provement of the latter due to its smaller particle size. This is consistent with the results of Ettre (3). Also, for nonporous supports it appears that an irregular surface may be preferable t o that of a smooth sphere. Upon changing the liquid load from 29 to 58 mg., several interesting changes occurred. First, the k values for acetone and benzene increased nearly fivefold using 60/80 mesh beads but only 50 to 60% on the other supports. ilt the heavier loading the N values for most supports were relatively unchanged, but the N values for the 60,430 mesh beads increased while that of the crushed Vycor decreased. A more careful determination of van Deemter plots should clarify these apparent anomalies. It is important to note that 30/60 mesh beads were unable to resolve the solutes from air, even a t the heavier loading. Even Teflon columns, for which inlet pressures and flow rates were virtually identical, gave better separations. This may reflect better wetting of Teflon by Carbowax or improved ef-
ficiency from a rougher surface of less uniform shape. These results appear to confirm loss of liquid a t the glass contacts. Finally, the k values for methylpentene on the Chromosorb column were unchanged, (and on the Teflon, only slightly changed) upon doubling the amount of liquid. This suggests that the bare surfaces of these supports or the surface of the liquid phase were responsible for retention of that solute. Altogether, the tabulated results indicate that great care must be exercised in comparing column performances. They also indicate how the relative efficiencies of columns can change relative to one another under closely comparable conditions. In cases where adsorption by the support can be neglected, it appears that support particles having irregular surfaces map be preferable to smooth spheres. Furthermore, a porous support may be preferable to a nonporous one when the larger specific surface area of the former does not lead to detrimental adsorption effects.
LITERATURE CITED
(1) Deemter, J. J. van, Zuiderweg, F. J., Klinkenberg, A., Chem. Eng. Sci. 5, 271 ilR5A). \ - - - - I .
(2) Duffield, J. J., Rogers, L. B., ANAL.
CHEM. 32,340 (1960). (3) Ettre, L. S., J . Chromatoo. 4, 166 (1960). ( 4 ) Hishta, C., Messerly, J. P., Reschke, R. F., ASAL. CHEM.32, 1730 (1960). ( 5 ) Hishta, C., Messerly, J. P., Reschke, R. F., Fredericks, D. H., Cooke, W. D., l b i d . , 880 (1960). ( 6 ) Littlewood, -4.,,B., in “Gas Chromatography, 1958, Desty, D. H., ed., Butterworths, London, 1958, p. 23. ( 7 ) Martin, R. L., . 4 ~ . 4 ~CHEM. . 33, 347 (1961). (8) Pollard, F. H., Hardy, C. J., in “Vapour Phase Chromatography,” Desty, D. H., ed., Buttern-orths, London, 1957, p. 120. RECEIVED for review September 22, 1961. Resubmitted June 4, 1962. Accepted August 6, 1962. Taken in part from a thesis submitted by Peter Hurwitz in partial fulfillment of the requirement for a Bachelor of Science degree at the Rlassachusetts Institute of Technology, May 1961. Work was sup orted in part by under the Atomic Energy Contract At(30-1)-905 and by the Air Force Office of Scientific Research under Contract AF 49(638)-333.
commission
Gas Chromatographic Analysis of Head Space Gas of Dilute Aqueous Solutions RICHARD BASSETTE, SUHEYLL OZERIS, and C. H. WHITNAH Departments o f Dairy Science and Biochemistry, Kansas State University, Manhattan, Kan +
b
Enrichment of head space gas in sulfides, carbonyls, esters, and alcohols prior to chromatographic analyses was accomplished by adding anhydrous sodium sulfate salt to dilute aqueous solutions. It was possible to analyze head space gas from aqueous solutions below the 1 p.p.m. level. In addition to retention times, evidence for identification of these trace organic compounds was obtained by eliminating peaks with selective qualitative reagents. Evidence for the identification of the 18 components of a complex mixture of sulfides, carbonyls, esters, and alcohols a t the 1 p.p.m. level is presented. An analysis of a solution containing 0.01 p.p.m. carbonyls was made to establish the limits of sensitivity of the method.
T
of head space gas of dilute aqueous solutions of organic compounds b y gas chromatography has been reported by a number of workers ( 2 , 4, 6). Bailey et al. ( 2 ) studied volatiles associated with cabbage flavor. Weurman (e), studying enzymatic formation of volatiles in HE ANALYSIS
1540
ANALYTICAL CHEMISTRY
raspberries, reported that head space gas analysis over aqueous solutions could be made quantitative. He related peak heights to concentrations over the range of 0.025 to 0.001% for isopropanol, ethylacetate, propionaldehyde, and acetone. Mackay, Lang, and Berdick (4) reported that direct analysis of head space gas of foods could be related to sensory evaluations. Among odors he chromatographed were peppermint oil, roasted and brewed coffee, banana, brandy, onion, and cigar and cigarette smoke. Teranishi, Buttery, and Lundin ( 5 ) successfully utilized a dual flame ionization detector with dual columns and temperature programming for direct vapor analysis of some food products. The analysis of volatile components of biological fluids such as milk, blood, and urine presents certain problems to the analyst. The extremely low levels of significant volatiles a t and below the part per million level necessitates a preliminary concentration step. There is often some question of the authenticity of volatile constituents of biological fluids removed by solvent extraction
or distillation. Proteins. carbohydrates, and lipids in biological fluids interfere with, if they do not prevent, the direct analysis of liquid samples. This study was undertaken to develop gas chromatographic methods for the separation and identification of volatile organic compounds a t concentrations of 1 p.p.m. or less. Previous identification techniques by using selective reactions to alter chromatograms (3) proved unsatisfactory for trace analyses. New selective reagents were considered for this purpose. Such techniques would make possible the direct chromatographic analysis of trace volatiles of biological fluids.
EXPERIMENTAL Apparatus.
CHROMATOGRAPHIC
UNIT. A Model A-90-C Aerograph fitted with a hydrogen flame detector kit and a 1-mv. Brown-Honeywell recorder was used t o obtain the chromatograms. The Aerograph electrometer was modified by shorting out one of a series of resistors in the recorder leads according to manufacturer‘s instructions (1) to increase sensitivity 16
p.p.m. of each of the organic compounds listed above. Procedure. HEADSPACEGASSAMPLINGFOR CHROMATOGRAPHIC ANBLYSES. Unless otherwise indicated, each sample of head space gas, salted out from a n aqueous solution, was prepared as follows: 1.2 grams of sodium sulfate was transferred into a serum vial followed b y 2 ml. of the solution t o be analyzed. After the vial was sealed with a serum cap, the solution was mixed on the mechanical shaker for 5 minutes. The vial was then placed in a 60' C. water bath a t a depth slightly above the level of liquid in the vial for 3 minutes. A 1-ml. sample of the head 3). space gas was obtained by inserting the MECHANICALSHAKER. A general needle of the gas-tight syringe through purpose, horizontal shaker (Eberbach the rubber serum cap and drawing the 8: Sons) was used. vapors into the syringe. The syringe Reagents. ACIDIC HYDROXYL-was evacuated and refilled five times, A M I K E was a solution containing removed from the vial, the volume 0.4 gram S H 2 0 H H&04 treated with adjusted to 1 ml., and the sample 2 nil. of S S a O H and diluted to 10-ml. injected into the chromatograph. volume with distilled water. Solutions of each class of sulfides, BASIC HYDROXTLAJIIXE was a SOlUesters, and alcohols, a t 1 p.p.m. contion prepared by dissolving 0.4 gram centration were analyzed, using the of S H 2 0 H H2S04 in 10 ml. of 1X head space gas technique. A sample XaOH. containing all of the components, each Aqueous solutions to be analyzed, a t 1 p.p.m. concentration, and one contained 1 p.p.m. of mch of the folcontaining only the alcohols and sulfides lowing : were analyzed. Carbonyls. Propanal. n-butanal, nREMOVAL OF CARBONYL PEAKS. pentanal. acetone, 2-butanone. 2-sen2-ml. sample of the carbonyl solution tanone, and 2-hesanone. was treated with 0.1 ml. of acid hySulfides. Allyl sulfide and diethyl droxylamine reagent in a serum vial and, sulfide. with the serum cap in place, was mixed Esters. Methyl formate, ethyl forfor 1 hour at room temperature. It mate, ethyl acetate. ethyl propionate, was then analyzed as head space gas. and ethyl butyrate. T o be sure t h a t sulfides, esters, and Alcohols. Methanol, ethanol, nalcohols were not affected by the acid propanol. and n-butanol. hydroxylamine reagent, a solution conA composite solution containing 1 taining all of the components of the
times. Conditions were: column teniperature, 100" C.; nitrogen outflow, 20 ml. per minute; nitrogen input, 18.7 p.s.i.g.; and hydrogen outflow, 20 ml. per minute. CoLchiN. A 10-foot, commercially prepared, l/s-inch stainless steel column packed with 20% Carbowae 20 M on 60 t o 80 mesh acid-washed Firebrick n a s used. SAJIPLIXG BOTTLES were serum vials, 15 nim. in diameter X 52 mni., of 5-nil. capacity with self-sealing rubber caps. SYRISGES mere a 1-ml. gas tight syringe (Hamilton S o . 1001) and a 10-111. microsyringe (Hamilton $01
composite except carbonyls a t 1 p.p,m. concentration, was analyzed after treatment with this reagent. The composite was subjected to the same treatment and analyzed as previously described. After the l-hour reaction period with acid hydroxylamine, head space gas samples for chromatographic analyses were obtained b y treating with sodium sulfate and warming as previously described. REMOVAL OF SULFIDE PEAKS.A 2-ml. sample of the sulfide solution was treated with 0.2 gram of niercuric chloride in a serum vial, sealed with a serum cap and mixed on the mechanical shaker a t room temperature for 1 hour. It was then analyzed as head space gas To establish the effect of mercuric chloride on carbonyls, esters, and alcohols, a n aqueous solution containing 1 p.p.m. of each of the components of these classes of compounds was analyzed after being treated with mercuric chloride. The composite sample was treated with mercuric chloride in the same manner and analyzed. After the 1-hour reaction period with niercuric chloride, head space gas samples for chromatographic analyses were obtained by treating with sodium sulfate and warming as previously described. REMOVAL O F ESTER.4ND CARBONYL PEAKS. A 2-ml. sample of the ester solution, treated with 0.1 ml. of basic hydroxylamine reagent in a serum vial with the serum cap in place, mas mixed for 1 hour at room temperature. It was then analyzed as head space gas. To establish the effect of alkaline hydroxylamine on sulfides and alcohols, a sample containing only the sulfides and alcohols was treated with the alkaline hydroxylamine and analyzed.
Table I. Peak Heights Obtained from Head Space Gas Analysis of Different Classes of Organic Compounds a t Levels of 1 p.p.m.
Peak heights Compounds Methyl forinate Propanal Rater
Rel. ret. time" 0.144 0.167 0.185
Components 448 704
0 186
1824
;6
Acetone Ethyl formate hIethano1
I
n-Butanal
;
I
I
Ethyl acetate 2-Butanone
j
Ethyl sulfide
I
i
0 193 0 250
All
Carbonyls 62 6
*c
1
I
I
0 250 0 276
*
960
0.276
3840 I
Sulfides
x attenuation factor)
Esters 432
56
peak.
Sulfides Alcohols
56
72
8
12
1696 3965
3776
Ethanol J 0.276 * Ethyl propionate 0.348 2272 2272 2-Pentanone 0.412 3040 2656 n-Pentanal n-Propanol 0 424 192 Ethyl butyrate 0.515 1984 1856 0 682 1040 832 2-Hexanone n-Butanol 0.788 160 Allyl sulfide 1 0 1152 1040 Retention time of allyl sulfide was 22 min. Braces indicate components usually not separated.
)
Alcohols
1360 1312
J
of full scale deflection
528
i 3136
0 250
(70
60
108
124
100
140 1280 *Includes this component in the
VOL. 34, NO. 12, NOVEMBER 1962
1541
Table II.
Effect
Compounds Methyl formate Propanal Water Acetone Ethyl formate Methanol n-Butanal Ethyl acetate 2-Butanone Ethyl sulfide
of Selective Reactions on Peak Heights of Different Classes of Compounds Analyzed by Head Space Gas Peak heights (yoof full scale deflection X attenuation factor) after reaction of aqueous solution with Acid hydroxylamine Esters Sulfides Carbonyls Alcohols Composite 368 400
) 5
i1
72
*b
! I
1296
*
1520
*
Mercuric chloride Carbonvls Esters Sulfides Alcohols Composite 408 680 * 56
-
I
1760
*I *
~
1184
* I
I
I
1552
i
2816
j
Sulfides Alcohols Composite
1760 I
* I
3008 I
3040
1120
1344
I
i
Esters
12
12
2080
2464
80
128
72
140 472
i
'I
2464
Basic hydroxylamine
156
I
I
i
!
J
Ethanol Ethyl propionate 1984 2144 2112 2144 2-Pentanone 2848 3008 n-Pentanal n-Propanol 120 192 120 168 Ethyl butyrate 1696 1664 1664 1856 2-Hexanone 944 1008 n-Butanol 124 160 120 140 Allyl sulfide 480 672 Braces indicate components usually not separated. *Includes this component in the peak.
176
424
0
The composite was treated and analyzed in the same manner. After the 1-hour reaction period with the basic hydroxylamine, head space gas samples for chromatographic analyses were obtained by treating with sodium sulfate and warming as previously described. COMPARISOX O F CONVEKTIONhL UID S.4MPLIh.G WITH HEADSP.4CE
LIQGAS
SAMPLIMG. A 1-11. liquid sample of the 1 p.p.m. carbonyl solution was analyzed by conventional methods. The peak heights of the resulting chromatogram were compared with peak heights obtained from analyses of t1q-o 1-ml, samples of head space gas of the same sample. I n one of the gas analyses sodium sulfate was not added and the subsequent shaking was eliminated. A hundred-fold dilution of the carbonyl solution (0.01 p.p.m.) was analyzed as head space gas. The chromatogram from this analysis was compared with a similar one of distilled water. RESULTS A N D DISCUSSION
I t was possible to demonstrate the presence of each component in each class of compounds at 1 p.p.m. concentration when analyzed separately except for 2-pentanone and n-pentanal (see Table I). Both of these carbonyls had relative retention times of 0.412. It is apparent that the peak height at the time these carbonyls should appear was about two times that resulting from single component peaks. A number of factors must be considered when comparing peak heights of
1542
ANALYTICAL CHEMISTRY
compounds within a class or those of different classes. Relatively small alcohol peaks probably resulted from the strong affinity of water for alcohol. A close association of these components apparently limited the liberation of alcohol into the vapor phase. Sulfides, on the other hand, are classified as insoluble in water and their resulting peak heights were relatively large. The relationship of solubility to release of volatiles into the vapors can be observed by comparing peak heights of equal length carbon chain aldehydes and ketones. Less soluble aldehydes yielded larger peaks. A lower response of the hydrogen flame detector to short chain compounds undoubtedly contributed to the smaller peak heights of lower molecular weight compounds. It would be difficult to separate the effects of solubility and detector response. Spreading of peaks in the chromatograms as a function of time reduced peak heights. This is undoubtedly one of the factors that produced reduced peak heights in the last component in each homologous series. The higher boiling points of the longer chain compounds undoubtedly added to this effect. As the head space was taken a t 60' C., higher boiling points of 2hesanone (b.p. 127' C.) and allyl sulfide (b.p. 139' C.) limited their vaporization. Analysis of the composite illustrates the lack of complete separation of some of the compounds of each class.
To show the presence of separate components in this mixture and give evidence for their identification, it was necessary to eliminate from separate samples all but one component of each overlapping peak. It was possible to demonstrate a close relationship between the peak heights of individual components and their summation in the composite sample. The data of Table I1 illustrate the effect of various chemical reactions upon samples and their subsequent analysis. Carbonyl compound peaks were eliminated when the sample was treated with acid hydrosylamine. The same treatment had little effect on ester peak analysis. Alcohols and sulfides, however, were reduced by one third to one half by this treatment. When the composite was treated with acid hydroxylamine, the carbonyl compound peaks were eliminated and the sulfide and alcohol peaks were reduced. Sulfide peaks were eliminated when treated with mercuric chloride except for ethyl sulfide which Fas reduced from 3776 to 156. It is possible that this remaining peak represents a n impurity (not sulfide), as allyl sulfide m-as completely removed. Mercuric chloride had no effect on carbonyls, esters, or alcohols as analysis of classes other than sulfides showed. When the composite mixture was treated with mercuric chloride, the sulfide peaks were correspondingly removed. Ester peaks were completely removed when ester-containing solutions were
treated with basic hydroxylamine. The increase in the ethanol peak probably occurred from the formation of ethanol during the hydrovylamine reaction with ethyl esters. It can be seen from analysis of the composite that carbonyl compound peaks as well as esters were removed by basic hydroxylamine. Thus i t is necessary to employ both acid and ba'ic hydroxylamine to establish carbonyls in the presence of esters. As in the case of acid hydroxylamine, sulfide peaks were reduced by the basic 11)-droj-larnine. lT7hen the composite was treated with alkaline hydroxylamine, only sulfide and alcohol peaks remained. By a process of elimination it was possible to demonstrate the presence of the alcohols except for ethanol which appears with ethyl sulfide. The analysis of a solution of sulfides and alcohols without hydroxylamine compared with the analysis of these classes m-hen treated with basic hydrovylamine showed a reduction in pt.ak heights of these constituents in the presence of hydroxylamine. On this hisis it 1s possible to interpret some of t h e otherir ise confusing reductions in p t 4 i heights. .\ comparison was made among the p t ~ i kheights from I-pl. of liquid sample, 1 nil. of conventional head space gas, and 1 nil. of head space gas after the sample 15-as viturated with sodium sulfate. From tlip data of Table 111 it iq :ippnrcnt that 1 ml. of head space gas rcsults in pcaks that are from two to over 10 times a; high HS those obtained
Table Ill. Comparison of Peak Heights of 1 pl. of Liquid Sample with 1 ml. of Head Space Gas Sample of Carbonyls in Water
Compounds Propanal Water Acetone n-Butanal 2-Butanone n-Pentanal 2-Pentanone 2-Hexanone Q
b
Rel. ret. timea 0 167 0 185 0 186 0 250 0 276 0 412
Peak heights (yo of full scale deflection X attenuation factor) Liquid, 1 PI., Head space gas, 1 nil. 1 p.p.m. I p.p.ni, 1 p.p.m.* 0.01 p.p.m.b H206 60 176 616 10 212 56 142 200 528 80 132 265 1312 22 7s 161 960 20 9ii 404 2656 36
0 412 0 6S2
10
832
120
12
Retention time of allyl sulfide was 22 minutes. Addition of 1.2 grams of Sa2SOJ to 2 nil. of sample
from 1 pl. of the liquid. Although 1 pl. of liquid approaches the practical limits of sample size in the l/s-inch packed column, larger gas samples could be used. hddition of sodium sulfate to the sample before remoT ing the gas sample further increased peak heights from 4 to 7 times. This can be seen in Table I11 by comparing the head space gas analysis with and without sodium sulfate. Results of the analysis of the 0.01 p.p.m. carbonyl solution by the head space gas technique are also presented in Table 111. Discernible peaks were obtained for each of the carbonyl components in the mixture. K i t h the sample size and conditions employed herein, it is felt that for the carhonyl
mixture 0.01 p.p.m. concentration is close to the limits of the method. LITERATURE CITED
(1) Aerograph Research Xotes, Summer Issue 1961, p. 3, Wilkens Instrument & Research. (2) Bailey, S. D., Bazinet. M. L., Driacoll, J. L., McCarthy, A. I., J. Food Sci.
2 6 , 2 (1961). (3) Bassette, R., Whitnah, C. H., ANAL. CHEM.32,1098 (1960). (4) Mackay, D. A. M., Lang, D. A. Berdick,'M., Ibid., 33, 1369 (196l'). (5) Teranishi, R., Buttery, R. G., Lundin, R. E., Ibid., 34,1033 (1962). (6) Weurman, C., J . Food Tech. 15, 531 (1961) RECEIVED for review February 12, 1962. Accepted August 29, 1962. Co?tribution Nos. 304 Dept. of Dairy Science, 19 Dept. of Biochemistry, and 139 .4nnual .4gricultural Experiment Station.
1
Gas Chromatography of Pyrolytic Products ot Purines and Pyrimidines EDWARD C. JENNINGS, Jr., and K. P. DlMlCK
Wilkens lnsfrument & Research, Inc., P. 0. Box 373, Walnut Creek, Calif.
b A simplified technique of pyrolysis has been developed for the microgram range which enables a rapid, reproducible means of characterizing nonvolatile mater;als b y gas chromatography using a hydrogen flame ionization detector. Previous work in the microgram range has been conducted using an Argon detector, and work has been done in the milligram range using a thermal conductivity de,ector which permits isolation of the pyrolytic products and identification o f the products b y infrared spectrophotometry. High temperature pyrolysis a t 1100" to 1200" C. has been found to give maximum yield o f pyrolytic products from the purines
and pyrimidines. Under these conditions various molecular structures show characteristic thermal fission patterns. The yield o f a major pyrolytic product may b e directly related to the total amount of material subjected to pyrolysis, for example, the amount o f acetone produced upon pyrolysis o f thymine. In several cases slight changes in molecular structure may b e detected in the kinds and amounts of the pyrolytic products as in the p y rolysis of cytosine and isocytosine. Identification of isolated purines and pyrimidines has been made rapidly from the characteristic thermal fission patterns.
I
of nonvolatile substances such as purines and pyrimidines has been made by the gas chromatography of their pyrolysis products. Previous studies include the pyrolysis of polymers in which a thermal conductivity detector was used and milligram quantities of material were pyrolyzed, (4, 6, 7, 8). Jones and Moyles ( 3 ) worked in the microgram range employing a n argon detector. Throughout our research the various requirements that would make the combination of pyrolysis and gas chromatography a useful analytical tool have been investigated, and a micropyrolytic chamber has been developed to meet these requirements. DENTIFICATION
VOL. 34, NO. 12, NOVEMBER 1962
1543
