Gas Chromatographic Analysis of Some Alcohol 3,5-Dinitro benzoates with Application to Analysis of Acetals W. G. GALETTO, RICHARD
E. KEPNER,
and A. DINSMOOR WEBB
Departments of Viticulture and Enology and of Chemisfry, University of California, Davis, Calif.
The gas-liquid chromatography of the 3,5-dinitrobenzoate derivatives of a variety of alcohols has been demonstrated using SE-30 silicone fluid as the liquid phase at a column temperature of 250" C. The effluent material from the peaks has been trapped and shown by infrared and thin layer chromatography to be the original derivative injected. Mixtures of the derivatives can be separated successfully without previous purification. The investigation of the structures of trace quantities of acetals by gas chromatography of the 3,5dinitrobenzoate and 2,4-dinitrophenylhydrazone derivatives of the component parts is described.
S
OUKUP, SCARPELLINO, AKD DANIELCZIK (5) have reported recently the
gas chromatographic separation of carbonyl 2,4-dinitrophenylhydrazones.Although the use of a flame ionization detector precluded collection of unaltered effluent samples and proof that intact 2,4-dinitrophenylhydrazoneswere being chromatographed, the excellent separations achieved suggested the possibility of employing preparative scale gas chromatographic techniques to purify and identify trace quantities of carbonyl derivative mixtures. In turn, if successful gas chromatography of unaltered carbonyl-2,4-dinitrophenylhydrazones were possible, it seemed that there might be equal success in gas chromatographic separation of 3,5dinitrobenzoates of alcohols. Analyses of trace quantities of acetals contained in complicated aroma isolates have always been difficult. It was observed (6) that higher boiling fractions of aroma material from Muscat grapes yielded acetaldehyde-2,4dinitrophenylhydrazone upon treatment with an acidic solution of 2,4-dinitrophenylhydrazine. As the high boiling point of the sample precluded the presence of free acetaldehyde, the evidence indicated that acetals were present. The gas chromatographic separation of alcohol 3,5-dinitrobenzoates and the analysis of acetals through hydrolysis and preparation of the carbonyl derivative followed by preparation of the 3,5dinitrobenzoate esters of the liberated 34
ANALYTICAL CHEMISTRY
alcohols and their identification by gas chromatography is the subject of this paper. EXPERIMENTAL
Apparatus. Two gas chromatographs were used in this investigation. The first was a Wilkens Instrument and Research, Inc., Hy-Fi Model 600D flame detector instrument operated isothermally a t 250" C., injector temperature 275' C., 19 ml./ minute Nz flow rate. The column employed was a 6 foot long, 1/8-inch o.d., stainless steel tube packed with 10% w./w. SE-30 silicone fluid on acidwashed chromosorb W. Detector signals were recorded with a MinneapolisHoneywell Electronik 16 recorder. A Loenco Model 70 Hi-Flex dual column, dual thermal conductivity detector instrument was used when it was desired to collect effluent samples from the instrument column. A l/*-inch 0.d. by 10 foot long stainless steel tube packed with 10% w./w. SE-30 silicon fluid on acid-washed Chromosorb W was used. Signals were recorded on a Minneapolis-Honeywell Electronik 16 recorder. The exit line from the sample side of the thermal conductivity detector was provided with a special heating coil which extended to the termination of the line outside of the instrument cabinet. Samples were collected in thin-walled glass capillary tubes ll/n-mm. 0.d. by 12 inches in length. Thin layer chromatography, using the techniques of Dhont and de Rooy ( I ) , and infrared spectra were used to check the purity and identity of the various derivatives before and after gas chromatography. Materials and Methods. The reagent from which 2arbonyl derivatives were obtained was prepared by dissolving 1 gram of 2,4-dinitrophenylhydrazine in 5 ml. of concentrated H2SOa. After dissolution, 6.7 ml. of HzO were added slowly; the solution was allowed to set overnight and filtered before use. Derivatives of known alcohols were prepared from 3,5-dinitrobenzoyl chloride by standard methods (4) and recrystallized to constant melting points. The derivatives were dissolved in benzene a t low concentration and injected into the Hy-Fi gas chromatograph individually for determination of retention times and as mixtures for determination of the completeness of separation. The derivatives in benzene were also injected into the Loenco
instrument and the effluent samples collected for investigation. Ethyl isoamyl acetal was prepared by reacting 0.05 mole each of anhydrous ethyl alcohol and isoamyl alcohol with 0.05 mole of redistilled acetaldehyde. Dry HC1 bubbled into the alcohol solution before addition of the aldehyde served as catalyst. After addition of the acetaldehyde, the flask was mechanically stirred for 1 hour a t room temperature. At this time the lower water phase was removed with a pipet and anhydrous K&03 was added. After filtration, the desired ethyl isoamyl acetal was separated from the other two acetals and the two alcohols by preparative scale gas chromatography on a polyneopentylglycoladipate column. Analysis of the ethyl isoamyl acetal was accomplished as follows: to 4 drops of the 2,4-dinitrophenylhydrazinereagent solution contained in a 3-ml. centrifuge tube was added with mixing 1 p1. of the ethyl isoamyl acetal. After 10 minutes the precipitate (2,4dinitrophenylhydrazone of the carbonyl portion of the acetal) was centrifuged to the bottom of the tube. The supernatant liquid was transferred by pipet to a 5-ml. pear-shaped flask, 1.5 mi. of H20 were added, the flask was fitted to a simple distillation head and condenser, and 0.8-1.0 ml. of the sample was distilled. To the distillate. cooled to 0" C.. was added 1.0 ml. of benzene containing 0.9 gram of 3,bdinitrobenzoyl chloride (9). The mixture was shaken and kept a t 0" C. for approximately 10 minutes. Two-tenths of a microliter of the benzene phase was injected directly into the Hy-Fi gas chromatograph for analysis. The 2,4-dinitrophenylhydrazone precinitate in the centrifuge tube was taken to dryness and &en dissolved in a few drops of benzene or methylene chloride. Five- to lo-& samples of the solution of the carbonyl derivative from the hydrolyzed acetal and of selected known carbonyl 2,4-dinitrophenylhydrazones were injected into the Hy-Fi gas chromatograph using the same column as was used for the 3,5-dinitrobenzoates, but operated isothermally a t 270" C. RESULTS AND DISCUSSION
The gas chromatographic retention times for 23 alcohol-3,5-dinitrobenzoates investigated are listed in Table I. A typical chromatogram showing the
0
$4
0
z 8 0
g
m 0 Y)
s 0
a
20.0
Figure 1. alcohols
15.0
7.5
Time, min.
5.0
2.5
Gas chromatogram of 3,5-dinitrobenzoate derivatives of known
separation of the 3,Bdinitrobenzoates of 12 different alcohols is presented in Figure 1. Investigation of single derivatives, recrystallized to constant melting point, showed that a single sharp peak with relatively little tailing was obtained for each derivative. When the Loenco gas chromatograph with the thermal conductivity detectors is used, it is found that separated peaks can be collected easily in capillary collection tubes. Rechromatography of a collected peak yields a single peak with the same retention time, indicating either that decomposition is insignificant or that the initial compound reacts cleanly under the heat of the injector to give a second substance which is stable. The infrared spectra determined on a number of the collected fractions were identical with the spectra for the corresponding alcohol-3.5-dinitrobenzoatesin the Sadtler (3) tables indicating that unaltered alcohol derivatives emerged from the column. Further confirmation of the identity of the material collected from the gas chromatograph was obtained by running thin layer chromatograms ( I ) , in which case it was found that the component before and after gas chromatography behaved identically. The minimum detectable quantity of derivative was not determined. However, only a very small fraction of the total derivative
obtained from the hydrolysis of 1 pl. of ethyl isoamyl acetal was required to give easily visible peaks on the chromatogram. The boiling points of the alcohols used to prepare the 3,bdinitrobenzoate deTable 1.
Gas Chromatographic Retention Times of Some Alcohol-3,5-Dinitrobenzoates
Alcohol Methyl Ethyl
?g"$
sec-Butyl Isobutyl
rivatives and the melting points of the derivatives used in this investigation are also listed in Table I. Since the column oven temperature (250' C.) is well above the melting points of all of the compounds studied, it is not surprising that there is 'no correlation between the melting points and the retention times. The nearly perfect correlation between alcohol boiling points and retention times is interesting, however, as it signifies that conversion of alcohols to 3,5-dinitrobenzoate esters does not alter their relative positions in series arranged according to vapor pressures. Analysis of 1 11. of the acetal, l-ethoxy1-isopentoxyethane, was accomplished by hydrolysis and identification of the products through gas chromatography of the derivatives. The acidic 2,sl-dinitrophenylhydrazine solution hydrolyzed the acetal very rapidly giving a copious precipitate of the carbonyl derivative almost instantaneously after the addition of the acetal to the reagent. A tenminute period of shaking and waiting is suggested simply to assure that all of the acetal is hydrolyzed. Simple distillation of 60-S0~0of the volume of the diluted hydrolysis mixture serves to separate most alcohols from the carbonyl derivative. Water-soluble alcohols with boiling points lower than that of water would be distilled over because of the low boiling points, while most higher boiling, less water-soluble alcohols would be carried across by steam distillation. It is t'rue, of course, that extremely ~at~er-soluble,very high boiling alcohols and glycols would not be recovered by this technique. Although no detailed study was made of the completeness of the reaction between
%pentyl 3-Methyl-2-butyl n-Butyl Active amy1 Isoamyl 2-Hexyl n-Amyl cis-3-Hexenyl 2-Heptyl n-Hexyl Isoheptyl n-Hept 1 n-Oct y?' Benzyl n-Nonyl 2-Phenethyl n-Decyl
B.p., "C. 64.7 78.5 82.3 97.2 99.5 108.4 119.3 114
117.7 128 130.5
140 138 156-157 160.4 157.2 168 176 195
205.2 213 220
231
3,5-DvB m.p., C. 106.5-106.8 93.0- 93.2 121.5-121.8 73.0 75.2- 75.5 85.5- 86.0 60.7- 60.9 76.0- 76.4 63.0- 63.2 84.0- 84.9 60.0- 61.0 37.5- 38.2 44.0- 45.0 48.5- 50.0 49.5- 50.0 57.9- 58.7 56.5- 57.0 46.0- 46.5 61 - 62 112 -113 52 - 53
107.3-107.8 56.5- 57.0
3,5-DNB retention time, min. 3.4 3.6
3.7 4.3
4.4
4.7 5.2 5.2
5.4 6.0 6.1 6.5
6.9 8.1 8.2 8.3
9.6 10.4 13.3
14.0
16.6 17.1 21.5
VOL. 38, NO. 1, JANUARY 1 9 6 6
35
Table II. Gas Chromatographic Retention Times of Alcohol-3,5-Dinitrobenzoates and Carbonyl-2,4-Dinitrophenylhydrazone from Hydrolyzed Acetal
Compound 3,5-DNB from acetal (Peak 1) 3,5-DNB from acetal (Peak 2) 2,PDNH from acetal Ethyl-3,5-DNB Isoamyl-3,5-DNB Acetaldehyde-2,4DNH
Retention time, min. 3.6 6.1 4.1 3.6 6.1 4.1
Table 111. Gas Chromatographic Retention Times of Certain Carbonyl-2,4Dinitrophenylhydrazones
Compound Acetaldehyde Propionaldehyde Acetone Isovaleraldehyde
Retention time, min. 4.1
4.9
5.1
6.6
the 3,5-dinitrobeneoyl chloride and the alcohols under the conditions used, it was observed that the conversion was complete enough to give two large peaks, in addition to the benzene peak, on gas-liquid chromatography of a portion of the reaction products from 1 111. of acetal. These peaks had retention times corresponding to
those for ethyl-3,5-dinitrobenzoate and isoamyl-3,5-dinitrobenzoate (Table 11). Injection of a benzene solution of 3,5dinitrobenzoyl chloride or 3,5-dinitrobenzoic acid gave no discernible peaks other than that of the benzene solvent. The retention time of the 2,4-dinitrophenylhydrazone derivative of the carbonyl portion of the hydrolyzed acetal is also given in Table 11. In Table I11 are listed, for comparison purposes, the retention times of four known carbonyl2,4 - dinitrophenylhydrazones which were run gas chromatographically under conditions identical to those used for the hydrolysis product. It is evident that the hydrolysis products from an acetal can be determined successfully in the form of the derivatives by means of gas chromatography. In order to confirm that 2,4-dinitrophenylhydrazone derivatives do pass through the gas chromatograph without decomposition, samples of known isovaleraldehyde - 2,4 - dinitrophenylhydrazone were injected into the Loenco instrument and collected after passage through the gas chromatograph. The derivative before and after gas chromatography showed identical behavior on a thin layer chromatogram. The fact that ~arbonyl-2~4-dinitrophenylhydrazones and alcohol-3,5-dinitrobenzoates can be separated easily by gas chromatography without decomposition suggests that a number of other derivative compounds also might be separated and identified in this manner. Perhaps of more significance,
however, is the fact that gas chromatography can be used as a method to obtain the derivative in a pure state when the quantity is too small to permit purification by recrystallization techniques. A small sample of a mixture of derivatives thus could be resolved into its components and the components tentatively identified through retention times. After collection of the separated components melting points can be determined using a micro hot stage and the identifications thus be further confirmed. Although this report is limited to a discussion of the successful application of these techniques to the analysis of trace quantities of complex acetals, it appears that functional group separations and analyses of very small samples of complex aroma or flavor extracts should be facilitated. LITERATURE CITED
(1) Dhont, J. H., de Rooy, C., Analyst 86, 74 (1961). (2) Lipscomb, W. N., Baker, R. H., J. Am. Chem. SOC.64, 179 (1942). (3) Sadtler, S. P., “Catalog of Infrared
Spectrograms,” Philadelphia, Pa. (4) Shriner, R. L., Fuson, R. C., Curtin, D. Y., “The Systematic Identification of Organic Compounds” 4th ed., p. 212, Wiley, Kew York, G..Y., 1956. ( 5 ) Soukup, R. J., Scarpellino, R. J., Danielczik., E.., ANAL.CHEM.36. 2255 (1964). (6) Webb, -4.D., Kepner, R. E., Food Res. 22, 384 (1957). RECEIVED for review September 7, 1965. Accepted November 15, 1965.
Gas Chromatographic Determination of Campesterol, P-Sitosterol and Stigmasterol ANDREW ROZANSKI Research Laboratories, The Upjohn Co., Kalamazoo, Mich. An accurate gas chromatographic method for the determination of campesterol, p-sitosterol, and stigmasterol is described. The sterols are separated as trimethylsilyl ethers on a stainless steel column, packed with 0.75-1 .O% silicone gum rubber on an acid-washed, silanized support, having about 4 0 0 0 theoretical plates. The quantification is carried out by measuring ratios of peak areas before and after addition of a standard amount of one of the sterols to the sample, or by using cholesterol as an internal standard. For the first time, the relative responses in the flame ionization detector of the trimethylsilyl ethers of these three sterols and cholesterol have been accurately determined and found to be significantly different. The method is
36
ANALYTICAL CHEMISTRY
easily used on a routine basis with precision and accuracy hitherto unattainable, and has been applied to the analysis of soybean sterols.
E
of the commonly occurring phytosterolscampesterol (24a -methyl - cholest - 5 en-3B-o1), 0-sitosterol (240-ethylcholest-5en-3P-o1), and stigmasterol (24p-ethyl-cholest-5,22-dien-3~-ol) - is important in a number of studies and Complete applications (7, 8, 19). analysis of a mixture of such closely related sterols had been very difficult before the successful application (25) of gas-liquid chromatography (GLC) to steroids. Their separation by paper (18) or thin-layer (4) chromatography XACT DETERMINATION
is difficult and a t best semiquantitative results might be expected. It is possible to determine stigmasterol in soybean sterols by two specific methods based on its higher degree of unsaturation-e.g., by an infrared (10) and by a radioactive isotope dilution technique (6). GLC is capable of separating campesterol, @-sitosterol, and stigmasterol and has been frequently reported (1, 2 , 7, 8, 11, IS, 15, 20, 21, 23). Derivatives such as esters (12-14, 22) and ethers (S), including trimethylsilyl (TMS) ethers (12, 24) have also been separated by GLC. Although this informative and inherently precise method has been often used for the separation and identification, attempts a t quantitative analysis (1, 7 , 8, 11, 14,
