high molar ratios of metal ions which may or may not be extracted from hydrochloric acid onto the column. The effects of anions which might act as complexing agents for iron(III), and hence interfere with its extraction, were studied (Table IV). The results indicate that very little iron(II1) is not extracted by the column under the loading and washing conditions used, and that the complexing agents tested do not significantly interfere with the extraction of iron(II1). We conclude that reversed-phase chromatography using 2-octanone as the stationary phase and Haloport-F
as the support is entirely adequate for the quantitative separation of small or large amounts of iron(II1) from other metal ions. This method should also be useful for the separation of other metal ions from hydrochloric acid solutions and for other separations of analytical importance. LITERATURE CITED
(1) Brandt, W. W., Preiser, A. E., ANAL. CHEM.25, 567 (1953). (2) Elbeih, I. I. M., Abou-Elnaga, M. A., Chemist Analyst 47, 92 (1958). (3) Fidelis. I.. Siekierski. S..J. Chromatoo. 5, 161 (1961). \
,
I
,
(4) Fritz, J. S., Abbink, J. E., Payne, M. A., ANAL.CHEM.33, 1381 (1961). (5) Hamlin, A. G., Roberts, B. J., Loughlin, W., Walker, S. G., Ibid., p . 1547. (6) Kuznetsov, V. I., J. Gen. Chem. U.S.S.R. 17,175(1947); C. A. 42,lS-e (1948). (7) Pierce, T. B., Anal. Chim. Acta 24, 146 (1961). ( 8 ) Siekierski, S., Fidelis, I., J. Chromatog. 4, 60 (1960). (9) Small, H., J. Inorg. Nucl. Chem. 19, 160 (1961). \----I
RECEIVED for review March 13, 1962. Accepted July 23, 1962. Contribution No. 1174. Work performed in the Amea L+boratory, U. S. Atomic Energy Commission.
Gas Chromatography of the n-Butyl-N-TrifluoroacetyI Derivatives of Amino Acids CLAIRE ZOMZELY,' GIN0 MARCO, and EDWARD EMERY laboratory o f Biochemistry and Nutrition, Monsanto Chemical Co., ,The n-butyl-N-trifluoroacetyl derivatives of individual amino acids or mixtures of amino acids can b e determined b y gas liquid chromatography. A mixture of 2 2 naturally occurring amino acids (after conversion to the double derivative) can be separated in 43 minutes or less with a single columr, using the F & M Model 500 Automatic Programmer with the Model 1609 hydrogen flame attachment. The column used is 2-meter stainless steel (0.63 cm.) packed with Gas Chrom A (60-80 mesh) and coated with 1 .O% neopentylglycol succinate polyester.
SEXE
studies have been reported the past five years on the application of gas liquid chromatography to the determination of amino acids. Progress in this area has been slow because amino acids, lacking volatility, must be converted to derivatives in which the carboxyl group, amino group, or both are removed or masked before they can be chromatographed in the gas phase. However, the use of gas chromatographic methods should permit more rapid analysis with smaller quantities of amino acids than is possible with the present liquid chromatographic methods. Gas chromatography of some aliphatic amino acids after conversion to the corresponding aldehydes with ninhy1 Present address, Department of Physiological Chemistry, University of California Medical Center, Los Angeles 24, Calif.
1414
ANALYTICAL CHEMISTRY
Sf. louis 66, Mo.
drin has been reported by Hunter, Dimmick, and Corse (5) and Zlatkis, Oro, and Kimball (16). Bier and Teitelbaum (3) successfully chromatographed several amines obtained from amino acids by decarboxylation. Bayer, Reuther, and Born (2) determined some aliphatic amino acids after conversion into the methyl esters. Bayer (1) also chromatographed the methanolyzate of albumin, in which he was able to show the presence of esters of alanine, proline, valine, leucine, isoleucine, and glutamic and aspartic acids. Youngs (14) chromatographed some amino acids after conversion to N-acetyl-nbutyl esters. Liberti (8) reported the chromatography of the methyl esters of a-hydroxy acids derived from amino acids by treatment with nitrous acid. Melamed and Renard (10) converted some aliphatic monocarboxylic amino acids to the corresponding a-chloro acids, then to the methyl esters with diazomethane for chromatography with silicone-stearic acid columns. Riihlmann and Giesecke (11) separated a mixture of the silylated derivatives of alanine, glycine, leucine, isoleucine, valine, glutamic acid, and phenylalanine by gas chromatography. The N-trimethylsilyl amino acid trimethylsilyl esters, obtained by the reaction of trimethylchlorosilane with amino acid salts or by reaction of N-trimethylsilyldialkylamines with free amino acids, were used in their study. Recently, Saroff and Karmen (12) and Johnson, Scott, and bfeister ( 7 ) reported the separation of the largest number of amino acids that have been chromatographed successfully. Saroff
and Karmen (12) chromatographed the N-trifluoroacetyl amino acid methyl esters on columns coated with polyethylene glycol adipate. However, cystine, histidine, and tryptophan derivatives were not eluted from the column, while the tyrosine derivative gave four peaks and the arginine derivative two peaks on chromatography. In addition, they were unable to separate the alanine-valine derivatives and the glutamic - serine derivatives. Johnson, Scott, and iMeister (7) separated the N-acetyl-n-amyl amino acid esters of 33 amino acids, including 18 of the common protein amino acids. Two separate columns were used to separate the less volatile amino acid derivatives in 45 minutes. Of the amino acids derived from proteins, only derivatives of tryptophan, histidine, and arginine were not eluted under the conditions employed. I n a more recent report (6) they presented data showing elution of arginine and histidine as well as chromatography on a single column using temperature programming. Unfortunately, tryptophan and cystine were destroyed in the esterification procedure which they employed. The present study concerns the chemical conversion of amino acids to the n-butyl-N-trifluoroacetyl derivative and the chromatographic conditions for separation of a mixture of 19 of the common protein amino acids. The nbutyl-N-trifluoroacetyl derivative is more volatile than the corresponding N-acetyl derivative, resulting in faster elution from the column on chromatography. Columns packed with neopentylglycol succinate coated on Gas
Chrom A were used and derivatives of 22 individual amino acids as well as mixtures of these amino acids have been prepared and separated. Certain aspects of quantitation are also discussed. EXPERIMENTAL
Apparatus. An F & M Model 500 Automatic Programmer with Model 1609 hydrogen flame detector attachment was used. The principal carrier gas was nitrogen, b u t helium is also satisfactory. Two-meter stainless steel coiled columns (i.d. 0.63 cm.) were packed with neopentylglycol succinate polyester (Wilkens Instrument and Research, Inc.) coated on a solid support consisting of Gas Chrom A (60- to 80-mesh) purchased from Applied Science Laboratories, Inc., State College, Pa A weighed amount of the solid support was added to a solution of neopentylglycol succinate in methylene chloride. The solvent was removed under slight vacuum with a rotary evaporator (30" C. water bath). The column, after packing, was conditioned for 12 t o 16 hours with nitrogen, a t a flow rate of 100 ml. per minute. Reagents. The amino acids were obtained from the Sigma Chemical Co. and Nutritional Biochemicals Corp. All amino acids, except glycine, were of the L-configuration. However, the chromatographic behavior of both L- and m-amino acids was compared and no differences were found. The I-butanol, methylene chloride, and dimethylformamide were obtained from the J. T. Baker Chemical Co.; the dibutoxypropane and trifluoroacetic anhydride, from Eastman Organic Chemicals. Procedure. PREPARATION OF nBUTYL-N-TRIFLUOROACETYL AMINO ACIDS. Esterification. The free amino acids are first converted to the n-butyl esters under very mild conditions by a modification of the method of Lorette and Brown (9), which was developed for the esterification of fatty acids. Individual amino acids or mixtures of amino acids were dissolved in a mixture of 1-butanol containing 5% anhydrous hydrogen chloride, dimethylformamide (10% of the volume of butanol), and &butoxypropane (used as a water scavenger). The dimethylformamide was necessary for esterification of the basic amino acids in this procedure. For samples containing 10 mg. or less of amino acids, 20 ml. of the acidified 1-butanol and 2 ml. of dimethylformamide were used (individual or mixtures), and the volumes of the acidified butanol and dimethylformamide were adjusted proportionately. Dibutoxypropane was added in threefold the amount required to react with the water formed in the esterification process. The Erlenmeyer flask, fitted with a CaCh drying tube and containing the reaction mixture, was placed in a glycerol bath a t 55" to 60" C. and heated, with magnetic stirring, for 3 hours. The alcohol was evaporated in vacuo a t 60" C. in a rotary evaporator. The final residue, consisting of the ester
hydrochlorides of the amino acids, WP.E neutralized with 1N sodium carbonate and extracted three times with 10 ml. of using a 50-ml. separatoryextract funnel.wBs The methylene evaporated in
vacuo at 300 c. in a rotaryevaporator. b additional 10 ml, of methylene &loride was added t o the residue as a precaution in removing any residual water and the evaporation procedure was repeated. Trifluoroacetulation. The residue resulting from the esterification reaction was dissolved in 2 to 5 ml. of methylene chloride containing 2% dimethylformamide. The trifluoroacetic anhydride was transferred in a closed system to a micro reaction flask by immersing the flask in a sodium chloride-ice bath and applying a slight vacuum, according to the method of Weygand and Geiger (IS). For samples containing 10 x g . or less of amino acid, 0.2 t o 0.5 ml. of trifluoroacetic anhydride was used; 1.5 ml. was used for amino acid mixtures containing 80 to 130 mg. of amino acid. After addition of the anhydride, the bath was removed and trifluoroacetylation carried out a t 28" C. (flask immersed in glycerol bath) for 30 minutes. The excess reagents were removed in vacuo and the residue was dissolved in acetone for chromatography. Chromatography. The column temperature was programmed from 75' to 220' C. T h e temperature of the injection block was 265' C. and t h a t of the detector was maintained at 250" C. The flow rate of the carrier gas was 128 ml. per minute throughout the program, as measured a t the outlet. The samples were introduced into the column in acetone with a 10-pl. No. 701 N Hamilton syringe (Hamilton Co., Whittier, Calif.). The column temperature was set a t 75" C. and maintained a t this temperature for 5 minutes after sample injection. Then the temperature program (previously set a t 5.6" C. per minute) was initiated. Twenty-one minutes later, the temperature program was adjusted to 7.9" C. per minute and the temperature allowed to increase to the maximum setting of 220" C. G a s Chromatographic Determination of Water. A 2-meter column (0.63-em. i.d.) packed with 30-mesh Teflon coated with 8 weight % Quadrol (N,N,X',N'-tetrakis-[2-hydroxypropyll-ethylenediamine)obtained from Wyandotte Chemical Corp., Wyandotte, Mich., was used in a PerkinElmer Model 154 Vapor Fractometer. The injection block was maintained a t 200" C. with the carrier gas, helium, a t a flow rate of 50 to 60 ml. per minute and a column temperature of 68" C. RESULTS
Figure 1 shows a typical chromatogram of 19 amino acid derivatives separated with a 2-meter column packed with Gas Chrom A and coated with 1% neopentylglycol succinate. The free amino acids were mixed, then esterified and trifluoroacetylated. The peaks on
the chromatogram were identified by checking the retention times with those of the individual amino acid derivatives chromatographed under the same conditions as the mixture. Supplementary amounts of individual amino acid derivatives were added to several fractions of the mixture and these fractions chromatographed to check for increased response of the particular component in the mixture. The electrometer input range or attenuation was set at 100. Attenuations marked on the chromatogram-e.g., X 6 G a r e those of the output signal from the electrometer. The areas of the peaks represent about 0.4 pmole of each amino acid. The chromatogram (Figure 1) shows that the detector response is not the same for equivalent micromoles of the various amino acids. For example, the peak areas, measured by a Model K-1 disk integrator, of tryptophan (56,400 counts), lysine (77,800 counts), and serine (110,000 counts) are less than for leucine (310,200 counts), aspartic (170,500 counts), and alanine (187,600 counts). A partial explanation is that the hydrogen flame detector is thought to respond primarily to the CH-bond and that various functional groupse.g., carbonyl, carboxyl, ether, and amine-cause a loss of response as discussed by Dewar (4). Therefore, the lysine derivative with two trifluoroacetylated amino groups would be expected to produce a smaller response than the derivative of the aliphatic amino acid, alanine, which has no interfering groups. Saroff and Karmen (12) in their work with the methyl trifluoroacetyl amino acids were also unable to obtain similar peak areas for equal amounts of different amino acids using a hydrogen flame ionization detector. They thought the results represented either unequal yields in the synthesis of the amino acid derivatives or a variation in the detector response to the different amino acid derivatives. One or both of these explanations are possible. The possibility of unequal yields is being investigated a t the present time in our quantitation studies. Table I shows the retention times of 22 n-butyl-N-trifluoroacetyl amino acids under the same conditions for chromatography as in Figure 1. DISCUSSION
The data presented in this study show that a single gas liquid chromatographic column can be used for the separation of a complex mixture of amino acids after conversion to the n-buty1-Ntrifluoroacetyl derivatives. The use of a n automatic programmed hydrogen flame detector instrument permits the resolution of a mixture of 19 of the common protein amino acids in 35 to VOL. 34, NO. 11, OCTOBER 1962
1415
w v)
z
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v)
w
a a
0 I-
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w Iw
P
MINUTES Figure 1.
Separation of mixture of 19 n-butyl-N-trifluoroacetyl amino acids
2-meter column packed with G a s Chrom A, coated with 1% neopentylglycol succinate. Flaw rate. 128 ml. per minute. Starting temperature 75' C . After 5 minutes, temperature program set a t 5.6' C. p e r minute; 21 minutes later, temperature program set a t 7.9' C. p e r minute to maximum of 220' C.
45 minutes, using a singlc packcd column. Although the chemical procedures involved in the conversion of the amino acids to the n-butyl-N-trifluoroacetyl derivatives require a longer time than reported in previous studies, the use of the mild esterification conditions preserves such amino acids as tryptophan, cystine, cysteine, arginine, and histidine. In addition, many samples of amino acid mixtures can be carried through the chemical treatments simultaneously. I n a preliminary report (16) chloroform had been used as the solvent for extraction of the butyl esters of the amino acids after liberation from the hydrochloride salt with aqueous sodium carbonate, as well as for aseotroping
Table I. Retention Times of n-ButylN-trifluoroacetyl Amino Acids
(Conditions of chromatography described in Figure 1 ) Retention time, Amino acid minutes Alanine 10 9 Sarcosine 11.7 Valine 12.4 Isoleucine 14.5 Glycine 15.7 Leucine 16.4 Proline 18.4 Serine
Threonine Methionine Methionine sulfoxide Aspartic acid Phenylalanine Cysteine Glutamic acid Histidine Ornithine Lysine Arginine
Tyrosine Tryptophan Cystine 1416
0
19.1 20.9 23.6 23.6 24.3 24.7 26.0 27.2 28 3 30 8 32.1 32.7 35.3 38.8 41.9
ANALYTICAL CHEMISTRY
residual water from the chloroform extract. More recently, we have found that methylene chloride is superior for this, since i t is a more effective drying agent. Aliquots of both chloroform and methylene chloride extracts of the amino acid butyl esters were checked for water content by gas chromatography before and after azeotroping. Only 75% of the residual water was removed from the chloroform extract after the first azeotroping, whereas 99% was removed from thc methylene chloride extract. I n our earlier studies, we used a Perkin-Elmer Model 154 Vapor Fractometer with hydrogen flame detector attachment for the amino acid gas chromatographic analysis. Since it was not possible to elute the complete spectrum of amino acid derivatives from alanine to cystine from a single column under isothermal conditions, we attempted to simulate linear programming by controlling the power input to the instrument. This method was successful, as reported previously ( I @ , but it was extremely difficult to reproduce the retention times of the amino acid derivatives. Therefore, when the F BE M Automatic Programmer with hydrogen flame attachment became available, the gas chromatographic analysis was carried out with this instrument. The retention times of mixtures of amino acid derivatives as well as individual derivatives are reproducible with the automatic linear program instrument. Preliminary quantitation studies shon-ed that conversion of the amino acids to the n-butyl ester form appears to be more than 90% complete, since tha aqueous sodium carbonate phase shows only trace amounts of ninhydrinpositive material. Also, infrared analyses of the crude-free ester preparations showed the absence of the free carboxyl group, indicating the absence of hydroly-
sis during the neutralization and azeotroping procedures. Initial studies of the esterification reaction using thin layer chromatography indicate a trace of unreacted amino acid and one major component, presumably the ester. Additional studies using this technique are in progress to define the quantitation of the chemical reactions. Studies in which purified derivatives of the amino acids are compared with the crude form as to detector response and extent of conversion are now in progress. While the reproducible separation of the common protein amino acids has been accomplished, quantitation of the method is necessary for application to the complete quantitative analysis of protein hydrolysates. Without quantitation, any gas chromatographic method can serve only a limited function as a tool for the determination of amino acids, peptides, and proteins in biochemical studies. ACKNOWLEDGMENT
The authors express their appreciation to Andrew Bybell and Robert L. McKinley for their excellent assistance in portions of the gas chromatographic work. LITERATURE CITED
(1)
Raver, E., "Gas Chromatoaanhv D. H. Desty, ed., F Press, Piew Yc
Teitelbaum, P., Ann. A'. Y . Acad. Sci. 72, 641 (1969). ( 4 ) Dewar, R. A , J Chromatog. 6 , 312 (1961). (5) Hunter, I. R., Dimmick, K. P., Corse, J. W., Chem. and Ind. (London) 1956, 294. ( 6 ) Johnson, D. E., Scott, S. J., Meister, A., Abstracts of Papers, Bm. Chem. Foc., Chicago, Ill., 1961, p. 48C.
( 7 ) Johnson, D. E., Scott, S. J., L‘leister, A,, ANAL.CHEM.33, 669 (1961). (8) Liberti, A., “Gas Chromatography
1958,” D. H. Desty, ed., p. 341, Academic Press, New York, 1958. (9) Lorette, K. B., Brown, J. H., Jr., J . Org. Chem. 24, 261 (1959). (10) Melamed, N., Renard, M., J . Chromatog. 4, 339 (1960)
P., Ibzd., 32, 162 (1960). (16) Zomzely, C., Marco, G., Emery, E., Abstracts of Papers, ilm. Chem. doc., Chicago, Ill., 1981, p. 48C.
(11) Ruhlmann, K., Giesecke, W., Angeus. Chem. 73, 113 (1961). (12) Saroff, H. A., Karmen, A,, Anal.
Biochem. 1, 344 (1960). (13) Weygandj F., Geiger) R.j Ber. 89, 647 (1956).
RECEIVED Februarj- 19, 1962. Accepted July 20, 1962. Presented in part before Division of Biological Chemistry, 140th Meeting, ACS, Chicago, Ill., September 1961
(14) Youngs, c. G., ANAL. CHEU. 31, 1019 (1959). (15) Zlatkis, A,, Oro, J. F., Kimball, ;1.
Operation of the Quantitative and Qualitative Ionization Detector and Its App ication for Gas Chromatographic Studies PETER
F.
VARADl and KITTY ETTRE
The Machlett Laboratories, Inc., Springdale, Conn.
b The combination of a thermionic ionization g a g e and a mass analyzer system in a single quantitative and qualitative (QQ) detector for gas chromatography is described. The principle of its operation and its design are discussed. This QQ detector is used in connection with a packed column, and its performance is demonstrated with experiments on organic and inorganic gases or vapors. Applications of the QQ detector are shown in cases where it was used for retention time marking, for detection of overlapping or hidden peaks, and for positive identification of unknown compounds.
I
DESIRABLE in gas chromatography to identify positively the gases or vapors of a sample emerging from the gas chromatographic column. A new trend is to combine a gas chromatograph, which is capable of only quantitative determinations, with a mass spectrometer unit used for qualitative identifications. The combination of a gas chromatograph with a mass spectrometer seems to be very advantageous. The separation of the components of a sample by the gas chromatograph eliminates the complicated computing work of a straight mass spectrometer analysis. It also makes possible the application of less sophisticated mass spectrometers, because in this combination they have to deal with only a single sample at a time. I n a previous paper (5) the advantages and disadvantages of such combinations were discussed. It was shown t h a t a gas chromatograph and a mass spectrometer, simply switched one after the other, are uneconomical and in many cases d o not perform satisfactorily. We proposed a new type of detector system in which the two units are not
in which the ionization voltage is kept below the ionization potential of the carrier gas but high enough to ionize the gases or vapors of the analyzed sample. The ions produced in the ion source are divided, because of the geometry of thz electrodes of the detector and the applied voltage. A part of the formed ions is collected immediately on one of the electrodes: the ion current measured here is proportional t o the quantity of the compound. This ion current can be monitored continuously on a recorder which prints the familiar quantitative chromatographic pattern. The other part of the ions not trapped here is directed into a mass analyzer electrode system, making possible, in many cases, the immediate detchrmination of the molecular weight and the characteristic ion pattern. and in other cases only a characteristic pattern for the qualitative identification of the compound. This ion current can be monitored simultaneously with the quantitative peak on the second rhannel
simply switched one after the other, or in parallel, but their combination is rather achieved in a single specially designed detector unit. The principle of this proposed new qualitative and quantitative (QQ) detector is based on the direct combination of a n ionization detector and a mass analyzer in a single unit wherein the quantitative measurements and the qualitative identifications are performed from the same sample and at the same place and a t the same time. We describe here a QQ detector which we developed and used in analytical problems : identification of unknown peaks in the chromatogram, retention time calibration (marker) , and analysis of overlapping peaks.
T IS HIGHLY
PRINCIPLE OF OPERATION
The gas stream emerging from the chromatographic column enters the specially designed ionization detector. The electron impact principle is utilized
-
I
SIS
If-
CARRIER GAS SUPPLY
Figure
1.
Schematic drawing of experimental system VOL. 34, NO. 11, OCTOBER 1962
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