ACKNOWLEDGMENT
The authors express their appreciation to E. M. VanderWall for performing the thermogravimetric analysis.
LITERATURE CITED
(1) Duval,
C., “Inorganic Thermogravimetric .4nalysis,” Elsevier, New York, 1953. (2) Hillebrand, W. F., Lundell, G. E. F., Bright, H. A,, Hoffman, J. I., ‘‘Applied
Inorganic Analysis,” Wiley, Sew York, 1953. (3) Kolthoff, I. M., Sandell, E. B., IND. ENG.CHEM.,ANAL.ED.1, 181 (1929). (4) Leddicotte, G. W., Natl. Acad. Sci.Natl. Res. Council NAS-NS 3020, 1960. (5) Lingane, J. L., Davis, D. G., Anal. Chim.Acta 15,201 (1956). (6) Maeck, W. J., Booman, G. L., Kussy, M. E., Rein, J. E., ANAL.CHEM.33, 1775 (1961). ( 7 ) Matusaek, J. M., Jr., Sugihara, T. T., Zbid., 33,35 (1961). ( 8 ) Morrison, G. H., Freiser, H., “Solvent Extraction in Analytical Chemistry,” chap. 13, Wiley, New York, 1957.
(9) . , Smith,. H.,, AXAL. CHEW 34. 191 (1962). (10) Strickland, J. D. H., Spicer, G., Anal. Chim.Acta 3,543 (1949). (11) Yamamura, S. S., Phillips Petroleum Co.. Idaho Falls, Idaho,. urivate communication, 1962.‘
RECEIVEDfor review May 25, 1962. Accepted August 8, 1962. Division of Analytical Chemistry, 142nd Meeting ACS, Atlantic City, N. J., Se tember 1962. Work performed under 8ontract At( 1@1)-205 to the Idaho Operations Office, U. S. Atomic Energy Commission.
Separation of Iron by Reversed-Phase Chromatography JAMES
5. FRITZ
and C.
E.
HEDRICK
lnstifute for Atomic Research and Department of Chemistry, Iowa State University, Ames, Iowa
b Iron(l1l) can b e separated from many elements b y extraction from 6M to 8M hydrochloric acid, using the technique of reversed-phase chromatography. The extraction is carried out b y passing the aqueous hydrochloric acid mobile phase through a column packed with Haloport-F, a dispersion polymer of tetrafluoroethylene, to which a stationary phase of 2-octanone is sorbed. Fluoride, phosphate, or citrate causes no interference. Traces of iron(l1l) can b e separated quantitatively from high concentrations of copper(1l) or zinc(ll), and traces of titanium(1V) can b e separated from large amounts of iron(lI1).
A
liquid-liquid extraction is useful in analytical chemistry, i t has several limitations. For example, many estractive separations are not complete unless several extractions with fresh solvent are employed. This can be a time-consuming operation. Solvent extractions can be carried out by adsorbing a n organic solvent onto a solid support. A column is then packed with this support, and a n aqueous solution containing solutes to be extracted is passed through the column. As the aqueous phase passes through the column, it constantly comes in contact with fresh organic solvent. Column extraction of this type seems to be equivalent to many batch extractions with portions of fresh solvent. Small (9) has developed a scheme of this type for the extraction of uranium and certain other elements from aqueous solutions. He uses a styrene-divinylbeneeene copolymer t h a t is surfacesulfonated to make the polymer somewhat less hydrophobic. When the resin is soaked in a solvent mixture such as perchloroethylene and tributyl phosphate, i t swells and becomes gellike. LTHOUGH
A column packed with this gel d l quantitatively extract uranium from nitric acid solution containing nitrate as a salting-out agent. We found the extraction of uranium to be complete, but were unable to remove the uranium quantitatively from the column with water. A faint yellowish band always remained which contained about 2 to 7y0of the uranium originally extracted. The only way that we could recover the uranium completely was to destroy the resin with a mixture of nitric and perchloric acids. A few other column extractions of this type h a r e been attempted. Siekierski and Fidelis (3, 8 ) have successfully extracted zirconium and rare earths from a strong aqueous solution of nitric acid. They used a column in which Hyflo-Supercel was the solid support, with tributyl phosphate as the organic solvent on the column. Pierce ( 7 ) extracted small amounts of copper onto a column packed with dithizone on silica gel. During the course of our work, a paper b y Hamlin et al. (5, appeared which described the extraction of uranium using a column packed with Kel-F, a fluorinated organic polymer, and coated with tributyl phosphate as the stationary phase. They gxtracted uranium from 5.5-\if aqueous nitric acid, separated i t from a number of other elements, and reported no difficulty in stripping the uranium from the column following the separation. We tried various solid supports for reversed - phase chromatographic systems. Silica and firebrick were unsatisfactory. Glass microbeads, coated with lauryltrimethylammonium chloride to render the surfaces hydrophobic, were receptive to organic solvents, and proved rather successful. The glass microbeads provided a very thin solvent layer and had excellent packing characteristics. The main
limitation, however, was the extremely low solvent capacity of a column of this type. A column of solvent-coated glass beads may be useful for the extraction of trace amounts of metal ions. Of the solid supports tested, Haloport-F was the best. This is a dispersion polymer of tetrafluoroethylene used mainly for gas chromatography packing. The resin is porous and has a high capacity for solvents, b u t does not swell appreciably in contact with solvents. I n the work reported, the extraction of iron(II1) from strong aqueous solutions of hydrochloric acid, using the technique of reversed-phase chromatography, was studied. Any one of several solvents is satisfactory as the stationary phase for this separation. Isoamyl acetate gave good results, but 2-octanone gave better. Iron(II1) is quantitatively extracted from 6 to 8M hydrochloric acid and can be separated from most other metal ions. Following the separation, the iron(II1) can be quantitatively removed fron the column b y stripping the stationary phase from the support with methanol and ethyl ether. EXPERIMENTAL
Reagents. E a s t m a n practical grade 2-octanone was distilled before use. Haloport-F ( F & M Scientific CO., Newcastle, Del.), a dispersion polymer of tetrafluoroethylene, was used witho u t further treatment. Hydrochloric acid solutions (6M and 8 M ) were prepared and equilibrated with 2-octanone b y moderate shaking for one minute in separatory funnels. The equilibrated solutions were stored under 2-octanone. Iron(II1) perchlorate was prepared b y oxidizing reagent grade ferrous ammonium sulfate with ammonium persulfate and extracting the iron(II1) into 2-octanone from 8M hydrochloric VOL. 34, NO. 1 1, OCTOBER 1962
141 1
acid. The iron(II1) was then stripped from the sulfate-free 2-octanone solution into water and brought to fumes with 1 t o 1 perchloric-nitric acid mixture to remove traces of 2-octanone. Metal ion solutions were prepared by dissolving reagent grade, chloride, perchlorate, or sulfate salts in water, and diluting to about 0.05M. Chromotropic acid disodium salt (Eastman practical grade, 230) was used without further purification. Baker reagent-grade 1,lo-phenanthroline was recrystallized three times from an ethanol-water solution before use. Sample Solutions. Solutions of metal ions were prepared b y mixing 5 ml. of 0.05.V iron(II1) perchlorate with 5 ml. of 0.05M solutions of other metal ions and evaporating the 10-ml. volume to about 2 mi. Stock solutions of 0.05N iron(III), titanium(IV), copper(II), and zinc(I1) were standardized, and diluted t o 5 X 10-4M for use in studies at high concentration ratios of metal ions. Apparatus. Standard, 1 X 15 em. coarse-frit chromatographic columns equipped with stopcock control were used. A Beckman D U spectrophotometer was used to determine the absorbance of samples analyzed colorimetrically. PROCEDURE
Column Packing. An amount of 2-octanone-treated Haloport-F sufficient to pack two 10- to 12-em. columns was prepared as follows. A 30-ml. coarse, sintered-glass filter crucible mas filled with t h e resin, and s with a n excess the resin w ~ stirred of 2-octanone. Excess solvent was removed by suction filtration, and the 2-octanone-coated resin was washed with about 15 ml. of 2-octanone-equilibrated 8M hydrochloric acid. The damp support was then slurried with fresh 2-octanone-equilibrated 8 M hydrochloric acid in a beaker and poured into a standard 1 X 15 em. chromatographic column equipped with stopcock control. The material was packed by gently tapping the column until no further settling was observed. A 15-crn.-long resin bed compacts to about 10 to 12 em. of packed support.
Table I. Solvent Capacity of Haloport-F
MI. solvent in 100 grams
Solvent Acetylacetone Benzene Chloroform Diisobutyl ketone Ethyl acetate n-Hexyl alcohol Isoamyl acetate Nitrobenzene 2-Octanone Toluene Tributyl phosphate +Xylene 1412
solvent-coated support 40.1 42.1 34.7 37.9 40.0 45.2 42.2 7.9 42.0 42.5 28.8
42.6
ANALYTICAL CHEMISTRY
Application of too much physical pressure to the 2-octanone-coated resin bed caused solvent “bleeding” and the accumulation of air pockets. Separation. Synthetic sample mixtures which had been previously boiled down to 2 ml. were treated with 3 t o 5 ml. of concentrated hydrochloric and washed onto t h e columns with 2-octanone-equilibrated 8 M hydrochloric acid. A flow of one drop of eluate every 1 to 1.5 seconds (2 t o 3 ml. per minute) was employed. After the last of t h e sample was on t h e column, t h e nonextracted metal ion was eluted completely b y dropwise addition of 10 to 15 ml. of 2-octanoneequilibrated 6 M hydrochloric acid. Iron(II1) was removed from the column by stripping the 2-octanone stationary phase with 20 ml. of methanol, followed by 20 ml. of ethyl ether, and then fuming the eluate with 10 ml. of perchloric acid and nitric acid (1 to 1). Eluates that contained titanium(1V) and molybdenum(V1) were fumed with 5 ml. of 1 to 1 perchloric-sulfuric acid mixture. Analysis. Iron(II1) and most other metal ions were determined by backtitrating excess E D T A with zinc(I1) at p H 6.5, using Naphthyl Azoxine S screened with Fast Green S F Yellowish (4). The color of the screened indicator changed from rust-brown to yellowgreen on titration of iron(II1) [250 pmoles of Fe(II1)-EDTA complex in 200 ml. of solution]. Samples containing 250 pmoles of uranium(VI), were diluted to 150 ml., buffered to p H 5 with pyridine, treated with 1 gram of ascorbic acid and a measured excess of EDTA, and boiled to a clear green. The solution was cooled, and back-titrated with lead perchlorate solution at p H 5, using Xylenol Orange as the indicator (2). Kitrates should be absent. Zirconium(1V) standards were fumed with perchloric acid in the same manner as the zirconium(1V) eluates to ensure complete breakdown of hydrated complexes. The titrations were carried out at p H 1 to 2, using Xylenol Orange as the indicator, a standard solution of bismuth(II1) nitrate as the back-titrant] and pyridine as the buffer. Chromium(II1) was determined by oxidizing it to chromium(V1) with ammonium persulfate, using silver as the catalyst. The chromium(V1) was then titrated with ferrous ammonium sulfate, using ferrous 1,lo-phenanthroline as the indicator. Molybdenum(V1) was determined by a precipitation titration of lead molybdate a t p H 4, using an 0.05M solution of lead perchlorate as the titrant and Xylenol Orange as the indicator. Samples of eluate and standards containing about 2 pmoles of titanium(1V) were analyzed spectrophotometrically by a slightly modified chromotropic acid procedure ( 1 ) . Titanium(1V) standards were prepared by diluting 1 t o 10 ml. of 5.92 X lOU4MTi(1V) sulfate to 50 ml. with 50% sulfuric acid after the addition of 5 ml. of aqueous 7% chromotropic acid, disodium salt. Enough concentrated sulfuric acid was
added to make the final solution 50% in H2S04. The absorbance was read at 520 mp. RESULTS AND DISCUSSION
Of several solvent supports studied, the polyfluorocarbon, Haloport-F, was selected because of its high solvent capacity, chemical inertness, resistance to swelling, and ease of regeneration by washing with methanol and ethyl ether. Table I shows that Haloport-F has good capacity for most solvents. Solvents with a low viscosity tend to be removed easily from the support by physical agitation. h solvent of medium viscosity seems to be the best. If the solvent used is too soluble in water, i t will be removed by passing the aqueous sample through the column. Presaturation of the aqueous solution to be used as the mobile phase with the solvent to be used as the stationary phase is helpful, and the solvent used should be somewhat polar. Haloport-F coated with a solvent such as o-xylene is so hydrophobic t h a t the resin particles cling together, and column extraction from an aqueous solution is impossible. I n practice] use of extremely hydrophobic solvents as stationary phases on Haloport-F caused clogging, channeling, and the formation of air pockets in the resin column, The slurries were not readily wet by aqueous solutions and tended to clump up in the column rather than to settle into the form of a homogeneous resin bed. Kuznetsov (6) found ketonic solvents to be the best for extraction of iron(II1) from hydrochloric acid. Methyl isobutyl ketone appears to be too soluble in water and of too low Yiscosity to be suitable as the stationary phase. We selected 2-octanone for the extraction of iron(II1) from hydrochloric acid solution. It is slightly viscous and forms a coherent, easily wetted column packing that does not lose solvent when washed with hydrochloric acid solutions under column flow conditions. Iron(II1) is extracted very rapidly to form a well-defined, sharp, yellow band v i t h no visible tailing. The band measures 1 em. in length, when 5 ml. of 0.05iZ1 feriic chloride [250 pmoles of F e ( I I I ) ] is washed onto the column with 8M 10 ml. of 2-octanone-equilibrated hydrochloric acid. The band lengthens to 1.3 to 1.5 em. when the column is subsequently washed with 10 ml. of 2-octanone-equilibrated 6.11 hydrochloric acid. The separation of iron(II1) from other metal ions by reversed-phase chromatography was tested by analyzing 1 to I mole-ratio mixtures of iron(II1) and other metal ions. The mixtures contained 5 ml. of 0.05M iron(II1) solution (250 #moles) and 5 ml. of a 0.05.21 solution of another metal ion (also 250
pmoles). These solutions were evaporated t o a small volume, treated with concentrated hydrochloric acid, and extracted by the column. Table I1 shons the results of these separations. Very fctv metal ions interfered in the separation of iron(III), although metal ions known to be extracted from hydrochloric acid were in general not included in this study. Tin(1V) interfered by partial extraction. No means were found for complete removal of tin(1V) from the column without removing the iron(II1). Vanadium(V) and molybdenum(V1) were partially extracted onto the column. Interference from these metal ions was avoided b y adding one or two drops of 30% hydrogen peroxide to the sample before i t was introduced onto the column to form the complex metal peroxide, which was then eluted by the hydrochloric acid. The hydrogen peroxide caused no difficulty in the extraction of the iron(II1). The extraction of iron(II1) b y the column was very complete. I n a typical run, analysis of the eluate after extracting 250 wmoles of iron(II1) on the column gave 0.161 pmole of iron(II1) in the eluate. When a typical column was washed with 10 ml. of 2-octanoneequilibrated 8111 hydrochloric acid, and no iron(II1) had been loaded onto the column, a blank value of 0.082 pmole of iron(II1) was found in the eluate. I n general, the losses of iron(II1) were SO small and so close to the reagent blank itself that small amounts of iron(II1) could be extracted successfully. Table 111 shows the results of a number of extractions of small quantities of iron (111) in the presence of large amounts of copper(I1) or zinc(I1). It also shows that quantitative recovery of small amounts of a metal ion, titanium(IV), is possible in the presence of large Copper(I1) amounts of iron(II1). and zinc(I1) were chosen because when present in large amounts they interfere in the normel determination of traces of iron(II1) by the colorimetric 1,lO-phenanthroline method. I n studies summarized in Table 111, the results are reported as an average of four determinations. The determinations were carried out b y adding 2.830 pmoles of iron(II1) to a strong solution of a copper(I1) or zinc(I1) salt so that the mole ratio of iron(II1) t o the other metal ion was approximately 1 to 1000. The solutions were then made 6 to 851 in hydrochloric acid and extracted on the column in the usual manner. Special care was taken to wash all the copper(I1) or zinc(I1) from the main yellow band of iron(III), which was clearly visible at the top of the column. Following the separation, the entire stationary phase was stripped from the support with methanol and ethyl ether, and the eluate was fumed down with a 1 to 1 mixture of
Table
II.
Separation of Iron(ll1) trom Other Metal Ions
Av . Ion Ion Fe+3, pmoles dev., added, found, Found pmole pmoles pmoles Added ~ 1 ~ 3 265 5* 265 5 10.0 203 5 203 5 Ba + 2 282 5b 283 0 1 0 5 289 5 290 0 Bi + 3 266 0 266 0 f.0 0 266 5 266 5 Cd + 2 265 5 265 2 -0 3 214 5 214 2 265 5 265 5 f.0 0 199 0 199 0 Ce + 3 Cr + 3 283 Oc 283 8 -0 2 291 5 290 8 c o +z 268 5 268 5 zko 0 291 5 291 8 c u +2 268 5 268 8 200 5 209.0 +0.3 285 5 285,5 Er +3 285 Ob 284 3 -0.7 332.0 rn + 3 266 0 +0.5 332 5 266 5 261.5 Pb +* 283 Oc 282 3 -0.7 260 5 239.0 Mg +2 266 0 265 7 -0.3 238 5 Mn + 2 265 5 265 2 223.3 223 0 -0.3 252. 7 284 Od 283 2 -0.8 252.0 -Mo is(HzOz) Ni + 2 262 0 268 5 268 8 +0.3 262.3 Rm +3 285 0 285 0 1 0 0 281 5 281 8 .~ :Sr +2 265. 5 b 365.5 io.0 277.2 2%. 2 Th + 4 266.0 265.5 -0.5 233.5 234.3 Ti+4 285. Oc 284 5 -0.5 285.5 285.7 283.8 -0.2 U-w 284. Od 213.0 212.6 236 0 270 5 270 0 10 0 V+6b+4(a2o2, 236 0 YL3 267 0 267 3 +0 3 292 7 298 0 i n +2 268 5 268 5 10 0 249 0 249 5 Zr - 4 267 W 267 2 +o 2 250 0 249 8 All data based on average of 2 determinations) except where noted. b Single determination. c rlverage of 3 determinations. d Average of 4 determinations.
Av. dev ., pmole
Other metal ion
~
~~
1 00
+o +o -0 +o -0 +o
5
0
3
0
7
3
-0 1 0 -0 +l
5 0 5 0 +o 5 +0.3 $0 7 +o 3
+o :i ii.0
~
+0.8
+0.2 -0.4 -0 5 +0 3
-to
5
-0 2
0
Table 111.
Separation of Copper(ll), Titanium(lV), and Zinc(l1) from Iron(lll) a t High Concentration Ratios
Fe+s-ion Av. trace ion, *moles Ion identity ratio Added Found c u +2 1:200 2.830" 2.833 c u +z 1 :1000 2,830" 2.813 Zn +2 1:200 2 . 830a 2.825 Zn +2 1: 1000 2. 830a 2.775 Ti +4 1000:1 2 . 960b 3.020 Trace ion determined was iron(II1). b Trace ion determined was titanium( IV).
N ~ of.
detns. 3 4 2 4 4
Av . dev., pmole +O. 003
-0.017 -0.005 -0.055 + O . 060
@
nitric and perchloric acids. The iron (111) was measured by high precision spectrophotometry using I, 10-phenanthroline. As seen from Table 111, iron(II1) was retained quantitatively by the column at molar ratios of 1 to 200 and was adequately retained a t ratios of 1 to 1000. The reversed-phase chromatography of iron(II1) is also useful in the removal of iron(II1) prior to the determination of metals present in the iron in trace amounts. This is also illustrated in Table 111, which shows the separation of titanium(1V) from large amounts of iron(II1).
A sample mixture containing 2.5 mmoles of iron(II1) and 2.96 wmoles of titanium(1V) was prepared. The mixture was passed through a 1 X 10 cm. column t o extract the iron(III), and the titanium(1V) mas washed through the column with about 10 ml. of 2-octanoneequilibrated 6 M hydrochloric acid. The effluent from the column was fumed down with sulfuric acid and analyzed by a spectrophotometric chromotropic acid procedure ( I ) .
Table IV. Effect of Anions on Extraction of Iron(lll)"
Iron(II1) added to column, 250 pmoles. Anion washed through column, 3000 pmoles in 15 ml. of 2-octanone-equilbrated 6M HCl. Total Fe+3 Net Fe+3 found found, in corrected eluate, for blanks, Acid added pmoles pmoles None (6M HCl) 0.109 (HC1 blank) HF, no Fe+3 on column 0.211 ( H F blank) Hydrofluoric acid 0.215 0 . 004b Citric acid 0.111 0.002 Phosphoric acid 0.152 0.043 Averages of 4 determinations. b HF blank applied in this case alone.
The recovery of titanium(1V) was slightly high. The results of Table 111 thus show that traces of both iron(II1) and other metal ions (not extracted from hydrochloric acid) can be determined quantitatively even in the presence of VOL 34, NO. 1 1 , OCTOBER 1962
1413
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). \----I
(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).
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 b y 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 b y 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
