~
boiling) peak with that of HMD, while complete separation of all three diamines was finally obtained on the DC 710 column. Figure 4 shows the separation obtained using an 8-meter column of 10% DC 710 on Chromosorb W 5% KOH. This separation was found to be entirely satisfactory for quantitative analysis, even though base line separation was not achieved a t the retention times chosen.
+
EXPERIMENTAL
The sources for the various packing substrates and partition solvents used in this work are listed below. Packing Substrates. Chromosorb. W. H. Curtin Co., Houston, Tex., Cetalog No. WHC-8786G7B, 30/60-
mesh size. Chromosorb W. W. H. Curtin Co., Houston. Tex.. Cataloe No. WHC8786G7D, 30/6b-mesh &e. Firebrick. Coast Engineering Laboratory, Hermosa Beach, Calif., GC-22 Super Support, 42/60-mesh size. Celite. Perkin-Elmer Corp., Norwalk, Conn., Catalog KO.154-0048. Partition Liquids. Carbowax 20M. Union Carbide Chemicals Co., New York 17, N. Y. Dow Corning 710 Fluid. Dow Corning Corp., Midland, Mich. Apiezon L Grease. James G. Biddle Co., Philadelphia 7, Pa.
The solid substrates were tested as received for inherent adsorption properties by packing into ‘/,-inch by 1meter aluminum U-tube columns. All gas chromatograms were run on a Perkin-Elmer Model 154-B Vapor Fractometer after the columns were conditioned for a t least 1 to 2 hours at about 200’ C. The solid substrates were coated with KOH by slurrying a weighed quantity with a methanolic solution of the KOH. The excess methanol was first removed by evaporation in a water bath with continuous stirring. The moist packing material was then dried in a circulating air oven for 1 to 2 hours a t 100’ C. The calculated quantity of partitioning phase was applied to the pretreated packing in a similar manner. Water was used as the solvent for Carbowax 20M, benzene for Apiezon L, and methylene chloride for the Dow Corning 710 fluid. After evaporation of the excess solvent, the packings were finally dried in a circulating air oven a t 120’ C. No particular precautions were used to protect the packings from COZ during their preparation or later use, so some of the KOH may have been present as the carbonate. ACKNOWLEDGMENT
The authors express their appreciation to M. B. Powell for her careful work in preparing the many gas chromatographic packings studied in this
work, and for running and recording the actual chromatograms. LITERATURE CITED
(1) . . Bens, E. M.. AN^. CHEM.33. 178 (1961) (2) Brooks, V. T., Collins, G. A,, Chent. and Znd. (London) 38, 1021 (1956). (3) Decors, A. W., Dinneen, G. V.,
:
“Solid Support for Gas-Liquid Chromatography of Strongly Basic Nitrogen Cbmuounds.” 2nd Bisnnual Internatj6nal G& Chromatography Symr m , Michigan State University, ast Lansing, Mich., June 1959. (4) Glueckauf, E., Trans. Faraday Soe. (5)51,Hardy, 34 (1955). C. J., Pollard, F. H., J .
Chromalog. 2, 1-43 (1959). (6) Honegger, V. T., Honegger, R., Nature 184, 551-2 (1959). (7) James, A. T., ANAL. CHEM.28, 1564 (1956). (8) James, A. T., Biochem. J . 52, 242 (1952). (9) James A. T., Martin, A. J. P., Smith, 6. H., Zbid., 52, 238 (1952). (10) Ottenstein, D. M., “Influence of the
Chromatographic Support on the Separation of Polar Compounds,” Seventh Detroit Anachem Conference, Detroit, Mich., October 1959. (11). Ring, R. D., Riley, F. W., “GasLiquid Chromatographic Analysis of Amine Mixtures,” Sixth Detroit Anachem Conference, Detroit, Mich., October 1958. RECEIVED for review July 25, 1960. Accepted May 23, 1961. Contribution 70 from Chemstrand Research Center. Inc.
Chromatog ra phic Separation and Identification of Normal Aliphatic Alcohols as Esters of p-Nitrophenylazobenzoic Acid by infrared and X-Ray Diffraction ROLF BOSVIK, KNUT V. KNUTSEN, and C. F. ERIK von SYDOW Swedish Institute for Food Preservation Research (SIK), Gothenburg, Sweden
b Alcohols in fruit flavors can be separated and identified as esters of p-nitrophenylazobenzoic acid by combined paper partition chromatography, infrared analysis, and x-ray diffraction. Both infrared spectra and x-ray diffraction patterns show marked differences among the esters.
A
are generally present in fruit flavors, although mostly in very small amounts. Paper partition chromatography is very useful for separation and identification of the alcohols, and several methods have been described. A general trend is to chromatograph compounds having colors of high intensity and distinctive melting points for a wide melting point LCOHOLS
1 162
rn
ANALYTICAL CHEMISTRY
range. Esters of p-nitrophenylazobenzoic acid (p-NPAZB acid) are easily prepared and seem to be suitable derivatives. Their use for characterization of alcohols using melting point data was first suggested by Hecker (I), and later Winter et al. (8) proposed chromatographic separation of the esters. The possible use of paper partition chromatography, which seemed to be the most effective and practical procedure, was investigated. Identification of compounds using R, values alone, is often insufficient due to the fact that the conditions during chromatography are hard to control, and that several compounds may have nearly the same Rf value. These difficulties can be reduced by using other supplementary methods. In the present
work infrared spectrophotometry and x-ray diffraction proved effective supplementary techniques. EXPERIMENTAL
Reagents. The p-nitrophenylazobenzoyl chloride (p-NPAZB chloride) used for the preparation of the esters was synthesized according to Hecker ( I ) , starting with the ethyl ester of p-nitrobenzoic acid. This ester was transformed into the nitroso compound, which was condensed with p-nitroaniline to give the ethyl ester of p-NPAZB acid. After saponification, the acid chloride was obtained after reaction with thionyl chloride. The alcohols used as references were commercial samples, purified, and checked in this laboratory by gas-liquid chromatography.
Table I.
R,
Valuer for Esters of p-NPAZB Acid
R,
Ester
Figure 1 . Paper chromatogram of esters of p-NPAZB acid
Apparatus. The infrared spectrophotometer used was a Perkin-Elmer Infracord 137. I n all cases the samples, 1 mg., were pressed in 300 mg. of KBr to give disks of 13-mm. diameter, and a pure K B r ~ d i s k was used as reference. The x-ray diffraction measurements were carried out on a Guinier w a y diffraction powder camera using Cu K a radiation. The specimen (0.1 to 0.2 mg.) was mixed with Si, used for calibration, and mounted on thin cellulose tape. Paper Chromatography. The reference e.sters were made by reaction of the alcohols with p-NPAZB chloride in benzene solution. The crude esters thus obtained were purified by passing them through a short column of .41?03, and by repeated recrystallization. The melting points agreed with those given by Hecker (f), with the exception of the pentyl ester, mhich melted at 115" C. The heptyl ester, which was not included in Hecker's work, melted at 108.5' C. The chromatographic paper used ww Number 2045 bM made by Schleicher and Schiill, Dassel/Kr. Einbeck, Germany. It was impregnated with N,N'dimethylformamide by immersing it for 20 minutes in a 30% solution in acetone, and afterward dried by pressing between filter papers. Ascending chromatography was used with 2,2,4trimethylpentane as the mobile phase. The atmosphere in the jar was saturated with both phases by evaporation from paper strips suspended inside the walls. The ester mixtures, dissolved in benzene, mere applied to the paper in strips along the start line, and the chromatograms were developed for 16 hours. Five to 10 pg. of each ester v-as used. Elution. Several series of esters were developed on the same paper. After a complete run, the paper was cut in strips containing spots of
With careful equilibration of the system at constant temperature (20" C.), and using amounts of 5 to 15 pg. of each ester, satisfactory separation was obtained (Figure I). Representative R,
identical B , values. The spots were eluted with acetone by descending chromatography. By this procedure the esters followed the front, allowing a minimum amount of solvent to be used, thus reducing the concentration of impurities from the solvent. By evaporation in vacuo, the solvent and the eluted stationary phase could be removed. Paper fibers were removed by filtration through ALOa. The purity thus obtained, was satisfactory for x-ray diffraction and infrared analysis. RESULTS
Paper Chromatography. Preliminary work indicated that the best separation was accomplished using impregnated paper with a low flow rate. Of several systems tried, the one using N,N'-dimethylformamide as the stationary and 2,2,4-trimethylpentane as the mobile phase, gave the best results. To ohtain the necessary low flow rates the paper was cut in tongues just in front of the start line in the following manner. The 12-em. wide paper, used for four parallel runs, was cut in four symmetrically placed rectangular tongues of 1-em. width, leaving three Zcm. wide open areas between the tongues.
Figure 2. Infrared spectra of narmal saturated estersof p-NPAZB acid
Crofyl
Figure 3. Infrared spectra of isopropyl, neopentyl, and crotyl esters of pNPAZB acid
EO."'
VOL 33, NO. 9, AUGUST 1961
1163
Table II. X-Ray Diffraction Powder Patterns of Esters of p-NPAZB Acid Methyl InSpactening A. sity vw S
m W
vw vs m vs v w m vs W
vs vw vw vw W
vw vw m W
13.97 6.96 6.54 6.30 5.58 5.15 4.43 3.61 3.57 3.45 3.32 3.29 3.16 3.07 2.96 2.64 2.59 2.42 2.39 2.30 2.17
Ethyl InSpactening sity A. S 16.16 S 8.03 W 6.04 vs 5.40 W 5.29 vw 4.78 4.61 vw vw 4.56 4.53 vw W 4.40 vw 4.07 vs 3.74 S 3.70 m 3.69 vs 3.66 S 3.56 m 3.49 3.41 S vs 3.19 m 3.16 m 3.08 m 3.07 vw 3.00 vw 2.89 vw 2.86 2.49 vw m 2.43 m 2.37 W 2.30 2.25 vw
Propyl Butyl In- Spac- In- Spacing tening tensity A. A. sity m s 8.52 17.68 m 6.12 m 8.80 m 5.90 m 6.22 m 5.71 S 5.76 W 5.19 8 5.53 s 5.30 5 4.90 W m 5.12 4.74 W w 4.87 4.50 v w 4.73 W 4.37 W v w 4.53 4.04 m v w 4.29 3.76 m 3.70 v w 4.09 m 3.60 v s 3.75 v s 3.72 W 3.54 m 3.69 W 3.48 m 3.66 W 3.33 m 3.59 S 3.23 m 3.21 m 3.55 w 3.48 W 3.16 w 3.41 W 3.10 W v s 3.23 3.06 v s 3.20 W 3.00 W v w 3.17 2.97 w 3.15 v w 2.90 W w 3.08 2.86 W v w 2.98 2.80 W 2.68 v w 2.77 w 2.36 v w 2.57 2.42 w 2.35 v w m w 2.32 2.39 v w 2.16 m 2.36
Pentyl Spacing sity A. m 14.91 W 9.93 W 6.08 S 5.54 W 5.30 R 5.07 W 4.58 m .4.42 4.31 vw 4.14 vw W 3.90 W 3.75 W 3.71 W 3.65 W 3.56 3.46 S vw 3.39 m 3.29 m 3.21 W 3.09 W 3.03 vw 2.97 W 2.93 W 2.86 W 2.82 W 2.76 2.71 W W 2.63 W 2.57 W 2.47 2.41 vw vw 2.38 W 2.16 Inten-
Intensity
Hexyl Spacing
m W W W
8 W W W
W W
W W
vw W W W W
m m m vw m vw W
vw vw vw vw vw vw vw
A.
15.70 10.42 6.07 5.77 5.38 4.93 4.48 4.42 4.36 4.21 4.06 4.00 3.94 3.75 3.72 3.63 3.56 3.46 3.41 3.35 3.29 3.22 3.07 2.87 2.78 2.75 2.65 2.43 2.38 2.33 2.16
Heptyl InSpacing tenA. sity m 16.06 11.06 W 8.31 W 6.11 m 5.74 m 5.52 vw 5.38 S 4.93 m 4.57 vw 4.51 m 4.43 W 4.39 m 4.29 W 4.11 W 3.91 m 3.75 vw W 3.70 3.60 W 3.55 m 3.45 m 3.41 m 3.33 m 3.23 5 3.03 W 2.97 W 2.94 vw 2.89 W 2.86 W 2.83 vw 2.80 W 2.77 vw 2.70 W 2.67 vw 2.65 vw 2.45 vw 2.38 vw 2.37 vw 2.17 W
Octyl InSqacten1% A. sity W
vw vw vw W W
m vw W W W
vw W
vw vw vw W
vw W
vw vw vw vw vw vw vw vw vw
17.48 11.65 8.68 6.96 6.07 5.66 5.30 5.07 4.89 4.49 4.39 4.25 4.12 3.98 3.73 3.59 3.52 3.46 3.40 3.35 3.25 3.19 2.97 2.89 2.81 2.74 2.38 2.33
Intensities given as: v s, very strong; s, strong; m, medium; w, weak; and v w, very weak.
values obtained under these conditions are given in Table I. The esters are easily visible, especially in ultraviolet light, in concentrations down to approximately 5 pg., and may be photographed for a permanent record. Infrared Spectrophotometry. There is no difficulty in distinguishing between the normal saturated CI to Cs esters (Figure 2). The differences, however, become smaller with increasing chain length. The CSto Ca esters may be identified by measuring the intensity of the 3.42-micron (>CH%)band, and by determining the position of the 13.6to 13.8-micron band. The latter shifts towards longer wave lengths with increasing chain length. The branched chaii and unsaturated e&rs show marked differences (Figure 31. Several prominent bands may be distinguished; for instance, in the isopropyl ester two characteristic bands due to the isopropyl group can clearly be seen, i.e., the 8.47- and 10.90-micron bands. Three weaker, but significant bands, appear a t 7.29, 7.67, and 8.93 microns. In the neopentyl ester, the 3.37-micron band due to -CHs stretching vibrations has a high intensity due 1164
ANALMICAL CHEMISTRY
to the three --CHa groups. Furthermore, the 8.21-micron band and possibly the 9.68-micron band are highly characteristic of the tertiary butyl group. Weaker bands appear a t 7.31 and 10.28 microns. The one unsaturated compound so far investigated, is the crotyl ester. Three typical bands can easily be seen, the 7.76- and 10.29-micron bands, both (hum) vibrations due to -CH=CHand the 10.84micron -Mvibration band. Weaker bands appear at 10.12 and 11.07 microns, both due to unsaturation. X-Ray Diffraction. The x-ray diffraction powder patterns of the normal esters of p-NPAXB acid are presented in Table 11. There are marked differences between the sets of spacings, which enables clear distinction among the various members of the series. DISCUSSION
Extraction of a steam distillate with benzene provides a suitable method for the isolation of alcohols in fruit flavor. The reagent, pNPAZB chloride, is added to the dried benzene solution, where the esters are formed
and later crystallked. However, compounds with other functional groups such as --SH and -NH, also react and form derivatives of p-NPAZB acid, so that a separation into classes on an Alsoscolumn is necessary. These classes can be characterized by their infrared spectra, after which a suitable paper chromatographic procedure, such as the one suggested in this work, may be chosen and the derivatives identified by their R, values and, after elution, by their x-ray diffraction powder patterns. The use of these three methods for the analysis of alcohols, and perhaps also thiols and amines, seems to be a n effective combination. ACKNOWLEDGMENT
We are indebted to Stig Aleby for help with the x-ray recordings. LITERATURE CITED
(1) Hecker, E., Ber. 88, 1666 (1955). (2) Winter, M., Willhalm, B., Hinder, M., PaUnv, E., Sundt, E., Raechstoffe u. Arm-& 8 , '252, 282 (1958); Perf&&?ry Essat. Oil Record 49, 250 (1958).
RECEIVED for review January 23, 1961. Accepted April 3, 1961.
