contains aspirin,sodium bi-carbonate and citric acid.

.4 Alka-Seltzer Ditto

5 Bufferin Anti-pain；contains aspirin, calicium carbonate, magnesium stearate, citric acid , etc,...

6 Piriton Expectorant linctus; contains Piriton, ammonium chloride, sodium citrate, citric acid, glycerin, etc.

7 Vicks Extra strength cough Formula 44 suppresent； contains sodium

citrate, dextromethorphan HBr, alcohol, etc.

8 Visine (A.O.) Tears drop； contains boric acid, sodium citrate, sodium chloride, etc.,

61

Each saraple was diluted to an appropriate

concentration with the moble phase, and 20 jul of the

diluted sample was injected into the column three times.

The amount of citrate was deduced from the calibration

graph. The results of the analysis are shown in Table 3.10

and the respective results obtained using the standard AOAC

method mentioned"* and the claimed label values, if any, are

also shown in Table 3.10 for comparison. Typical

chromatograms for some of the samples were shown in Figures

3.12a to Figure 3.12c. 擎

The contents of citrate found by the proposed method

agree well with the respective claimed label value, and

those obtained using the standard method except for sample

No. 3 and 7.

62

Table 3.10 Assay of content of citrate in drug sample

. citrate content Sample No. Proposed method A.O.A.C. Claimed

spectroscopic label ^ „ method value Amount Mean*

1 41.96 , 41.52 41.87 % 42.3 43.26 % 42.14 (0.76)

2 41.22 , 40.08 41.10 % 42.17 43.02 % 41.99 (2.34)

3 208 ,218 ,213 213 mg/tablet 196 207 (2.35)

4 1.14 , 1.10 1.17 g/tablet 1.21 1.20 1.27 (7.69)

5 84.2 , 83.0 83.3 mg 78.8 82.69 (0.96)

6 9.54 , 9.48 9.41 mg/ml 9.32 9.66 9.21 (1.19)

7 39.5 , 39.0 38.7 mg/ml 35.4 36.6 37.7 (2.33)

8 12.6 , 12.9 13.0 mg/ml 12.2 13.5 (3.85)

* Relative standard deviation, in percent, shown in parentheses.

I

63 t

t •(

<=c

I I '1 ,..r ！ i'''； I .... “.'：：'‘M' I'"' ••： •'.! ....•: •：：•；• ’.j::..丨• ，. 广丨..丨丨厂,I. •.丨•

o：' ‘ • •‘， •.，. t .1 r 八 j\ J 寸....；•;;：;• o . . 0_. f ^^

\ ： “ , “ ‘ “ . , ,

.-.I •I •…•:•_:‘ ！,：| • i.r", ‘ Y. I. .• • •

丨丨 : r ^ 丨:, ：

I . ,:...• . . •.….I H I： . •:.

^ .». I I' \

t »

I- .• • • - I:..�

Figures 3.12c Chromatograms of sample No. 7 (left) and sample No. 8 (right)

64

‘ ！：

，J

1.1" 1 I ..• “

_

• O C .. . . - . • "...1 .-.I ft ':f[ C. •••',. •••i ...., •..'：. II ' n. "• • - •“ “

'•'•••I ‘：：!；,! , " • I . ., '••；' ••” . .."• 11:. ‘ . .:•«• 卜 _ II -^ j

I'.. ‘ 1.

卜...丨 I..... I • a 1-丨：：丨： ‘：!：：

“ L I , •.-•.I 丨•.丨 -'."••I "..'I 丨,丨！ “ ij. ,:...• -._.... 1:11 1.1


65

1.1, •• •:, kX •：：! “

t-

<=c t-1 \0 ‘ H ....J

- ‘：：! — -.：| .,：：' It , C::' -' 1.1.丨 . “ .. -̂.I •• “ 11:.. n. \ l! ' I ..

o . A 巧 • ‘ v l • ..I I ..:‘. ^̂ ^̂ ^̂ m

I..,.,, .... 广 1:1. ... r, ••I / •I •‘ .1

I « . ’• • 1 L y

r...

I...... I"' I.…‘ • •••• a:

•..I. a.:

. Ml I: I ••••H r I • •* I ._

•1 I ti I t „T “

.J.


66

3.5.9 Recovery tests

In order to test the reliability of the method,

recovery tests were also carried out by spiking known

amounts of standard citrate to each sample before any

treatment and the percent recovery was then calculated.

The percent recovery of the proposed method ranged

from 95.0 to 106 percent ( see Table 3.11 ) and may be

considered very good.

Table 3.11 Recovery test for the proposed method

Sample Add//igmr^ Found/jiigmr* Recovery ( % )

1 80 77.8 97.3

2 80 76.0 95.0

3 80 76.5 95.6

4 80 77.3 96.6

5 80 84.0 105

6 80 79.0 98.8

7 80 84.8 106

8 80 77.7 97.1

a. average of duplicate result

67

3.6 CONCLUSION

An accurate, simple and efficient high performance

liquid chromatographic method for the determination of

citrate in pharmaceutical preparations has been developed.

The proposed method employs ion-interaction chromatography

with an indirect method of detection, where salicylate is

used as a co-anion to an ion-interaction reagent,

tetrabutylammononium ion, and produces a constant

background of UV absorption. The UV detector will respond

to the presence of citrate by producing a negative peak.

Hence, the method does not depend on any specific

functional group of the analyte. This method has been

applied successfully to many types of pharamceutical

preprations with good precision and accuracy.

68

REFERENCES

1. D.D. Duxsury, Food Processing, 1991, 5, 83

2. The Merck Index, 11th edition, 1989, 2328

3. Branen, Food Additives 1990, 144

4. Official Methods of analysis-AOAC, 1990, 746

5. S. Girotti, R. Budini, E. Gattavecchia and D.

Tonelli, Anal, Chim, Acta, 1981, 124, 215

6. H.E. Indyk and A. Kurmann, Analyst, 1987, 112, 1173

7. S.B. Salama and S. A. Awad, Microchem. J., 1988,

37(1), 13

8. M.F. El-Taras and E. Pungor, Anal, Chim, Acta, 1976,

82, 285

9. A. Olin and B. Wallen, Anal. Chim. Acta., 1983,

151, 65 �

10. G. Gran, Analyst, 1952, 77, 661

11. Analytical Abstract May 1988 5C16 & 5F41

12. L. Che, J. Wang, J. Liu and L. Zhang Fenxi Huaxue,

69 *

1990, 18(6), 575

13. R.J. Schwarzenbach, J• Chromatogr., 1982, 251, 339

14. A. Clement and B. Loubinoux, J. Liquid

Chromatography, 1983, 6(9), 1705

15. R.H. Evans, W.V. Van Soestbergen and K.A. Ristow,

J,A.O,A,C,, 1983, 66(6), 1517

16. S.H. Ashoor and M.J. Knox, J. Chromatogr,, 1984, 299,

288

17. D. Cox, P. Hanison G Jandik and W. Jones, Food

Technology, 1985, 7, 41

18. V. Hong and R.E. Wrolstad, J,A.O.A,C., 1986, 69(2),

208

19. E.D. Coppola and M.S. Stan, J,A.O.A.C., 1986,

69(4), 594

20. F. Caccamo； G. Carfagnini, A.D. Corcia'^Xxnd R.

Samperi, J. Chromatography, 1986, 362, 47

70

21. D.B. Gomis, M.J. Moran Gutierrez, M. D. Gutierrez

Alvarez and A. S. Medel, Chromatographia, 1987, 24,

347

22. E. Burke, S.R. Zimmerman, D.S. Brown and D.R. Jenke,

J. Chromatogr, Sc,, 1988, 26(10), 527

23. D.B. Gomis, M.J. Moran Gutierrez, M.D. Gutierrez

Alvarez and J.J. Mangas Alonso, Chromatographia,

1988, 25, 1054

24. M.C. Gennaro and P.L. Bertolo, J. Chromatography,

1989, 472(2), 433 25. W.R. Jones, P. Jandik and M.T. Swartz,

J.Chromatography, 1989, 473, 171

26. R.M. Marce, M. Calull, R.M. Manchobas, F. Bonull and

F.X. Rius, Chromatographia, 1990, 29, 54

27. Y. Zhu, L. Zhu and X. Zhuang, Fenxi Huaxue, 1990,

18(3), 263

28. E. Mentasti, M.C. Gennaro, C. Sarzanini, C. Barocchi

and M. Savigliano, J• Chromatography, 1985； 322, 177 (

29. E.D. Coppola, E.G. Conrad and R. Cotter, J,A,0.A,C,,

1978, 61, 1490

71

30. W. Distler, J. of Chromatography, 1978, 152, 250

31. Giddings, Editor, Advances in Chromatography Vol. 27

1987

32. B.P. Downey and D.R. Jenke, J. Chromatogr. Sci,,

1987, 2 5 , 519

33. H. Small and T.E. Miller, Anal. Chem., 1982, 54, 462

i • 34. N. Raghavan and D.R. Jenke, J. Chromatogr. Sci.,

1985, 22, 75

35. W^Xx. Barber and P.W. Carr, J. Chromatography, 1984,

301, 25

36. B.A. Bidlingmeyer, J. Chromatogr, Sci., 1980, 18(10),

525

37. D.P. Wittmer, N.O. Nuessle and W.G. Haney, Anal,

Chem ” 1975, 47, 1422

38. C. Horvath, W. Melander, I. Molnar and P. Molnar,

Anal. Chem., 1977, 49, 2295

39. C. Horvath, W. Melander, I. Molnar and P. Molnar,

J. Chromatography, 1976, 125, 281

72

40. J.L.M. Venne, J.L.H.M. Hendrikx and R.S. Deedler


41. J.C. Kraak, K.M. Jonker and J.F.K. Huber,


42. N.E. Hoffman, and J.C. Liao, Anal. Chem,, 1977, 49,

2231

43. P.T. Kissinger, Anal. Chem., 1977, 49, 883

44. B. A. Bidlingmeyer, S.N. Deming, W.P. Price, Jr .B.

Sachok and M. Petrusek, J. Chromatography, 1979, 186,

419

45. H. Small, Ion Chromatography, 1989, Plenum, • 1 I New York, p.54

t

73

CHAPTER 4

DETERMINATION OF CAMPHOR AND MENTHOL BY

HPLC USING INDIRECT CONDUCTOMETRIC DETECTION »

4.1 INTRODUCTION

Camphor is an excellent plasticizer for cellulose

esters and ethers. It is used in manufacturing of

plastics, especially for celluloid; in lacquers and

varnishes; in explosives; in pyrotechnics; as moth

repellent; in embalming fluids; as preservative in

pharmaceuticals and cosmetics.' Applied externally, camphor

acts as a rubefacient and mild analgesic and is employed in

ointment as a counter-irritant in fibrositis, neuralgia,

and similar conditions.2 Camphor Injection was used as a

restorative in collapse because of its stimulating effect

on the cerebral cortex and medullary vasomotor and

respiratory centres.^ A great range of preparations are

used to relieve upper respiratory tract congestion and

obstruction in simple infections. It is also used in

eardrops and earwax softeners as a weak antiseptic and a

mild anesthetic intended to suppress itching. At

concentrations of 0.1 - 3 %, camphor depresses cutaneous

receptors, thereby relieving itching and irritation. At

higher concentrations of 3 - 11 %, camphor acts as a

counterirritant because it stimulates cutaneous receptors.

74 t ‘ ；

Camphor is safe and effective for use as an external

analgesic at these concentrations but can be very dangerous

if ingested.4

Menthol possesses similar usage as camphor." It may

also be used safely in small quantities as a flavoring

agent and has found wide acceptance in candy, chewing gum,

cigarettes,cough drops, toothpaste, nasal sprays, and

liqueurs. Menthol may give rise to hypersensitivity

reactions including contact dermatitis. There have been

reports of instant collapse in infants following the local

application of menthol to their nostrils. Ingestion of

menthol is reported to cause severe abdominal pain,

vomiting, drowsiness and coma.^

Both camphor and menthol are UV inactive and cannot be

determined by HPLC using UV~detection. They are neutral

compounds and hence cannot be detected using direct

conductometric detection. The objective of this work is to

develop an HPLC method for the simultaneous determination

of camphor and menthol using indirect conductometric

detection.

’丨

、

75

4.2 BRIEF REVIEW OF THE ANALYTICAL METHODS FOR

THE DETERMINATION OF CAMPHOR AND MENTHOL

Few methods are reported in the literature for the

simultaneous determination of camphor and menthol. The

only popular method is the gas-liquid chromatographic

method using flame ionization detector ( FID ) and no

article reporting the determination by HPLC method probably

due to the fact that camphor and menthol are UV inactive

where UV detection is one of the most popular method of

detection.

4.2.1 Gas - liquid chromatographic method i

Most of the methods used for the simultaneous

determination of camphor and menthol depend on the gas-

liquid chromatographic separation by choosing different

stationary phases with FID and either helium or nitrogen as

carrier gas.̂ "'̂ All these methods require a careful choice

of column and therefore only a few columns can be used for

the simultaneous determination of these two compounds.

4.2.2 GC - FTIR spectrometry

Sample containing camphor and menthol has also been

reported to be separated by a fused-silica column coated

with SE-30 with helium as carrier gas, FID, and FTIR

spectrometric detection with a CdHgTe detector.This kind

of analytical method is not common due to the high cost of

instrumentation.

76

4.3 THEORY OF DETECTION

4.3.1 Theory of conductometric detection*"^^^

When electrolytes dissolve in solvents of high

dielectric constant such as water they dissociate into

their constituent ions and the solutions are electrically

conducting. The electrical properities of the solution

obey Ohm's law in that the resistance to current flow may

be defined as

V = iR ( Eq. 4.1 )

where V is the voltage and i is the current that flows

through an element of solution. The resistance R is a

function of temperature and the concentration of the

electrolyte. The resistance of an element is proportional

to its thickness, 1, and inversely to its cross-sectional

area, A. Therefore

R = al/A ( Eq. 4.2 )

where o is defined as specific resistance and is in ohm cm.

If several ions in a solution can conduct, they

can be thought of as conductors in parallel, with the net

resistance R of the composite being related to the

separate resistances Rj； R?, R3

77

1 / R = 1 / R I + I / R 2 + I / R 3 + … ( E Q . 4 . 3 )

Since the reciprocal of resistances are additive,

electrolytes can be conveniently treated by using the

conductance, G, which is the reciprocal of resistance. The

specific conductance, denoted k, is therefore

k = 1/a = 1/AR ( Eq. 4.4 )

The conductance of the ions produced by one gram

equivalent of electrolyte at any concentration can be

evaluated by considering a cell with electrodes placed one

cm apart but of sufficient area as to just enclose the

whole volume containing the one equivalent of electrolyte.

The conductance of such an assembly is termed the

equivalent conductance and is denoted r . Equivalent

conductance r and specific conductance k are related in

the following way

r = 1000 k / C ( Eq. 4.5 )

where C is the concentration of the electrolyte in

equivalents per liter, and has the unit of cm2 equiv' ohm''.

Combining equations ( 4.4 ) and ( 4 . 5 ) gives the following

equation which relates equivalent conductance to measured

conductance, G

G = r C / 1000 K ( Eq. 4.6 )

78

where K = 1 / A and is called the cell constant and G has

the unit of ohm"' or mho. If G is expressed in /imhos, equation ( 4 . 6 ) becomes

G = r C / 10-3 K ( Eq. 4.7 )

The equivalent conductance of an electrolyte may be

considered to consist of the ionic conductance of the

cation and that of the anion r"

R = T + + T"- ( Eq. 4.8 )

At infinite dilution, the ions migrate independently

of each other and the limiting equivalent conductance r� is

thus related to the limiting ionic conductance T+Q and T� b y

r 。 = T + O + T-� ( Eq. 4.9 )

事

In ion chromatography, the concentration of the

eletrolytes are usually in the range of millimole and

therefore can be considered as at infinite dilution.

Equation ( 4 . 7 ) then becomes

G = r � C / 10-3 K

= ( r + � + r � ） C / 10-3 K ( Eq. 4.10 )

Consider an electrolyte E which contains a single

79

species and which can dissociate partially to give anions

and cations. When E acts an element and under column

equilibrium conditions, the background conductance of the

eluent as indicated by the conductivity detector can be

deduced according to Fritz et as

GBackgrouncl = ( ^ + 厂。’̂ ) C^l^ / lO'' K ( E q . 4 . 1 1 )

where C^is the concentration of the eluent species and is

the fraction of the eluent species which is present in the * ionic form.

4.3.2 Indirect conductometric detection

The mobile phase employed for this work is a 30 %

aqueous acetonitrile containing a background conducting

species, in this case, trisodium citrate.

When the column is in equilibrium state, there

exists a dynamic equilibrium for the adsorbed citrate ions

and the sodium ions between the mobile phase and the active

sites of the polar cyano column. The interactive forces

are mainly polar interations with a little non-polar

interations being involved because of the ionic character

of the conducting species. Hence, a continuous background

signal is maintained.

Since camphor and menthol contain no charge, we can

assume that the interaction with the column active sites

80

for these compounds is mainly hydrophobic interaction.

When the sample containing camphor and menthol enter

the column, some of the active sites will be occupied by

these compounds and some citrate ions will be displaced and

leave the column. In order to maintain the charge balance,

it is believed that sodium ion will also be displaced, when

the analyte leaves the column, citrate ions and also sodium

ions from the mobile phase will re-enter the column and

hence a sudden drop of the background conductance will

result and a negative peak is therefore detected by the

detector.

«

81

4.4 EXPERIMENTAL

4.4.1 Apparatus

4,4.1.1 The liquid chromatograph

The high performance liquid chromatographic system was

composed of a solvent delivery module ( Beckman llOB ) with

an Free-flow pulse dampener ( Alltech Associates ) ； a

conductivity detector ( Wescan model 21511001 ) with a

temperature controller ( model 24 02 0001 ) and column

compartment ( model 26650051 )； an injection port (

Rheodyne ) with a 20 or 100 /il sample loop ； an

adsorbosphere CN column of length 250 mm, internal diameter

of 4.6 mm and particle size of 5 /xm ( Alltech Associates ),

protected by a lOmm x 4. 6min guard column packed with the

same material as the column; and an integrator ( Hitachi 1

833A Data Processor ).

4.4.1.1.1 Parameter setting of the detector

The following settings for the

conductivity detector were kept unchanged

during the experiment.

Parameter Settings

Detector Range 1

Column Temperature 30 °C

82 r

Peaks were detected as negative changes in

conductance, and the detector-integrator connections

were reversed in polarity to give positive display of

peak on the integrator.

4.4.1.2 The gas chromatograph for the counter check eras-

liquid chromatographic method

The standard method employed for counter checking the

sample components is the method currently in use by the

Hong Kong Government Laboratory in the daily analysis of

pharmaceutical products submitted to the Laboratory. ̂^

The instrument consisted of the following

components :

Gas Chromatograph : HP 5890 Series II

Column : Supelcowax TH 10 Fused Silica

Capillary column, 30 m, 0.53 mm

internal diameter, 1.0 jim film

thickness.

Detector : Flame Ionization Detector (FID)

Integrator : HP 3396A Integrator

83

4.4.1.2.1 Operational conditions

Carrier gas : Helium

Flow 一 rate : 7.5 ml / min

Injector Temp. : 23 0 °C

Detector Temp. : 250 �C

Column Temp. : Temp, programming

Initial Temp. : 70 °C

Hold : 0 min.

Ramp : 2 0 °C / min

Final Temp. : 170 �C

Hold : 38 min.

4,4.1.4 Glassware

All volumetric flask and pipettes were of grade A and

grade B and were calibrated to the relevant BS standard

before use.

4.4.2 Reagents and materials

All reagents used were of analytical reagent grade and used without further purification.

4.4.2.1 Water f Ultra pure water )

All water used during the experiment was distilled

water purified by the Millipore Milli-Q50 ultra pure water

84

system ( Millipore, France )• The water was deionized and

filtered through the 0.1 /m filter from the system. The

resistivity of water produced was 18 Mn / cm.

4,4.2.2 Standard solutions

10 mg/ml Camphor and menthol standard solutions were

prepared by dissolving exact amount of each compound into

the mobile phase and the solutions were further diluted to

the required concentrations by mobile phase.

4.4.3 Aqueous mobile phase

Various mixture of the mobile phase were prepared by-

dissolving appropriate amount salt into 30 % aqueous

acetonitrile solution. The mobile phases prepared were

then filtered through a Millipore HA type filter of 0.45 ^m

pore size to remove any trace of solid particles. The

filtered mobile phases were then degassed by shaking in an

ultrasonic bath ( Branson 1200 ) for at least half an hour

before being used.

4.4.4 Column conditioning

I

The column was conditioned each time when a new mobile

phase was used. This normally took two hours to do so.

Whenever the new mobile phase was changed from one

85 , r

system to another , the column had to clean thoroughly by

20 column volume of water ( this usually took two hour at

a flowrate of 1 ml / min ) followed by another 20 column

volume of 1 : 1 mixture of methanol and water so as to

ensure that the salts adsorbed on the column bed were I

completely removed.

4.4.5 Sample treatment

Generally the liquid samples were diluted to

appropriate concentrations with the mobile phase. For the

cream base samples, an accurate amount of sample was

weighed and dissolved in mobile phase by shaking in an

ultrasonic bath ( Branson 1200 ) for fifteen minutes. Any

undissolved particles were filtered and the solution was

transferred to a calibrated volumetric flask and made up to

the mark by mobile phase.


Samples were introduced into the column through the

external loop injection valve by using a 1 ml syringe

fitted with a 0.2 jLtm disposable filter.

86

4.5 RESULT AND DISCUSSION

The objective of the following work was to find out

the optimum condition for the proposed liquid

chromatographic method. Calibration graphs were then

obtained from the standard camphor and menthol solutions.

A series of sample were then taken for analysis to evaluate

the practicability of the proposed method. Finally the

analytical results obtained were compared with those

obtained by a developed official method in order to check

the accuracy of the proposed method.

4.5.1 Choice of background conducting species

Different kind of conducting species of 1 mM were

mixed with 30 % aqueous acetonitrile to produce different

mobile phases. They were then passed in turn to the column

for conditioning, and 10 ixg each of camphor and menthol

were injected after equilibrium had been reached. The peak

area and retention time were then recorded in each case and

the results are summarized in Table 4.1.

I

87 *

Table 4.1 Effect of conducting species in the mobile phase on the sensitivity and retention on camphor and menthol

Retention time Conducting Peak area

• ( min ) species (1 mM) " “ “ Camphor Menthol Camphor Menthol

‘ Na Benzoate 1827 2658 6.86 7.22

K hydrogen 2896 4669 6.78 7.17 phthalate

Na salicylate 2097 2733 6.7-3 7.09

Na citrate 8226 12231 6.75 7.13

Na oxalate 4902 5171 6.76 7.13

Na sorbate 3339 3770 6.69 7.08

Na acetate 1363 2217 6.71 7.09

Na formate 3415 4878 6.73 7.09

Na tartrate 5498 7944 6.63 6.98

Trimethyl- 5964 7507 6.77 7.16 ammonium chloride t

Methylammonium 5108 8212 6.69 7.06 chloride

Na : sodium K : potassium

88

From Table 4.1, we can find that tri-sodium citrate

gives the highest signal with similar separation ability (

difference in retention between camphor and menthol is

equal to 0.4 minutes ) . Therefore, we can conclude that

trisodium citrate is amoung the best conducting species and

should be chosen as the conducting background species.

It is obvious that with different conducting species,

the retention ability differs very little as you can see

from the difference in retention between camphor and

menthol. It can be inferred that the retention mechanism

does not depend very much on the polar conducting species,

the major function of the species is to provide the

conducting background.

Tri-sodium citrate gives larger signal than that of

formate or acetate, it seems contradicting since formate

and acetate is smaller in size and possesses larger ionic

mobility and hence larger conductance. The reason for the

cause is that citrate possesses 3 sodium and when one

citrate ion enters the active site in the column bed, 3

sodium ions will follow in order to maintain the charge

balance and therefore result in a larger conductance

change.

i.

89

4.5.2 Effect of background conducting species concentration on peak intensity and retention time

Different concentrations of sodium citrate in 3 0 %

aqueous acetonitrile were prepared and 20 fig of camphor and

menthol were injected into the column after equilibrium has

been reached. The peak area and retention time were taken

and was summarized in Table 4.2.

Table 4.2 Concentration effect of background conducting species on peak intensity and retention time

Concentration Peak area Retention time

of sodium ( min )

citrate ( mM ) " ‘ ‘ “ Camphor Menthol Camphor Menthol

1 4831 6307 6.39 6.96

2 11672 15003 • 6.42 7.01

3 18299 24347 6.48 7.06

4 28968 35708 6.69 7.31

It can be seen from the table that higher the

concentration of the conducting species, larger the

detector response and this means that more conducting

species were displaced from the column by the analyte.

The effect of the concentrationas on retention time of

90

the analyte is not obvious. It only shows a little

increase in retention with increasing concentration of the

conducting species. One possible reason may be due to the

fact that as the concentration of the conducting species

increases, the mobile phase will become more polar and

therefore, the analyte are then pushed to the column bed

since it is less polar. As a result, the analyte are

retained more strongly as increasing conducting species

concentration. It should be stressed that this effect is

not prominent and the separation mechanism is based mainly

on the hydrophobic interation.

4.5.3 Effect of changing the water / acetonitrile ratio

The retention behaviour of camphor and menthol on the

column using mobile phases with different water /

acetonitrile ratios was studied and the results are shown

in Table 4.3. 1 mM of sodium citrate is employed.

From the table, we can observe that with increasing

acetonitrile content, the elution time will be decreased

and this suggested that hydrophobic interaction of the

analytes with the column bed is mainly due to hydrophobic

interaction between the analytes and the column bed. j

It is probably correct to choose a lower fraction of

acetonitrile in order to give a good separation but

actually we must consider the practicability because too

91

small a fraction of acetonitrile in mobile phase will have

problem in dissolving the sample in the mobile phase for

analysis. Therefore, 30 % acetonitrile was chosen as the

analytical condition.

Table 4.3 Concentration effect of acetonitrile on retention time

Concentration of Retention time ( min )

acetonitrile ( % ) Camphor Menthol

10 6.98 7.70

20 6.87 7.50

30 6.37 6.96

40 5.95 7.31

3.5.4 Preparation of calibration graphs

The calibration graphs for camphor and menthol were

obtained by plotting the peak area against the

corresponding concentration of camphor and menthol, which

was made by the appropiate dilution of the standard - stock

solution. The data for the calibration graphs are listed

in Table 4.4' and Table 4.5. The calibration graphs are

shown in Figure 4.1 and 4.2.

92

Table 4.4 Data for the calibration of camphor

Concentration ( /xg/ml ) Peak area ( x lO^counts )

50.85 8.889

101.70 17.904

203.40 34.663

406.80 65.631

610.20 97.988

813.60 129.530

1017.00 162.558

Linear working range : 50-1000 jug / ml

Slope : 157.9 ml / /xg

Intercept : 1.613

Correlation coefficient : 0.9999

Conditions of the chromatograph

Detection temperature : 3 0° C

Mobile phase : 4 mM NaCit. in 30 % mM

aqueous acetonitrile

Flow 一 rate : 1.0 ml / min

Operating pressure : 2,000 psi

93

2 0 0 . 0 0 〕

三 /

150.00 一 /

1 ： / W 100.00 - /

！： / 50.00 - /

••00 —[―I~I~[―i~I~rn~I~I~|—I~|~1~I~1I~~[―Irn 0.00 500.00 1000.00


Figure 4.1 Calibration graph for the determination of camphor

94

Table 4.5 Data for the calibration of menthol

Concentration ( jug/ml ) Peak area ( x lO^counts )

50.55 13.169

101.10 20.096

202.20 39.901

404.40 75.469

606.60 111.042

808.80 146.853

1011.00 182.643

Linear working range ： 50-1000 /xg / ml

Slope : 177.2 ml / fig

Intercept ： 3.53 0

Correlation coefficient : o.9999

Conditions of the chromatoaraph

Detection temperature : 3 0° C

Mobile phase : 4 mM NaCit. in 30 %

aqueous acetonitrile

Flow - rate : 1.0 ml / min

Operating pressure ： 2,000 psi

95

200.00 -J ‘

/ 150.00 - J

I / 一 100.00 - /

！ / 50.00 一 /

/ 0.00 I I I I I I I I I I I I I I I I I I I I I I I I I

0-00 500.00 1000.00


Figure 4.2 Calibration graph for the determination of menthol

96

4.5.5 Precision test for the proposed method

In order to test the reproducibility of the proposed

method using the optimised system setting and mobile phase,

aliquots of camphor and menthol with a known concentration

are injected ten times into the column. The relative

standard deviation ( R.S.D. ) of the peak area for camphor

and menthol were calculated and were both found to be 0.9

% ( Table 4.6 ) . This means that the proposed method is

stable and good for analytical purposes.

•

Table 4.6 Precision test of the proposed method

20 ug Peak Area ( counts ) camphor/menthol

injected Camphor Menthol

1 28968 35708

2 29017 35999

3 28966 35567

4 28263 35678

5 . 28997 34967

6 29115 35632

7 28778 35890

8 28909 36087

9 28663 35478

10 28563 35778

R-S.D. 0.9 % 0.9 %

97

4.5.6 Determination of camphor and menthol in

pharmaceuticals

The pharmaceutical products for this project were

purchased from Watson's medicine department store and a

brief description of the samples are shown in Table 4.7.

Each sample was diluted to an appropriate

concentration with the mobile phase, and 20 /xl of the

diluted sample was injected into the column three times.

The amount of citrate was deduced from the calibration

graph. The results of the analysis are shown in Table 4.8

and the respective results obtained using the standard

• methodi3 and the claimed label values, if any, are also

shown in Table 4.8 for comparison. Typical chromatograms

for some of the samples were shown in Figures 4.3a to

Figure 4.3c.

The contents of citrate found by the proposed method

agree well with the respective claimed label value, and

those obtained using the standard method.

«

98

Table 4.7 Description of pharmaceutical preparation

Sample Sample Ingredient no.

1 White flower Wintergreen oil, menthol oil. Eucalyptus oil, camphor, etc.

2 Axe Brand Ditto universal oil

3 Kwan Loong Menthol, camphor, methyl oil salicylate, Eucalyptus oil,

white oil, etc.

4 Golden lion Ditto shield medicated oil

5 Tiger oil Ditto

6 Banjemin Camphor, spirit turpentine, Jaminton Eucalyptus oil, liquid healing oil paraffin.

7 Zheng Gu Shui Camphor, menthol, Croton Tigliim, Inula Cappa, etc.

8 Ammeltz Methyl salicylate, thymol, camphor, menthol, etc.

9 Radian-B Menthol, camphor, methyl salicylate, ammonium salicy-late, etc.

*

10 Vicks Menthol, camphor, . Eucalyptus oil, etc.

99

Table 4.8 Assay of content of camphor and menthol in drug sample

Sample Camphor and menthol content No.

Proposed Standard Label value method method

1 4.89 % ( C ) 4.81 % ( C )

14.6 % ( M ) 14.4 % ( M )

2 4.93 % ( C ) 4.91 % ( C ) 5 % ( C )

19.6 % ( M ) 20.5 % ( M ) 20 % ( M )

3 . 9.73 % ( C ) 10.1 % ( C ) 10 % ( C )

25.6 % ( M ) 25.9 % ( M ) 25 % ( M )

4 9.75 % ( C ) 9.89 % ( C ) 10 % ( C )

19.6 % ( M ) 19.2% ( M ) 20 % ( M )

5 17.0 % ( C ) 17.6% ( C ) 17.5% ( C )

7.7 % ( M ) 7.9% ( M ) 8.0% ( M )

6 2.96 % ( C ) 3.10 % ( C ) 3 % ( C )

nil % ( M ) nil ( M ) nil ( M )

7 2.04 % ( C ) 1.99 % ( C ) 2 % ( C )

2.89 % ( M ) 2.91 % ( M ) 3 % ( M ) 8 51.0 mg/ml 50.2 mg/ml 52 mg/ml

( C ) ( C ) ( C ) 51.5 mg/ml 51.3 mg/ml 52 mg/ml

( M ) ( M ) ( M ) 9 0.58 % ( C ) 0.58 % ( C ) 0.6 % ( C )

1.43 % ( M ) 1.39 % ( M ) 1.4 % ( M )

10 4.51 % ( C ) 4.53 % ( C ) 4.73 % ( C )

2.50 % ( M ) 2.49 % ( M ) 2.6 % ( M )

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102

4.5.7 Recovery tests

In order to test the reliability of the method,

recovery tests were also carried out by spiking known

amounts of standard camphor and menthol to each sample

before any treatment and the percent recovery was then

calculated.

The percent recovery of the proposed method ranged

from 95 to 105 percent ( see Table 4.9 ) and may be

considered very good.

Table 4.9 Recovery test for the proposed method

Sample Amount added Amount found Percentage No. ( jLtg / ml ) ( jLtg / ml ) recovery 1 100 ( C ) 96.7 ( C ) 97 ( C )

200 ( M ) 197.8 ( M ) 99 ( M ) 2 100 ( C ) 95.9 ( C ) 96 ( C )

200 ( M ) 200.8 ( M ) 100 ( M ) 3 100 ( C ) 102.9 ( C ) 103 ( C )

200 ( M ) 198.0 ( M ) 99 ( M ) 4 100 ( C ) 96.9 ( C ) 97 ( C )

200 ( M ) 196.8 ( M ) 98 ( M ) 5 100 ( C ) 95.2 ( C ) 95 ( C )

200 ( M ) 193.7 ( M ) 97 ( M ) 6 100 ( C ) 99.6 ( C ) 100 ( C )

200 ( M ) 198.1 ( M ) 99 ( M ) 7 100 ( C ) 104.7 ( C ) 105 ( C )

200 ( M ) 199.6 ( M ) 100 ( M ) 8 100 ( C ) 98.0 ( C ) 98 ( C )

200 ( M ) 194.9 ( M ) 97 ( M ) 9 100 ( C ) 95.1 ( C ) 95 ( C )

200 ( M ) 191.9 ( M ) 96 ( M ) 10 100 ( C ) 103.9 ( C ) 104 ( C )

200 ( M ) 198.0 ( M ) I 99 ( M )

average of duplicate result

103

4.6 CONCLUSION

An accurate, simple and efficient high performance

liquid chromatographic method for the determination of

camphor and menthol in pharmaceutical preparations was

developed. The proposed method applied reversed bonded

phase chromatography, employing an indirect method of

detection, where sodium citrate is used as a conducting

species produces a constant background of conductivity.

The conductivity detector will respond to the presence of

camphor and menthol by producing negative peaks. Hence,

the method does not depend on any specific functional group

of the analyte. This method has been applied successfully

to many types of pharmaceutical preprations with good

precision and accuracy.

I.

104

REFERENCES

1. The Merck Index, 11th edition, 1989, Merck & Co., Inc.

2. J. E. F. Reynolds, Editor, MARTINDALE, the Extra

Pharmacopoeia, 28th edition, 1982, The Pharmaceutical Press, London

3. R. D. Mann, Modern Drug Use, An Enquiry On Historical

Principles, 1984, MTP Press, U.S.A.

4. Handbook of Nonprescription Drugs, 9th edition, 1990,

American Pharmaceutical Association

5. Q. Wu and N. Qiu., Zhongguo-Yaoke-Daxue-Xuebao, 1989, 20 ( 1 ), 13

6. L. Cen and Z. He, Fenxi-Ceshi-Xuebao, 1993,

12 ( 2 ) , 30

7. G. S. Sadana and A. J. Potdar, Indian Drugs, 1991,

28 ( 8 ), 382

8. Y. Luo, R. Liu, F. Wu and P. Han, Yaowu-Fenxi-Zazhi,

1992, 12 ( 4 ), 240

9. H. Gao, Z. Sha, and W. Sun, Yaowu-Fenxi-Zazhi,

1989, 9 ( 6 ), 323

105

10. X. He, Yaowu-Fenxi-Zazhi, 1987, 7 ( 3 ) , 176

11. M, Sugrue and J. J. Brennan, Anal. Proc, ( London ),

1987, 24 ( 6 ), 191

12. Y. Gohee and J. Alary, Spectra-2000, 1985,

13 ( 108 ), 33

13. H. S. I. Tan, P. A. Kemper and P. E. Padron,

J. Chromatogr., 1982, 238 ( 1 ), 241

14. H. Small, Ion Chromatography, 1989, Plenum Press,

New York, p.107

15. J. S. Fritz, D. T. Gjerde and C. Pohlandt, Ion

Chromatography, 1982, Dr. Alfred Huthig,

Heidelberg, p.89

16. J. G. Tarter, Editor, Ion Chromatography, 1987, Marcel

Dekker, Inc., New York and Basel

17. J. S. Fritz, D. T. Gjerde and R. M. Becker, Anal.

Chem., 1980, 52, 1519

18. J. S. Fritz and D. T. Gjerde, Anal. Chem., 1981, 53, 2324

106

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Documents

u · 3.6 Signal of 2 /xg citrate at various wavelengths 56 3.7 Data for the calibration of citrate 58 3.8 Precision test of the proposed method 60 3.9 Description of pharmaceutical