Br. J. Anaesth. (1986), 58, 345-352

A STATIC CALIBRATION METHOD FOR THE GASCHROMATOGRAPHIC DETERMINATION OF PER CENTCONCENTRATIONS OF VOLATILE ANAESTHETIC AGENTS

W. M. GRAY

The ability to perform accurate determinations ofvolatile anaesthetic agents in gas mixtures is anessential requirement for the modern anaestheticslaboratory. One analytical method capable of veryhigh accuracy is interferometry (Edmondson,1957), and this technique has been used for theaccurate determination of per cent concentrationsof volatile anaesthetics (Paterson, Hulands andNunn, 1969; Kay, Cohen and Wheeler, 1982).However, precision interferometers are delicate,expensive and specialized instruments, and notevery anaesthetics laboratory can justify thepurchase of one.

In comparison with such interferometers, gaschromatographs are robust, inexpensive andversatile instruments, and are commonly found inanaesthetics laboratories. Although they arecapable of great precision, they are not widely usedfor accurate determinations of volatile agents, themain reason for this probably being that gaschromatographs require frequent calibration, andit is difficult to obtain or prepare gas mixtures forwhich the concentration of the volatile agent isknown with sufficient precision to allow accuratecalibration.

Standard gas mixtures can be prepared by staticor dynamic methods (Nelson, 1971; Barratt,1981). Static mixtures are simple to prepare, butsuffer from the potential disadvantage that theactual concentration obtained will be less than thetheoretical value because of adsorption on (orsolution in) the materials on the inside of the vesselcontaining the mixture. This effect is especiallyserious for components present in trace amounts(Saltzman, 1961; Groth and Doyle, 1968), but canalso be significant for concentrations in the per

W. M. GRAY, B.SC, PH.D., West of Scotland Health Boards,Department of Clinical Physics and Bio-Engineering, 11 WestGraham Street, Glasgow G4 9LF; and University of Glasgow,Department of Anaesthesia, Western Infirmary, Glasgow G i l6NT.

SUMMARYA method is described for preparing staticstandards of per cent concentrations of volatileanaesthetic agents, that is designed to minimizeadsorptive losses in the container used for thestandards. The extent to which this aim has beenrealized was investigated for atmospheres ofhalothane, enf/urane and isoflurane, both bymonitoring the relative concentration within thecontainer over a period of 30 min after thestandard was prepared, and by measuring theabsolute concentration of the volatile agents. Gaschromatography was used for both sets ofmeasurements, with liquid standards being usedfor the absolute measurements. No discernibleloss of volatile agent occurred over the 30-minperiod, and the measured concentrations agreedclosely with the predicted values. The standardmixtures prepared by the method were suitablefor calibrating a gas chromatograph, and allowedaccurate determinations of volatile anaestheticsto be made. The relative standard error of thepredicted concentration for a single standardmixture was less than 0.5%, and the calibrationaccuracy could be further improved by taking themean chromatograph response to several'standardmixtures prepared in succession.

cent range if the component concerned has arelatively high boiling point, as is the case withvolatile anaesthetic agents (Stoelting, Ellis andLongshore, 1973). For trace concentrations ofanaesthetic agents, the effects of adsorption can beovercome by the use of dynamic methods (Barratt,1981; Gray and Burnside, 1984), but thesemethods are less suitable for preparing higherconcentrations of volatile agents because of thedifficulty of achieving the required rate ofevaporation of the liquid. Thus, there is a need for

346 BRITISH JOURNAL OF ANAESTHESIA

a reliable static method for preparing standards ofper cent concentrations of volatile anaestheticagents for gas chromatograph calibration.

This paper describes such a method. Precautionshave been taken to minimize adsorptive losses, andthe method has been assessed to determinewhether any significant losses occur.

DESCRIPTION OF METHOD

ApparatusThe standard mixtures were made up in a gas

sampling bulb (fig. 1), constructed of glass and ofnominal volume 500 ml (Alltech Associates). Theends of the bulb were fitted with PTFE stopcocks,and the side socket held a standard 0.25-inchcylindrical septum (Thermogreen half-hole type(Supelco)). Liquid anaesthetic agent was injectedthrough the septum from a 100-ul Hamiltongas-tight syringe, fitted with a 5-cm long, 0.15-mmbore needle (22 s gauge), and samples werewithdrawn from the bulb through the septum into

a 50-ml Hamilton gas-tight syringe, fitted with aHamilton two-way valve and a 5-cm long, 0.4-mmbore needle (22 gauge).

Preparation of standards

The bulb was flushed for several minutes withthe required carrier gas and then the gas flow wasturned oft" and the stopcocks closed. The requiredvolume of liquid agent was injected to the bulbfrom the 100-ul syringe. Following the evaporationof this liquid, which occurred within about 1 min(and could be hastened by holding the bulb in thepalm of the hand), the gases were mixed by rapidlyrotating the bulb to and fro in the hand. Theneedle of the 50-ml syringe was then inserted tothe bulb and the syringe flushed once bywithdrawing about 50 ml of the mixture into thesyringe and expelling it back into the bulb. Theturbulence produced by returning this samplepromoted further mixing. A sample of about 50 mlwas then withdrawn into the syringe for analysis.

Following the evaporation of the injected liquid,

FIG. 1. Gas sampling bulb in which standard mixtures were prepared, with 100-jil syringe used to injectthe volatile liquid and 50-ml syringe used to extract a sample of the mixture.

CHROMATOGRAPH CALIBRATION FOR VOLATILE ANAESTHETICS 347

the gas inside the bulb was at a pressure aboveatmospheric, by a fraction approximately equal tothe fractional concentration of the volatile agent inthe mixture (between 3% and 4% for a liquidinjection of 100 ul (see table III)). However,when the sample was withdrawn into the 50-mlsyringe, the pressure was reduced below atmos-pheric, since the total volume increased by about10%. Therefore, in order to avoid dilution of thesyringe contents when the two-way valve wasopened to allow some of the mixture to be injectedto a gas chromatograph, it was necessary first tocompress the gas in the syringe by pushing in theplunger by about 10% (5 ml) with the two-wayvalve closed.

In this laboratory, standard mixtures aregenerally prepared by injecting a full 100 |il ofliquid agent to the bulb. Henceforth, the term"standard mixture" will refer to a mixtureprepared in this way.

Calculation of concentration

The concentration of volatile agent x in astandard mixture (Cx) is given by:

where nx (number of moles of x in the bulb) and nc(number of moles of carrier gas in the bulb) aregiven by:

_VxQd)xpx(gBd-l)xl0r*

(litre)n

c ve (litre mol"1) xfwhere the symbols on the right sides of theseequations are as denned in table I. The value of/, the factor to correct the molar volume of thecarrier gas from the reference conditions of 20 °Cand 101.325 kPa to ambient temperature andpressure, is given by the following equation, on theassumption that the gas obeys the ideal gasequation (Zemansky and Dittman, 1981):

, (273.15+T(°C)) 101.325/ = . _ x-293.15 P(kPa)

Cx(%) = xlOO

where T and P are ambient temperature andpressure, respectively.

Table I also lists the values of the quantitiesrequired to calculate Cx for halothane, ennuraneand isoflurane, including those found by thecalibration procedures now to be described.

TABLE I. Data required to calculate concentration! of halothane, enflurane and isoflurane in standardmixtures. Values are mean ± SEM {inhere no SEM is given, the SEM is considered negligible in comparisonwith other contributions to the total SEM). jSEM quoted is for a single injection (see text). Number ofdeterminations are for data from present study. References concern data not determined in present study.Densities are at nominal 20 °C (actually between 19.5 "C and 20.5 °Q. %At 20.5 °C and 101.325 kPa,assuming ideal gas behaviour. * Calculated from value of universal gas constant and ideal gas equation

(Zemansky and Dittman, 1981)

Quantity and symbol

Volume of liquid delivered by100-ul syringe (Vz)

Density of liquid halothane (Ph»j)Density of liquid ennurane (/?„,,)Density of liquid isoflurane (A»O)Molar mass of halothane (M^jMolar mass of enflurane (Af^,)Molar mass of isoflurane (Afli0)Volume of sampling bulb (KJMolar volume of carrier gas$ (t/JFactor to convert vc to ambient

temperature and pressure ( / )

Value

99.70±0.36t

1.8693±0.0006gml-'1.5221 ±0.0004 g ml-'1.5035 ± 0.0006 gml-'

197.39 g mol"1

184.50 g mol"1

184.50 g mol"1

0.57180±0.00002 litre24.055 litre mol"1

See text

No. ofdeter-

minations

30

555

———5

——

Reference

—

———

Grant (1978)Grant (1978)Grant (1978)

—*

—

348

Volatile agents usedThe method has been developed and assessed

using three of the most widely used volatileanaesthetic agents: halothane (Fluothane, ICI),enflurane (Ethrane, Abbott) and isoflurane (Aer-rane, Ohio).

CALIBRATION

MethodsVolume of sampling bulb. This was found bydetermining the weight of distilled water requiredto fill the bulb, and dividing this by the density atthe appropriate temperature (Diem and Lentner,1970).

Liquid densities. These were measured byweighing samples dispensed into 10-ml volumetricflasks (Vuline or Volac) manufactured to BS 1792Class A accuracy, that is with a maximum error of±0.025 ml (British Standards Institution, 1982).The dispensing and weighing process was carriedout twice with each of five flasks, and the averageweight for each flask used in the final calculation.All measurements were carried out at temperaturesbetween 19.5 °C and 20.5 °C.

Volume delivered by 100-pi syringe. Analyticalgrade 1,1,1-trichloroethane (BDH) was used as atest liquid and its density determined as describedabove. The liquid delivered by the syringe wascollected in a 7-ml glass vial fitted with anopen-hole screw cap and a Teflon-lined siliconeseptum (Alltech Associates), and weighed. Thiswas done 30 times. The delivered volume wasobtained by dividing the mean weight by thedensity.

ResultsThese are shown in table I. The following pointsshould be noted:

Liquid densities. The value found for halothane(1.8693 ±0.0006 g ml"1) agrees with that reportedby Bottomley and Seiflow (1963) (1.8692 g ml-1 at20 °C). The values for enflurane and isoflurane areconsistent with the values quoted in the manufac-turers' data sheets for the specific gravity at 25 °Crelative to water at 25 °C (1.517 for enflurane and1.496 for isoflurane).

Volume delivered by 100-id syringe. The valuequoted for the error in this volume is the standard

BRITISH JOURNAL OF ANAESTHESIA

error of the mean (SEM) corresponding to a singleinjection (i.e. it is the standard deviation (SD)),since this is the relevant statistic for estimating theuncertainty associated with the composition of astandard mixture prepared from a single liquidinjection. The error in the estimate of thedelivered volume is the dominant factor inlimiting the accuracy with which the compositionof a prepared mixture is known, and indeed theerrors of the other quantities in table I arenegligible in comparison.

ASSESSMENT OF METHOD

MethodsSample analysis

Samples were analysed by means of a PyeUnicam PU 4500 gas chromatograph using a 2-mlong 4-mm i.d. glass column packed with 80-100mesh Chromosorb WHP coated with 10%silicone OV101. The carrier gas was oxygen-freenitrogen (BOC) 40 ml min" Sand a flame ionizationdetector at 250 °C was used. The output signal wasfed to a Hewlett-Packard HP 3390A reportingintegrator, and peak area used as a measure of theresponse of the chromatograph.

Time variation of standard concentrationsFor each volatile agent, a standard mixture in

medical quality air (BOC) was prepared. A samplewas withdrawn from the bulb and analysedimmediately after all the liquid had evaporated(time zero) and at 2.5-min intervals for a further30 min. The samples were taken with a 100-ulgas-tight syringe similar to that used to inject theliquid. The syringe was flushed three times beforethe sample was taken, and the transfer of thesample from the bulb to the chromatograph wassufficiently quick to avoid any sample loss from thesyringe. (No measurable loss occurred overtransfer times of up to 1 min.) The oventemperature was 200 °C for the chromatographicanalyses, giving a retention time of 0.5 min for allthree agents.

Linear regression analysis with time as theindependent variable and peak area as thedependent variable was performed for each agentand the value of the slope tested for significantdeparture from zero, using a two-tailed Student'st test and a critical P value of 0.05 (Chatfield,1983).

CHROMATOGRAPH CALIBRATION FOR VOLATILE ANAESTHETICS 349

Measurement of standard concentrationsThe concentration of each agent in the standard

gas mixture was measured by gas chromatography,against a liquid standard consisting of a nominal1 % v/v solution of the agent in analytical grade1,1,1-trichloroethane. The liquid standard wasprepared by injecting 100 ul of the volatile agentfrom the calibrated 100-ul Hamilton syringe intoa BS 1792 Class A 10-ml volumetric flask andmaking the volume up to 10 ml with the solvent.Mixing was achieved by energetic manual shakingof the stoppered flask. One-microlitre aliquots ofthis standard were injected through the injectorport septum of the chromatograph using a 1-ulplunger-in-needle type SGE syringe. The gassamples were removed from the bulb with the50-ml Hamilton syringe and injected to thechromatograph by means of a Valco gas samplingvalve fitted with a sample loop of nominal volume100 ul. Five separate gas mixtures were made upfor each agent, and the sample from each analysedin duplicate. An aliquot of the liquid standard wasanalysed before each duplicate gas analysis.

The chromatograph oven was set at the highesttemperature that would allow satisfactory separa-tion of the anaesthetic agent from the solvent peaksfor the liquid standard. This was 55 °C forhalothane (retention time 1.5 min) and 70 °C forenflurane and isoflurane (retention time 0.9 min).The injector temperature was 150 °C.

The number of moles of anaesthetic agent in thegas sample was obtained by multiplying thenumber of moles in the liquid sample (obtainedfrom the standard concentration, density, molarmass and injected volume) by the ratio of the peakarea for the gas sample to that of the liquid sample,and the concentration in the gas sample calculatedby dividing this number of moles by the totalnumber of moles in the sample; this latter valuewas obtained from the volume of the samplingloop, on" the assumption that the gas displayedideal behaviour (Zemansky and Dittman, 1981).The whole calculation was performed using themean values of the relevant quantities. Thevolumes of liquid standard injected and of the gassampling loop were measured as follows:

Calibration of 1-fil syringe. This was performedgravimetrically, using trichloroethane and a closedvial, as described above for the 100-ul syringe.However, because of the small volume, weighing

was performed for 10 pooled injections, and thiswas done five times.

Calibration of sampling loop. Samples from astandard mixture of enflurane were injected to thechromatograph either from a 250-ul Hamiltongas-tight syringe through the injection portseptum or via the gas sampling valve from the50-ml syringe, and the loop volume obtained bymultiplying the injected volume by the ratio ofpeak areas for the loop and syringe samples.Preliminary measurements established that theloop volume was approximately 130 ul, and thedefinitive syringe injections were made using thisvolume (as indicated on the syringe graduations).The actual volume delivered by the syringe for thissetting was determined gravimetrically as describedabove for the 100-ul syringe. The chromatographicprocedure differed from that described previouslyin that an empty glass column, 2 m long, 2 mm i.d.,and a carrier gas flow of 10 ml min"1 were used,to avoid injecting to the high pressure that resultswhen a packed column is used and that wouldcause significant sample retention in the syringeneedle. The replacement of the packed column bythe empty one reduced the pressure at the top ofthe column from 80 kPa to 0.7 kPa above ambientpressure. The use of an empty column waspermissible because the volatile agent was the onlyconstituent of the mixture to which the flameionization detector was sensitive. However, toavoid the possibility that the sensitivity of thedetector might be altered by different amounts ofoxygen in the syringe and loop samples, thestandard mixture was made up in oxygen-freenitrogen rather than in air.

Statistical methods. The standard errors of thepredicted and measured concentrations wereestimated by quadrature addition of the relativestandard errors (RSE) of the component variables(Chatfield, 1983):

= [ I RSEJJIt should be noted that the appropriate standarderror of the volume of liquid injected in thepreparation of the gas standards is l / \ /5 times theerror associated with a single injection, since themean chromatograph response for five separatelyprepared standards was measured. Hence theerrors for the predicted concentrations for a single

350 BRITISH JOURNAL OF ANAESTHESIA

injection will be V5 times greater than those intable III.

Results

Time variation of standard concentrations

The results (table II) showed no indication ofany systematic trend in the concentrations withtime, and the regression analyses for all three

TABLE II. Summary of results for time variation of concentrationswithingas sampling bulb. Statistics for each agent were calculatedfrom the chromatographpeak areas expressed as % of the mean area

for that agent over the 30-min period of measurement

Slope ofregression line

Anaesthetic Range Coefficient (% min~l)agent (%) of variation (%) (±SEM)

HalothaneEnfluraneIsoflurane

99.6-100.499.1-100.699.7-100.4

0.260.400.19

-0.0094 ±0.00750.019 ±0.011

-0.0041 ±0.0058

agents resulted in slopes the magnitudes of whichwere small and not significantly different fromzero. While it is logically possible that, for aparticular agent, any systematic trend could havebeen masked by an opposing drift in the detectorsensitivity, the chance of this happening for allthree agents seems small enough to be discounted.The coefficients of variation over the 30-minperiod were similar to the value of 0.36% thatwould be expected solely as the result of randomvariation in the volume delivered to the chromato-graph by the 100-nl syringe (table I). (In fact, themeasured coefficient of variation should besomewhat greater than this, because of theadditional variation associated with the analysisprocess; however, estimates of standard deviationfrom a sample of 10 are subject to large samplingvariations, and there is no inconsistency in theabove finding.) Thus, the values shown in table IIshow no evidence of any variation in theconcentrations within the sampling bulb over the30-min measurement period.

TABLE III. Predicted v. measured concentrations for gasstandards (mean± SEM)

Anaestheticagent

HalothaneEnfluraneIsoflurane

Concentration (%)

Predicted

3.844±0.0073J17±O.0O63.277±O.0O6

Measured

3.845 ±0.0263.284 ±0.0233.261 ±0.022

Measurement of standard concentrations

The measured concentrations agreed with thepredicted values within the limits of experimentalerror (table III). For each agent, the two valuesdiffered by less than 1.5 times the SEM of themeasured value.

DISCUSSION

It is a fundamental requirement of any proposedstatic method of preparing gas standards thatsignificant adsorptive losses do not occur over thetimes for which it is planned to store the mixture.The sampling bulb used in the present method waschosen because it was constructed of relativelyinert, non-adsorbent, materials and thereforeshould have provided as much freedom fromadsorptive effects as it was possible to obtain. Theresults from the first part of the assessment showthat no significant lossess occurred over a periodof 30 min after evaporation was complete forhalothane, enflurane and isoflurane. The secondpart of the assessment demonstrated that, inaddition, no measurable loss occurred whileevaporation was proceeding or during transfer ofsamples to the gas chromatograph. Taken together,both parts show that, over a period of 30 min afterthe standard was prepared, the actual concentra-tions were identical to the predicted concentra-tions, within the limits of experimental error.Although it is likely that the mixture would remainstable for considerably longer periods, there is noneed to store it for such times because of the easewith which a fresh mixture can be prepared.

The main route by which loss of vapourmolecules would be expected is solution in theTeflon stopcocks and silicone rubber septum ofthe sampling bulb. The solubilities of volatileanaesthetics vary considerably. The values for themore commonly used agents for rubber are shownin table IV. Within the range of values shown,enflurane and isoflurane are relatively insoluble,halothane is moderately soluble and methoxyflur-ane and trichloroethylene are very soluble.Therefore, if it is planned to prepare standardmixtures of either methoxyflurane or trichloro-ethylene using the present method, it would beadvisable to check the stability of the mixtures.(Provisional work in this laboratory indicates thattrichloroethylene standards are stable over a30-min period.)

It has been possible to keep the present method

CHROMATOGRAPH CALIBRATION FOR VOLATILE ANAESTHETICS 351

TABLE IV. Rubber /gas partition coefficients at room temperaturefor some commonly used volatile anaesthetics. All values from Eger(1981), except that for trichloroethylene, which is taken from

Steward and colleagues (1973)

Agent

EnfluraneIsofluraneHalothaneMethoxyfluraneTrichloroethylene

Partitioncoefficient

7462

120630830

very simple because only small sample volumesare required for gas chromatograph calibration.This allows the use of a small container andobviates the need, present in applications requiringlarge sample volumes (Nelson, 1971; Barratt,1981), for providing a diluent gas to replace thesample removed. Furthermore, the compactnessof the sampling bulb allows mixing to beperformed easily by hand.

The accuracy with which the composition of thestandards is known depends on the accuracywith which the quantities listed in table I aredetermined (systematic errors), and on therepeatability of the injections of liquid agent to thesampling bulb (random error). Although thesyringe used for these injections had a specifiedaccuracy and repeatability of better than ± 1 %,greater accuracy can be achieved by means ofindividual calibration, and this procedure alsogives an improved estimate of the repeatability ofthe injections. One other source of error in thepredicted concentration which has not yet beenconsidered is variation in the liquid density causedby departures from the temperature at which thedensity was determined. The coefficient ofexpansion of the agents investigated is around0.0025/°C (referred to the density at 20 °C) and,thus, to achieve maximum accuracy it would benecessary either to control the laboratory tempera-ture or to make appropriate corrections for thetemperature variation. The estimated relativestandard error for the predicted concentration ofa single standard mixture prepared in thislaboratory is less than 0.5 %; greater calibrationaccuracy could be obtained by taking the meanvalue of the chromatograph response to severalmixtures prepared in succession, and so reduce theerror associated with variation in the injectedvolume.

The principle of the present method is not new

and indeed it has been used previously in atechnique for the preparation of methoxyfluraneand halothane standards for gas chromatography(Allott, Steward and Mapleson, 1971; Jones,Molloy and Rosen, 1971). The method describedhere differs from the previous one in two mainrespects: first, the mixing vessel is much morecompact (500 ml instead of 10 litre), and allowsmixing to be achieved in less than 1 min, insteadof the 10-15 min required by the earlier methodand, second, the amount of volatile agent dispensedinto the mixing vessel is measured volumetricallyrather than gravimetrically. There is no doubt thatgravimetric dispensing is fundamentally moreaccurate, since it avoids the uncertainties associatedwith syringe calibration, random variation ininjected volume and temperature variation ofliquid density; however, volumetric dispensing iseasier to carry out and therefore more suitable forroutine use and, as used here, provides sufficientaccuracy for most purposes. It would be a simplematter to change to gravimetric dispensing ifgreater accuracy were required.

The main innovations in the present work werethe measures taken to estimate the accuracy of themethod and to check the method by comparisonwith liquid standards. Although the earliermethod provided standards with only a very slowloss of halothane (the 95 % confidence interval was0.20-0.48% per day), no estimate of the absoluteaccuracy was given, nor was the method checkedagainst an independent standard. One potentialsource of error in any method of gas analysis isdifferential sample loss during transfer to themeasuring instrument. In the earlier method,all-glass syringes were used for sample transfer,and there would therefore seem to have been thepossibility of some sample loss by diffusion pastthe plunger. The use of a gas-tight syringe with aPTFE plunger tip avoids diffusive loss in thepresent method. However, gradual loss of sampledoes occur as the result of solution of the volatileagent in the PTFE. In order to prevent thisbecoming appreciable, the syringe should be filledto capacity and the sample analysed within 10 min.

The present method is used regularly in thislaboratory to provide standard mixtures forcalibrating a gas chromatograph which is thenused to analyse the output of anaesthetic vaporizers.This procedure is carried out either to check thevaporizer calibration for clinical purposes or toallow the vaporizer to be used as a source of avapour mixture of accurately known composition

352 BRITISH JOURNAL OF ANAESTHESIA

for the assessment and calibration of volatileanaesthetic monitors. A new generation of theseinstruments is now becoming available (Diprose,Epstein and Redman, 1980; Kay, Cohen andWheeler, 1982), and a future publication willpresent an assessment of one of them carried outin this manner. The availability of a simple,reliable and accurate calibration method allows thecapabilities of gas chromatography to be fullyrealized for such applications, and makes it thetechnique of choice for the determination ofvolatile agents in this laboratory.

ACKNOWLEDGEMENTSI am grateful to Dr D. Weatherill for encouraging me toundertake this project, to Dr J. O'Sullivan for informing meof the existence of the Alltech gas sampling bulb and to MrK. B. Carter for useful discussions about the calibration ofvapour analysers. I also wish to thank Mrs Joan Stewart for hertechnical assistance.

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Diem, K., and Lentner, C. (eds) (1970). Documenta Geigy.Scientific Tables, 7th edn, p. 230. Macclesfield: GeigyPharmaceutical.

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Steward, A., Allott, P. R., Cowles, A. L., and Mapleson,W. W. (1973). Solubility coefficients for inhaled anaestheticsfor water, oil and biological media. Br. J. Anaesth., 45, 282.

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