Analysis of Mixtures of Amino Acids by Gas Phase Chromatography

Zur Struktur der Inhaltsstoffe von Cecropia adenopus Martius, 3. Mitt. Richard Neidlein , Erich Koch. Archiv der Pharmazie 1980 313 (6), 498-508 ...
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should make possible the analysis of higher-boiling and more complex aldol condensation products with equal speed and precision. LITERATURE CITED

(1) Desty, D. H., “Vapour Phase Chromatography,” Academic Press, S e w York, 1957. (2) James, A. T., Biochenz. J . 52, 242 I 1 8.52’1 \----,

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Figure 3. Chromatogram of products from aldol condensation of propionaldehyde and isobutyraldehyde Column, paraffin on Celite 545, 2 meters X 4.7 mm. per minute. Recorder attenuation in parentheses A. 2,4-Dirnethyl-2-pentenal (8) B. 2-Methyl-2-pentenal (8) C. lsobutyraldehyde ( I )

Temperature, 120’ C.

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Aiaalyst (3) James, -4.T., Martin, A. J. P., A. T., Martin, A. J . P., Bio77, 915Is,(1952). (4)Jame , chem. J. 50, Si? (1953). Smith, (5) James, A . T., 1\lartin. -2. J. P.. G. H., Ibid., 52, 238 (1952). (6) James, D. H., Phillips, C. S. G., J . Chem. Soc. 1953, 1600. ( 7 ) Lambdin, IT.J., Kourey, R. E., Yarborough, V. A., unpublished data. (8) Phillips, C. S. G., Discussions Faraday Soc. 1949. s o . 7 . 241. (9) ITarren,‘ G,-JT.,’Haslrin, J. F., Kourey, R. E.,. Tarborough, T’. h., A h - a ~ . CHEM.,submittc:d for publication.

RECEIVED for review -4ugust 22, 1958. Accepted January 12, 1959.

Analysis of Mixtures of Amino Acids by Gas Phase Chromatography C. G. YOUNGS Prairie Regional laboratory, National Research Council o f Canada, Saskatoon, Sask., Canada

b A number of amino acids were quclntitatively separated b y gas phase chromatography, The amino acids were converted to their N-acetyl butyl esters prior to chromatography and the esters fractionated on a column of firebrick coated with a hydrogenated vegstable oil. Quantitative determinations of glycine, alanine, valine, leucine, isoleucine, and proline were obtained both for synthetic mixtures of the pure acids and for protein hydrolyzates. Several additional peaks were obtained during the analyses of the protein hydrolyzates. These materials, which were probably additional constituent amino acids, came off the column after the above six amino acids.

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PHASE chromatography has made possible the quantitative separation of the components of numerous complex mixtures. The rapidity, ease of operation, and the small sample size required by this new technique have opened many new avenues of research in fields such as fats and oils. I t s extension to the

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separation of mixtures of amino acids would greatly facilitate much of the work being done on proteins from a nutritional, biochemical, or chemical aspect. The separation of amino acids by gas phase chromatography is, in effect, trvo problems. The acids must be converted quantitatively to a volatile derivative and a suitable column and operating conditions must be found n hich give a sharp separation of these derivatives. K o r k is being done on the conversion of amino acids to aldehydes with ninhydrin and separation of the aldehydes by gas phase chromatography (2, 7 , 8 ) . Bayer. Reuther, and Born ( 1 ) have prepared the mcthjl esters of several amino acids by the Fischer method (6) and qeparated the esters by gas phase chromatography. None of these reports give quantitative data. Both the preparation of aldehydes and esters pose difficult problems in quantitative recovery of the derivatives. Cherbuliez et a / . (3, 4 ) have reported that LYacetylation of amino acid esters overcomes a number of the difficulties inherent in the Fischer ester distillation

method and this paper deals with the gas phase chromatography of these latter derivatives. EXPERIMENTAL

The X-acetylated ethyl esters of a mixture of glycine, alanine, valine, and leucine were prepared and injected into a Beckman GC2 chromatographic unit, Reasonable retention volumes were obtained a t 220’ C. with a good separation and symmetrical peaks. The .Y-acetylated ethyl ester of glycine tended to crystallize from the mivture of acetylated esters and a homogenous sample could not be taken for injection. This problem was overcome by conversion to the butyl esters of the amino acids rather than the ethyl esters. K h e n this was done the derivatives of all the amino acids remained as a viscous oil and by using the .&--acetylated butyl esters, a satisfactory chromatographic procedure was developed. Of the columns tested. the one consisting of hydrogenated vegetable oil on firebrick gave the best resolution with these derivatives. Preparation

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N-Acetyl

VOL. 31, NO. 6, JUNE 1959

Butyl

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Figure 2.

Trace for analysis of gelatin hydrolyzate

Trace for analysis of synthetic mixture

Esters.

Twenty milligrams each of glycine, alanine, valine, leucine, and isoleucine were suspended in 50 ml. of butyl alcohol and the solution saturated with anhydrous hydrogen chloride. Approximately one-half t h e butyl alcohol was removed by distillation at atmospheric pressure over a period of 45 minutes. T h e bulk of t h e water formed in t h e reaction was also removed during this distillation. T h e remaining butyl alcohol was removed by vacuum distillation leaving the ester hydrochlorides as a viscous sirup. Twenty-five milliliters of acetic anhydride were added to this sirup and after 1 hour at room temperature the unreacted acetic anhydride was removed by vacuum distillation. The lT-acetyl butyl esters remained as a viscous oil which was injected directly into the chromatographic unit. The entire procedure was repeated twice with the same amounts of amino acids to determine the degree of precision obtainable. This v a s followed by a misture containing 30y0 glycine, 10% alanine, 5% valine, 20% leucine, 2070 isoleucine, and 15y0proline. T v o grams of gelatin rrere hydrolyzed by reflusing for 20 hours with 50 ml. of 6;\r hydrochloric acid. The excess acid was removed by vacuum distillation and a portion of the resulting hydrolyzate substituted for the amino acid mixture in the above procedure. -1. Kjeldahl nitrogen determination was run on the hydrolyzate because the water was not completely removed by vacuum evaporation. The N-acetyl butyl esters were also prepared from samples containing 100 mg. of hydrolyzate with 10 nig. of valine and with 10 mg. of leucine. Gas Phase Chromatography. A Beckman GC2 chromatographic unit was used. T h e column consisted of a 6-foot length of l/r-inch copper tubing containing approximately 20 grams of 20- t o 40- mesh firebrick coated n i t h hydrogenated vegetable oil. Safflower oil, hydrogenated t o a n iodine value of less t h a n 1 mas used, and the ratio of solid support to liquid phase TTas 4 1020

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Figure 3. Trace for analysis of gelatin hydrolyzate plus 9.1 % leucine

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to 1. The oven temperature was 220' C. and helium was used as the carrier gas n i t h a flow rate of 80 ml. per hour measured a t the outlet of the detector cell. The sample size injected was 10 microliters. The emergence time of the last peak was approximately 1 hour. RESULTS AND DISCUSSION

Figure 1 shows the trace obtained from the analysis of a mixture containing equal amounts of glycine, alanine, valine, leucine, and isoleucine. The order of emergence of glycine and alanine is reversed from that expected on the basis of molecular 11eight, and leucine and isoleucine emerged as a single peak. The areas under the peaks were determined as the peak height times the width a t half the peak height. The areas were corrected for the difference in molecular weight betn-een the amino acids and their Xacetyl butyl esters and the per cent composition was calculated from the corrected areas. The results are given in Table I for three runs on the same

mixture and another mixture containing the same five amino acids plus proline. The agreement between the values calculated from the areas under peaks and the actual values is good. The maximum deviation from the actual values was i.1070. This is of the same order as the errors involved in determining the areas under the peaks. Figure 2 s h o m the trace obtained from the analysis of a gelatin hydrolyzate. Figure 3 is the trace for the hydrolyzate plus 9.1% leucine. The areas under the peaks n-ere determined as before and corrected to an amino acid basis. A correction was then made for the difference in sample size betn-een the two runs by totaling the areas under all the peaks except leucine for each trace and correcting the areas for one so that these totals ryere the same for both. The difference in areas for the leucine peaks on these final corrected values was taken t o represent 9.1% and the percentage represented by each peak calculated. These psrcentages n-ere brought t o a basis of

16% nitrogen from the value of 9.6% found on a Kjeldahl determination on the hydrolyzate. These final values are given in Table I1 with those determined in the same manner by adding valine rather than leucine. Table I1 includes the values reported by Greenberg (6) for the composition of gelatin; they are in good agreement with the duplicates reported here. Gas phase chromatography of the 1vacetylated butyl esters of amino acids can be used for the quantitative determination of these six amino acids. Horn far the method can be extended to include additional ones remains to be seen. A number of peaks were obtained beyond proline for the gelatin hydrolyzate and work is continuing to identify these and to see if quantitative data can be obtained. It is likely that there will be difficulty M-ith acids such as cystine, tyrosine, arginine, histidine, and lysine. LITERATURE CITED

(1) Baver. E.. Reuther. I