Gas Chromatographic Head Space Techniques for t Quantitative Determination of Volatile Components in Multicomponent Aqueous Solutions RICHARD E. KEPNER,' HENK MAARSE, and JACOBUS STRATING Central Institute for Nutrition and Food Research T.N.O., Utrecht, The Netherlands, and National Institute for Barley, Malt, and Beer T.N.O., Rotterdam, The Netherlands
b A method is 'described for the quantitative determination of the concentrations of volc tile components in dilute aqueous solutions containing several components without previous isolation or concen,tration. By the inclusion of an interiial reference compound and the injection of head space gas samples into Gas chromatographs fitted with ionization detectors, rapid and highly reprodiJcible analyses can be made for concentrations down to below the part per million range. Some factors aflecting the vapor pressures of volatile components in the aqueous solutions are mentioned. Par-
ticular attention i s paid to the analysis of fermentation products and the problems associated with high ethanol content. The advantages of quantitative head space techniques over other methods of analysis are indicated.
G
AS chromatography with ionization-
type detectors has been used for several years for the examination of head space vapor samples from a variety of food materials (1, 2, 4, 6,8, 10, 11, 13, 14) and from dilute aqueous solutions of volatile organic compounds (3). An attempt to utilize head space
techniques for the quantitative evaluation of the concentrations of volatile compounds in the solution itself has been reported by Weurman (14). A recent paper (28) gives additional data on single-component aqueous systems using essentially the technique reported by Weurman. Direct analysis of the head space can afford the fastest and undoubtedly the most precise measure of the true aroma composition of a material (8). It is often desirable, however, to alter the ratios of the 1 Permanent address, Department of Chemistry, University of California, Davis, Calif.
X
p1
Y
t
8 Lt t
P
5 t
Sample equilibrated a- 3 6 ' C. for 45 minutes and 6-ml. head space sample injected.
Attenuations
X 1 and a t range 1 OD except where otherwise indicated VOL. 36, NO. 1, JANUARY 1964
77
I:
7
I
8
Time in min.
Figure 2. Chromatogram of fully carbonated beer Sample saturated with sodium chloride, equilibrated at 2 0 ' C. for 30 minutes, and 20-ml. head space sample injected. Attenuations at range 1 indicated across top of chromatogram
Holland) instrument equipped with a flame ionization detector and a n F&M Model 609 flame ionization gas chromatograph. The Becker instrument was fitted with a Cmeter X 2.8-mm. i.d. stainless steel column (as a loop) packed with 25% by weight of 1,2,3tris (2-cyanoethoxy)propane (TCEP) on acid-washed Chromosorb White, 60to 70-mesh, and operated at 55' C. with Nz, Hz, and 0 2 flow rates of 40, 20, and 100 ml. per minute, respectively. The F & M unit was fitted with a 4-meter X 3-mm. i.d. coiled copper column filled with the same packing material and operated at 65" C. with corresponding flow rates of 33, 30, and 120 ml. per minute, respectively. SYRINGES.Ten-milliliter metal syringes fitted with a spacer to permit reproducible delivery of a 6-d. gas sample and 20-ml. glass syringes with a metal plunger were used. The syringes were cleaned between experiments by placing them in a vacuum oven a t 80" C. and 2 mm. of Hg pressure for 30 minutes. HEADSPACESAMPLEFLASKS.Infusion flasks of 120- or 360-ml. capacity were used when 6- or 10- to 20-ml. head space samples, respectively, were taken to be injected into the gas chromatographs. Materials. The compounds used for preparing the calibration curves EXPERIMENTAL and as internal standards were Apparatus. GASCHROMATOGRAPHS.normally redistilled through a Vigreux column and the purity was checked Two commercially available instruby gas chromatography before use. ments were used, a Becker (Delft,
volatile components in a head space sample by choice of a different equilibration temperature, by saturation of the solution with an inorganic salt (3), etr., to facilitate the determination of trace components. Under such circumstances the head space analysis no longer represents the true aroma of the material, but still makes possible the determination of the concentrations of the volatile components in the solution under investigation. The presence of large amounts of ethanol presents special problems in determination of volatile components in solution by head space techniques because of its effect on solubilities and hence vapor pressures of the volatile components and because of its tendency to tail badly on the gas chromatographic stationary phases. This paper presents the results of investigations n-hich were initiated to develop methods of analysis using head space techniques for the precise quantitative evaluation of the concentrations of volatile constituents in solutions, with particular reference to fermentation products with a wide range of ethanol concentrations.
78
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ANALYTICAL CHEMISTRY
The rum and beer samples were standard commercial products. Procedure. PREPARATION OF HEADSPACESAMPLE. A 50-ml. aliquot of the solution to be analyzed was placed in a head space sample flask, which was then fully immersed in a n upright position in a thermostated water bath. If required for analysis, the vapor pressures in the flask were increased by addition of sufficient (NH&S04 or NaCl to saturate the solution being analyzed. Bath temperatures from 10" to 36' C. were used. An equilibration time of 30 to 45 minutes, dependent on the bath temperature, has been found necessary to ensure that the system is at equilibrium before the head space sample is removed. The sample was carefully swirled manually several times while the flask was in the bath. At the end of the equilibration period the flask was raised so that the serum cap was just above the surface of the water and the surface of the cap blotted dry with filter paper, 4 syringe was brought to the same temperature as the sample during this equilibration period by placing it in a separate chamber in the bath. The syringe was slowly filled and emptied back into the sample flask four times to minimize losses resulting from adsorption on the inner surfaces of the syringe. The sample to be injected into the gas chromatograph was collected in the fifth filling of the syringe.
A 6-ml.head space [;ample was always used in the investigations involving rum, diluted rum, or 15% aqueous ethanol solutions, while head space samples up to 20 ml. were used in the investigations with beer and 5% aqueous ethanol solutions. INTERNALSTANDARDS. Pure compounds used for reference as internal standards were chosen to have retention times as similar as possible to the retention times of the peaks being analyzed and to appear a t an open spot on the chromatogram. The reference compound was dissolved n aqueous ethanol of approximately the same ethanol concentration as the solution to be analyzed. The concentration of the standard was adjusted so that the peak height was about half full seal: a t very similar attenuation to that used far the peaks with which it was to be compared. Two ways of adding internal standard solutions to the samples were used with equal success: (1) 2.00 ml. of the solution was mixed with 50.00 ml. of the sample and (2) 2.00 ml. of the solution was pipetted into a 250-ml. volumetric flask and mihde up to volume with the sample, and a 50-ml. aliquot was used for the analysis. CALIBRATION CURVES. Solutions for the preparation of calibration curves were obtained by addition of a constant amount of internal standard and increasing measured amounts of an aqueous ethanol solution of the component under investigation directly t o a series of separate samples of the fermentation product and also to a series of aqueous ethanol solutions having the same ethanol concentration as the fermentation product. Each solution was analyzed on the gas :hromatograph by head space techniques. The method of quantitative estimation of peak areas suggested by Carroll (6), the product of peak height tirr.es retention time, was utilized in the calculations. The concentration, in prtrts per million, of a component in the solution was plotted against the ratio of the product of the peak height times the retention distance for the component over the value for the internal reference compound a t the predetermined sensitivities. In the preparrition of the calibration curves by the addition of a component to the fermentation product, the concentration of the component in the fermentation oroduct itself was calculated from the increases in the ratio per increment3 of the component added. In all calculations corrections were applied for the dilution effect of the addition of the internal reference solution and of solutions of the various components. RESULTS AND DISCUSSION
The results of the present investigations on rum (7557, ethanol content) and on beer (5%, ethanol content) show clearly that with careful selection of experimental cor.ditions quantitative head space analysis can be applied to a wide range of fermentation products. Figures 1 and 2 present typical chro-
Figure 3.
Comparison of calibrations in 5% aqueous ethanol vs. beer Internal standard. 4.5 p.p.rn. 4-heptanone 1. Isoamyl acetate in beer 2. Isoamyl acetate in 5% aqueous ethanol 3. lsobutanol in beer 4. Irobutanol in 5% aqueousethanol
matograms of diluted rum (containing 15% ethanol) and a Dutch beer saturated with sodium chloride, respectively, with the peaks representing the added internal standards indicated by broken lines. The identities and the Kovats indices (9) of certain components in a number of the peaks are listed in Table I. The identities of the peaks as indicated were determined by standard methods of trapping, rerunning, and comparing retention data on several stationary phases, measurements of infrared spectra of the isolated compounds, and/or chemical identification. These experiments also gave the necessary information as to the purity of the individual peaks utilized in the present investigation. The applicability of the method as a whole is of course limited to single-component gas chro-
matographic peaks. Usually in the case of complex mixtures, a number of different gas chromatographic stationary phases will have to be used. The results discussed below pertaining to rum, diluted rum, and 15% aqueous ethanol solutions were obtained on the Becker instrument and those pertaining to beer and 5% aqueous ethanol on the F & M instrument under the experimental conditions described. Factors Affecting Choice of Equilibrium Conditions. The temperature a t which test samples are equilibrated affects the system in several ways. Raising the temperature increases the possibility of artifact formation and may do so more for beer than for rum because of differences in content of thermolabile compounds. On the other hand, such a rise will result in an VOL 36, NO. 1, JANUARY 1 9 6 4
79
Table I. Identity and Kovats Indices of S o m e Components in Rum and Beer Chromatograms Peak Kovats Peak Kovats index NO. Compound index No. Compound 1 Acetaldehyde 9 Isoamyl acetate 978 1350 2 Ethyl formate 1080 10 1-Butanol 1350 1098 3 Methyl acetate 11 PHeptanone 1389 4 Ethyl acetate 1139 12 2-Methyl-1-butanol 1399 5 Ethanol 1162 13 Isoamyl alcohol 1404 6 Ethyl propionate 1197 14 3-Heptanone 1420 7 Ethyl butyrate 1262 15 Ethyl caproate 1439 8 Isobutanol 1289 16 %-Amylalcohol 1448
increase of the vapor pressure in the head space, thus increasing the possibility of measuring the concentrations of trace components in the sample. The increase of vapor pressure is different, however, for compounds of different physical properties. When chromatographic parameters are such that a high boiling ester appears on the ethanol tailing curve, an equilibration temperature of 10' C. has been found the most satisfactory. ,4t the lower temperature the vapor pressure of the ethanol is decreased by a much greater fraction than that of the high boiling ester, thus permitting a much more accurate measurement of the concentration of the ester. The measurement of the concentration of a trace component in solution may also be facilitated by saturation of the sample with an inorganic salt carefully chosen for the purpose. Separate head space analyses of a 15-p.p.m. isoamyl alcohol solution in 5% aqueous ethanol showed four- and sevenfold increases in peak heights when saturated with NaCl and (NHJ2S04, respectively. Comparable effects were observed with the ethanol peak in fermentation samples but again evidence was obtained which showed that different types of compounds are affected to different degrees. The position of the component on the chromatogram may also influence the choice of the inorganic salt which is used. Use of Internal Standards. Preliminary investigations in quantitative head space analyses by simple measurement of peak heights gave a t best =I= 15% reproducibility under the conditions of our experiments. This poor reproducibility is undoubtedly due in part to such factors as changes in sensitivity in the gas chromatograph, the difficulty of reproducing flow and temperature conditions exactly, and gas leaks in the syringe or a t the serum cap during injection of the large head space samples. Such difficulties have been overcome to a major extent by the inclusion of a reference compound dissolved in the solution being analyzed. To be suitable for use as an internal standard a compound must be sufficiently soluble and stable in the appropriate solutions and 80
ANALYTICAL CHEMISTRY
must appear a t an open spot on the chromatogram reasonably close to the peak under investigation (7). A variety of compounds have been considered and tested for this purpose and ketones have been found satisfactory in all respects. In the present investigations on beer and rum using a TCEP column a t 55" C. either 3-heptanone or 4 heptanone can be used, while a t 65" C. 4-heptanone is preferred (see Figures 1 and 2). Calibration curves of p.p.m. us. area ratio covering the concentration ranges of components as normally observed in a fermentation product, have been prepared as described. Under t.he experimental conditions with the ranges of concentrations used the area ratios
can in general be determined with a reproducibility to * 5 % or better. The results of analyses presented are always based on the average area ratio obtained from several replicate (two to four) runs on a sample. Figures 3 and 4 present examples of calibration curves for beer and rum components, respectively. Ethyl caproate in beer (concentration 0.2 to 0.3 p.p.m.) us. 4 heptanone measured a t the attenuations indicated in Figure 2 also gives a calibration curve of similar reliability. Table 11, which presents a typical set of data for the preparation of a calibration curve, clearly illustrates the different degrees of reproducibility in analyses by measurement of peak heights only and by measurement of the area ratios. The large variation in head space sample size that can be used successfully in conjunction with the calibration curves is also illustrated in Table 11. Influence of Ethanol Concentration on Head Space Analyses. Besides water, ethanol is the main component of fermentation products. I t s concentration has a great influence on the vapor pressures of the other volatile components in the sample. The data in Table I11 illustrate the effect of diluting a rum, which contains 75% ethanol, to 50, 15, and 7.5% ethanol content by the addition of
Figure 4. Comparison of calibrations in 1570 aqueous ethanol vs. rum Internal standard, 15 p.p.m. 3-heptanone 1. 2.
Isoamyl alcohol in diluted rum (containing 15% ethanol) Isoamyl alcohol in 15% aqueous ethanol
Table It.
Solutiono NO.
1
2
4
Isoamyl acetate added, p.p.m. 0.00
0.99
2.97
Run SO.
Isoamyl Acetate Calibration Curve in Beer
Head space 4-Heptanone sample, Pk. ht., mm., Ret. dist., atten. X 6 4 mm. ml.
Isoamyl acetate (Pk. ht. x ret. dist.) Isoamyl acetate 4Heptanone (pk. ht. X Pk. ht., mm., Ret. dist., ret. dist.) mm. atten. X 6 4
Calcd. isoamyl acetate concn., p.p.m.
1 2 3 4
20 20 20 20
134 92.5 147
160 112.5 180 171
2s 1
144.5
365 366 366 373
282 281 286
0.92 0.94 0.94 0.91 Av. 0.93
3.10
5 6 7
10 10 10
82.5 77.5 76.5
374 376 382
139.5 123 117
2S7 289 294
1.29 1.22 1.18 Av. 1.23
4.04
12 13
7 8 7 7
31.5 71
370 371 373 381
75 156 99.5 140
285 285 2S6 292
1.84 1.69 1.68 1.66 Av. 1.72
5.92
14
15
45.5 65
a Each solution prepEred by pipetting 2.00 ml. of Pheptanone solution (567 p.p.m. in 5% aqueous ethanol) and appropriate aliquots of isoamyl acetate solution in 5Tcaqueous ethanol into 250-ml. volumetric flasks and filling t o volume with beer.
water. The undiluted and the three diluted rum samples were analyzed by head space techniques and the vapor pressures of the components measured in terms of peak heights on the chromatograms. Additional peaks were resolved in both the 50 and 15% dilutions. Essentially all compcnents showed a marked increase in pe:tk height for the sample containing 5057, ethanol, while the higher boiling components continued to show increases in peak heights for the sample containng 15y0ethanol. Further dilution down to 7.5y0ethanol content resulted in decreases in vapor pressure for all components, as evidenced by decreased peak heights on the chromatogram, although the peaks were in general still higher than in undiluted rum. Without any detai1:d consideration of the complex interactions (hydrogen bond formation, other solubility factors, etc.) contributing to Ihe results, it is feasible to use this dilution technique to determine the concentrations of volatile components ir rum by quantitative head space aralyses. An important advantage of the moderate dilution of beverages of high alcohol content is the better resolution and more accurate determination of the components with greater retention times than ethanol which results from decrease in the ethanol peak and subsequent tailing as the other peaks increase in size. The most favorable degree of dilution for a given type of fermentation product must be determined experimentally to facilitate the desired analysis.
Table 111.
Effect of Dilution on Vapor Pressures of Certain Volatile Constituents in Rum
Peak height," mm. , in rum with ethanol content Peak
75 %
7.5% Compound (undiluted) 50% 15% I iicetaldehvde 175 380 205 135 2980 1700 3280 4800 2 Ethyl formate 43 24 50 8 Isobutanol 17 73 50 56 9 13 Isoamyl alcohols and-2-methyl1-butanol 15 Ethyl caproate 10 169 318 227 Peak heights 6-ml. head space samples from rum equilibrated at 36" C. for 45 min. calculated t o attenuation x1 at range 109. Simple peak measurement, a3 compared to peak height X retention distance used elsewhere, was sufficiently accurate t o demonstrate concentration differences resulting from dilutions. SO.
Influence of Components Other than Ethanol on Head Space Analyses. The effects on the composition of a head space sample which may be exerted by the many other types of substances (carbon dioxide, proteins, sugars, phenols, etc.) which will be present in a fermentation product are difficult to predict. T o check for such effects calibration curves were prepared for isoamyl acetate and isobutanol in beer and in 5% aqueous ethanol (Figure 3) and for isoamyl alcohol (Figure 4 ) and ethyl caproate in diluted rum (15% ethanol) and 15% aqueous ethanol. Inspection of the results shows that the peak heights for isoamyl acetate, isobutanol and isoamyl alcohol are lower in the aqueous ethanol solutions than in the fermentation products for comparable concentrations. Ethyl caproate gave the same calibration curve in
diluted rum and in 15% aqueous ethanol. Consequently the data were not plotted in Figure 4. The differences thus appear t o be greater in beer than in diluted rum, which may well reflect the difference between a distilled and a nondistilled fermentation product in this regard. The results thus indicate that for precise quantitative head space analyses the calibration curve should always be prepared in the solution under investigation, using the exact experimental details by which the component will be determined. Advantages of Head Space Techniques for Quantitative Analysis. Determination of the concentrations of volatile components in fermentation products by gas chromatography or any other method has been necessarily preceded by isolation and concentration processes such as stripping, extraction, steam distillation, fracVOL. 36,
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tional distillation, and all possible combinations of these. These processes are time-consuming and often result in artifact formation and always in ratio changes, thus making accurate and reproducible quantitative analyses extremely diacult if not virtually impossible. On the other hand, analyses by quantitative head space techniques are rapid and can be done directly on the sample itself to measure concentrations down to below the part per million range when the gas chromatographic and head space sampling conditions are chosen carefully. There is very little possibility of artifact formation and reproducible ratio changes of the components in the vapor can be used to facilitate a given analysis. By the addition of an internal reference compound and the preparation of calibration curves in the fermentation product under the identical conditions
used for the analyses, a reproducibility of i 5 % or better can generally be obtained by head space techniques. ACKNOWLEDGMENT
The authors are indebted to Cornelis Weurman for many helpful suggestions during this investigation and to Truus de Nie for carrying out some of the experimental work. One of the authors (R. E. Kepner) expresses his sincere appreciation to the Central Institute for Nutrition and Food Research T.N.O., for hospitality and support during this investigation. LITERATURE CITED
(1) Bailey, S. D., Bazinet, M. L., Driscoll, J. L., McCarthy, A. I., J . Food Sci. 26, 2 (1961). (2) Bailey, S.D., Mitchell, D. G., Bazinet, M. L., Weurman, C., Ibid., 27, 165 (1962).
(3) Bassette, R., beria, S., Whitnah, C. H., ANAL.CHEM.34, 1540 (1962). ( 4 ) Bavisotto, V. S., Roch, L. A.,
Heinisch, B., Am. SOC.Brewing Chemists Proc. 1961, 16. (5) Buttery, R. G., Teranishi, R., ANAL. CHEM.33. 1441 (1961).
(1962). " ' (9) Kovats, E., 2. Anal. Chem. 181, 351 (1961). (10) Mickay, D. A. M., Lang, D. A.,
Berdick, M., Proc. Sci. Sect. Toilet
Goods ASSOC., No. 32, 7 (1959); ANAL.
CHEM.33, 1369 (1961). (11) Nawar, W. W., Fagenon, J. S., Food Technol. 16, No. 11, 107 (1962). CHEY. (12) Ozeris, S., Bassette, R., SKAL. 35, 1091 (1963). (13) Teranishi, R., Buttery, R. G., Lundin, R. F., Zbid., 34, 1033 (1962). (14) Weurman, C., Food Technol. 15, 531 (1961). RECEIVEDfor review May 9, 1963. Accepted September 18, 1963.
Prediction of Response Factors for Thermal Conductivity Detectors BUFORD D. SMITH and WARREN W. BOWDEN' School of Chemical Engineering, Purdue University, lafayefte, Ind.
b This paper develops the equations whereby response factors for thermal conductivity detectors on gas chromatography instruments can b e predicted without recourse to calibration curves. The response to a given compound can b e represented by dE/dyl and predicted from -dE =
dyz
dE dT dh dT Z &
where E is the out-of-balance bridge voltage, yz i s the mole fraction of the noncarrier gas compound in the detector, 7' is the sensing thermistor temperature, and h is the thermistor surface heat transfer coefficient. The dE/dT and dT/dh derivatives are calculated from the instrument characteristics while dh/dyz depends essentially on the thermal conductivity of the binary gas mixture in the detector. Predicted relative response factors (ratios of two dE/dy's) were within 10% of experimental values for pentane, benzene, and sulfur dioxide samples. Predicted values a t very low values of yz are more accurate since at infinite dilution the physical properties of only the carrier gas are involved.
T
the development of the theory whereby the relative response factors for a thermal conductivity detector can be predicted without the prior determination of caliHIS PAPER PRESENTS
82
ANALYTICAL CHEMISTRY
bration curves. It will be shown that if certain physical properties-thermal conductivity, viscosity, heat capacity, molecular weight, boiling temperatures, and density-are known for each gas which passes through the detector, it is possible to predict relative response factors with considerable accuracy. The prediction method requires a knowledge of numerical values for the following resistances and constants; all the resistances in the detector bridge circuit, the temperature-resistance relationship for the two thermistors, the physical dimensions of the thermistors and their mounting wires, the electrical conductivity of the thermistor mounting wires, the voltage applied to the bridge, and the span constant, s, which is defined by P = sE/a. Nominal values of the bridge resistances are available from wiring diagrams and thermistor catalogs (3, 7). If this prediction method proves useful in practice, the instrument manufactures could aid by providing actual values rather than nominal values for each individual instrument. For example, the thermistors are usually closely matched in each instrument but the only resistance values given in the catalog ('7) for the thermistors used in 20% the instrument were 8,000 ohms a t 25" C. Another item which the manufacturers could supply with each individual instrument is the surface area of the thermistors.
*
ANALYSIS OF DETECTOR RESPONSE
The response of the instrument to any given component can be defined as the derivative d E / d y , where y~ refers to the mole fraction in the detector of the component other than the carrier gas. The response can be obtained from the following product of derivatives:
The dE/dT represents the rate of change in the out-of-balance voltage with respect to the sensing thermistor temperature, T . The dT/dh describes the way the thermistor temperature changes with respect to the heat transfer coefficient between the thermistor and the flowing gas mixture. Both d E / d T and dT/dh can be calculated from the instrument characteristics and therefore curves for these derivatives us. the proper variable (which will be shown to be T ) could be supplied by the manufacturer with each individual chromatography unit. The last derivative, dh/dyz, shows how the heat transfer coefficient, h, varies with composition of the gas mixture flowing past the sensing thermistor. The coefficient h mill be shown to depend essentially on the thermal conductivity, k , of the gas mixture which can be predicted with existing correlations if certain physical properties of the pure gases are known. 1 Present address, ChemicalEngineering Department, Rose Polytechnlc Institute, Terre Haute, Ind.
