yi (mix .)
L L1
R
T TIL
V L
VL,, V L r
VRi’
equivalent activity coefficient of t’he i t h solute on a coupled or a heterogeneous column. = total length of the column. = lengt’h of column 1 containing liquid 1 measured from the column inlet. = moles of the i t h solu1;e. = number of moles of partitioning liquids 1 and 2, respec tively . = number of moles of the i t h solute disscdved in solvent 1 and the number of moles dissolved in solvent 2. = vap’w pressure of pure i t h solute at the temperature of the column. = universal gas constant. = column temperature ( ” K.). = volume of the immobile liquid in the column. = mo1.u volume of the liquid partitioner. = volume of the immobile liquids of kinds 1 and 2 in the entire column or in the column segrxents, = corrected retention =
X, XI,
x*
volume of the i t h solute. = mole fraction of solUte i. = mole fraction of liquids 1 and 2 respectively. LITERATURE CITED
(1) Araki, T., Goto, R., Ono, A., Nippon Kagaku Zasshi 81, 1318 (1960).
(2) Ascoli, F., Pispisa, B., Servello, F., J. Chromatog. 6, 544 (1961). (3) TBarnard, J. A., Hughes, H. W. D., h a t u r e 183, 250 (1959). (4) Dal Nogare, S., Juvet, R. S., Jr., “Gas Liquid Chromatography,” Interscience, New York, 1962. (5) Fredericks, E. M., Brooks, F.R., ANAL. CHEM.28, 297 (1956). (6) Gatrell, R. L., Ibzd., 35, 923 (1963). (7) Giddings, J. C., Zbid., p. 353. ( 8 ) Haahti, E. 0 . A., Vanden Heuvel, W. J. A., Homing, E. C., Anal. Bzochem. 2, 344 (1961). (9) Hildebrand, G. P., Reilley, C. N., ANAL.CHEM.36, 47 (1964). (10) Hildebrand, J. H., Scott, R. L., “The Solubility of Nonelectrolytes,” 3rd ed., Reinhold, New York, 1950. (11) Juvet, R. S., Jr., Chiu, J., Simborg, D. W., Dept. of Chemistry and Chemical Engineering, University of Illinois, Urbana, Ill., 1962. (Simborg, D. W., B. S. Thesis, University of Illinois.) (12) Kaiser, R., “Gas Chromatographie: Theorie, Apparaturen, Anwendung,” Akademische T’erlagsgesellschaft, GeestPortig K.G., Leipzig, 1960. (13) Keller, R. A., Stewart, G. H., ANAL. CHEM.34, 1834 (1962). (14) Keller, R. A., Stewart, G. H., J . Chromatog. 9, 1 (1962). (15) Kirkwood, J. H., Oppenheim, I., “Chemical Thermodynamics,” McGraw-Hill, Xew York,-1961. (16) Lewis, G. K.,Randall, M., revised by Pitzer, K. S.,Brewer, L., “Thermo-
dynamics,” 2nd ed., McGraw-Hill, New York, 1961. (17) McFadden, MT.H., ANAL.CHEM.30, 479 (1958). (18) McNair, H. M., Ph.D. Thesis, Purdue University, Lafayette, Ind., 19Fj9. (19) Martin, R. L., ANAL.CHEM.33, 347 (1961). (20) Matauda, T., Yatsugi, H., Bunseki Kagaku 11, 1116 (1962). (21) Pauling, L., “The Xature of the Chemical Bond,” 3rd ed., Cornell Univ. Press, Ithaca, N. Y., 1960. (22) Pierotti, G. J., Deal, C. H., Derr, E. L., Porter, P. E., J . Am. Chem. Soc. 78, 2989 (1956). (23) Porter, R. S., Hinkens, E. L., Tornheim, L., Johnson, J. F., 144th Meeting, ACS,.Los. Angeles, Calif., March, 1963. (24) Prigogine, I., with Bellemans A,, Mathot, V., “The Molecular Theory of Solutions,” Interscience, Sew York, 1957. (25) Prigogine, I., Defay, R. (trnsl. by Everett, D. H.), “Chemical Thermodynamics,]’ Longmans Green, London, 1954. (26) Primavesi, G. R., Nature 184, 2010 (1959). (27) Reilley, C. K.,Hildebrand, G. P., Papa, L. J., Sotreman, W. E., Jr., 144th Meeting, ACS, Los Angeles, Calif., March 1963. (28) Rohrschneider, L., Z . Anal. Chem. 170, 256 (1959). (29) Singliar, M., Bobak, A., Rrids, J., Lukacovic, L., Ibid., 177, 101 (19dC)). (30). Waksmundski, A., Suprynowicz, Z . , Pietrusinska, T., Chemie Analityczna 7, 1043 (1962). (31) Zhukhovitskii, -4. A,, Se!enkina, M. S., Turkel’taub, S . M., Russian J . Phys. Chem. English Transl. 36, 519 (1962). RECEIVED for review November 20, 1963. Accepted April 1, 1964. Work supported in part by the Petroleum Research Fund, 1305-D7 (An International Award).
Gas Chromatographic Analysis of Amino Acids as N -T rif III o roa cetyla mino Acid Methyl Esters PHILIP A. CRUlCKSHANKl and JOHN C. SHEEHAN Research Institute for Medicine and Chemistry, Cambridge 42, Mass.
b
Trifluoroacetylated amino acid methyl esters have been analyzed by gas liquid chromatography. Procedures for rapid esterification and acylation of individual or niixtures of amino acids have been developed for 21 naturally occurring amino acids. The derivatives were separated on a 2-foot stainless steel column packed with neopentyl glycol succinate coated on Gas Chrom P. Preparaticin of derivatives and chromatography lhas required approximately 2 hours. Determination of amino acid ratios in Ipeptides as well as qualitative determination of amino acids in proteins has been demonstrated.
T
o FACILITATE the qualitative and semi-quantitative analysis of amino acids from peptide hydrolysates, a study
of volatile amino acid derivatives for gas chromatography has been undertaken. Reactions for high yield conversion of 21 amino acids to N-trifluoroacetylamino acid methyl esters and chromatographic conditions for separating these derivatives have been developed. The procedure is characterized by a rapid conversion of all common amino acids to volatile derivatives and a superior separation of all derivatives on a single chromatographic column using a temperature gradient. Use of dimethyl sulfite as the esterifying reagent has afforded amino acid methyl ester hydrochlorides in nearly quantitative yield with a reaction time as short as 30 minutes. Acylation has been effected in 10 minutes by the direct reaction of the amino acid ester hydrochlorides with
trifluoroacetic anhydride. With a chromatography time of 7 5 minutes, a complete amino acid analysis can be carried out in approximately 2 hours. Single derivatives have been obtained for all amino acids except arginine (two minor contaminants in the major component), including the sensitive amino acids tryptophan and cystine. Column and operating parameters have been selected that afford a resolution of all derivatives satisfactory for determining peak areas, thus allowing calculation of amino acid ratios in samples. Recent spectacular advances in the chemistry of amino acids, proteins, and
Present address, Chemical Research and Development Center, FMC Corp., Princeton, N.J. VOL. 36, NO. 7, JUNE 1964
1191
peptides can be traced to the development of chromatographic techniques for amino acid analysis during the last 18 years. Procedures based on two dimensional paper chromatography and/or electrophoresis ( 3 ) and on ion exchange columns (8, 11) have afforded complete amino acid analyses in 24 hours. With the introduction of gas chromatography as a n analytical tool, the extremely rapid analysis of minute quantities of volatile substances became possible; application of this technique to amino acid analysis would be valuable in many instances. Although most amino acids do have a small but measurable vapor pressure ( 4 ) ,the volatility is considered too low for direct gas chromatographic assay. A suitably volatile derivative which can be prepared easily from all commonly encountered amino acids was needed. A number of investigations have been reported during the past 6 years concerning the search for a satisfactory derivative. The greatest buccess has been attained with N-acylamino acid esters. Johnson, Scott, and hieister (6) have prepared N-acetylamino acid n-amyl esters of 33 amino acids, and have successfully separated these derivatives; no derivatives have been obtained for cystine and tryptophan, however. Zomzely, Marco, and Emery (16) utilized N-trifluoroacetylamino acid n-butyl esters in their study. Derivatives for 22 amino acids commonly encountered in peptide or protein hydrolysates were separated, but with poor resolution between aspartic acid and phenylalanine. The time required to prepare the S-trifluoroacetylamino acid n-butyl esters was somewhat long (about 4 hours). Several groups have investigated trifluoroacetylated amino acid methyl esters (2, 9, 13, 14) with only moderate success. For a more complete summary of studies concerning application of gas chromatography techniques to amino acid analysis, the reader is referred to the paper by Zomzely et al. (15).
DEGREES CENTfGRADE
BO
1 192
ANALYTICAL CHEMISTRY
A
I I5 I
I35 I
I70
I
I
95 I
Yydroxyproline ryrorine
-
Swine
60 -
!420
loot
B
9
tlu!
mine
Tyrosinr
Q
ao -
60
flydror
-
rolfne
40-
I
20 -
-
EXPERIMENTAL
Reagents. All amino acids were of the highest purity available from the California Corp. for Biochemical Research (Los hngeles, Calif.), and were of the natural configuration. T h e proteins were obtained from Worthington Biochemical Corp. (Freehold, S . J.). Dimethyl sulfite was prepared in our laboratory (6). Trifluoroacetic acid and trifluoroacetic anhydride were obtained from Matheson, Coleman and Bell Division. Supports and stationary phases for gas chromatography were obtained from Applied Science Laboratories (College Park, Pa.) or from Analytical Engineering Laboratories (Hamden, Conn.). Procedure. The quantities of reagents indicated below were used for
215
1001
I
70
i
I
60
I
I
50
i
i
40
I
I 30
I
-
I
20
I
,wNu rEs Figure 1. A.
E.
Separation of derivatives of hydroxy amino acids
Derivatives prepared by acylation of free amino acids followed by esterification with diazomethane Derivatives prepared by esterification with dimethyl sulfite-methanolic hydrogen chloride followed by acylation
2-foot column, 1.5-mm. Ld., packed with Gas Chram P coated with 5% neopentyl glycol succinate. Argon Row rate 18 ml./min. Starting temp. 6 5 ' C. with 1.5" C./min. temperature program initiated immediately; after 20-min. program rate increased to 2 ' C./min., and after 42.5 min. fo 4' C./min. Sample Size: 2 pl. of an acetonitrile solution 0.01M in each amino acid derivative. Detector response: 1 X 10-7 ampere
samples containing 100 pmoles or less of amino acids. For 1.arger amounts of amino acids, the reagent volumes were increased proportionately. Reduction of Cystine. When conversion of cystine to cysteine was desired, the mixture of amino acid hydrochlorides was dissolved in 4.0 ml. of water and neutralized with a few drops of saturated aqueous sodium bicarbonate. Ethanethiol (0.1 ml.) was added and the mixture was heated under refius for 5 minutes. A4fter acidification with hydrochloric: acid the excess reagent and the soh-ent were evaporated with a jet of nitrogen. Sodium chloride present in the residue was removed by filtration after dissolving the amino acid hydrochlorides in methanolic hydrogen chloride. Est,erification of Amino Acids. The amino acid sample was suspended in 5.0 ml. of methanol which was then saturated with anhydrous hydrogen chloride (the mixture was cooled in an ice bath during this procedure). Dimethyl sulfite (1.0 m1.11 was then added and the solution was heated under reflux on a steam bath (the apparatus was protected from moisture by a calcium sulfate drying tube). After 30 minutes the methanol and excess dimethylsulfite were removed under reduced pressure and the residue of amino acid methyl ester hydrochlorides was dried under high v:tcuum. Trifluoroacetylation of Amino Acid Methyl Ester Hydrcichlorides. Trifluoroacetic anhydride (1.0 ml.) was added to the flask containing the amino acid methyl ester hyclrochlorides, and the resulting solution was heated under reflux on a steam bath for 10 minutes (apparatus protected from moisture with a calcium sulfate drying tube). The excess trifluoroacetic anhydride and the trifluoroacetic acid were evaporated with a stream of dry nitrogen, and the residue was redissolved in trifluoroacetic anhydride for chromatography (0.1 ml. for a sample containing 1 to 6 pmole of each amino acid). Trifluoroacetylation of Amino Acids. The dried sample of amino acids and/or amino acid hydrochlorides was dissolved in 1.0 ml. of anhydrous trifluoroacetic acid. Trifluoroacetic anhydride (1.0 ml.) was added and the solution was heated under reflux for 5 minutes; the solvent and excess reagent were then evaporated with a jet of dry nitrogen. Esterification of Trifluoroacebylamino Acids. The residue of trifluoroacetylamino acids was dissolved in 2.0 ml. of dry methanol and :i solution of diazomethane in ether was added in excess. The excess dimomethane was immediately destroyeci by a drop of glacial acetic acid, and the solvents were evaporated with nitrogen. The residue was then diss,olved in acetonitrile for chromatography (0.1 ml. for a sample containing 1 t o 6 winole of each amino acid). Apparatus. A Jarrell-iish Model 700 Universal Chromatograph equipped with a n automatic teniperature program control1t:r and a n argon diode ionization detector (H3 or Srw 8ource for ionizing radiation) was used.
The column was a 2-foot '/*-inch 0.d. (approximately 1.5-mm. i.d.) stainless steel tube containing as packing 80- to 100-mesh Gas Chrom P coated with 5oj, w./w. neopentyl glycol succinate. The support was coated by suspending in a chloroform solution of the polyester and evaporating the solvent under reduced pressure on a rotary concentrator. This coated support was preconditioned by heating to 100' C. at a reduced pressure of 0.1 mm. or less. The packed column was conditioned further a t 225' C. with a n argon flow of 18 ml. per minute. Chromatography. T h e column oven of the gas chromatograph was allowed to equilibrate a t 65' C. -4 2-pliter aliquot of a solution of the amino acid derivatives (1 to 10 pmole of each amino acid per ml.) was injected into the flash heater held a t 295" C. An argon flow rate of 18 ml./minute was maintained. Upon injection of the sample a programmed temperature increase of 1.5' C./minute was initiated; after 20 minutes, the rate was increased to 2" C./ minute, and after 42.5 minutes to 4' C./ minute .to a final temperature of 210' C. A total of 75 minutes was required for the complete analysis. Thin Layer Chromatography. Glass plates for thin layer chromatography were coated with Anasil €3 (Analytical loo
r------
90 -
Engineering Laboratories). The plates were developed with ethanolmater (60:40), phenol-water (75:25), chloroform-methanol (90: lo), or chloroform-acetone (90: 10). Staining was effected with ninhydrin ( 7 ) or with tbutyl hypochlorite followed by starchiodide reagent (10). DISCUSSION
Preparation of Derivatives. Trifluoroacetylamino acid methyl esters were chosen for this study since i t was felt t h a t these derivatives would have the greatest volatility and would best retain the inherent differences of t h e precursor amino acids. Two basically different approaches to preparing t h e derivatives were investigated: the amino acid sample (either free or as hydrochlorides) was first acylated followed by esterification with diazomethane; and the amino acid hydrochlorides were esterified prior to the acylation step. Direct acylation of the amino acid sample was effected by reaction with trifluoroacetic anhydride in trifluoroacetic acid solution for 5 minutes on a steam bath. After removal of escess reagent and solvent, the residue of
B
L 4
C
80 -
70-
3
60-
%
2
50-
3
)z
'
cl 40-
k!
30-
20-
' O t L__
L - J - - L - u 30 25 20
MJNUTES Figure 2.
Separation of glycine, threonine, and leucine derivatives
All columns 1.5-mm. 1.d. and packed with 5% neopentyl glycol succinate on 80- to 1 00-mesh Gas Chrom P 2-foot column; initial temp. 65' C. with temp. increase of 1.5' C./min.; argon flow rate 1 8 ml./min. 8. Same condition as A except argon flow rate decreased to 7 ml./min. C. 3-foot column; initial temp. 70' C. with temp. increase of 1.5' C./min.; argon flow rate 1 8 ml./min. Other conditions same as given in Figure 1
A.
VOL. 36, NO. 7, JUNE 1964
1193
Table I. Thin Layer Chromatography Results
Es erifica- TLC tion time,a solmin. ventb >15 1 >15 2
Rf Acylation TLC R f Tfa amino Rf time,O solderivaacid ester min. ventb tive >5 3 0 92 0 31 0.47 Alanine >5 4 0 16,O 4gC Arginine 0.17 0.35 AsDartic arid >15 1 0 17 0.69 >5 3 0 90 Cysteine 15-30 1 0.18 0.47 >5 3 0.73 Cystine 1 0.08 0.38 >5 3 0.86 >15 15-30 Glutamic acid 1 0.34 0.59 >5 3 0.81 Glycine 1 0.18 0.38 >5 3 0.77 >15 Histidine 15-30 >5 4 0 52 2 0.24 0.65 Hydroxyproline 1 0.43 0.59 ... . . .d >15 Isoleucine 1 0.62 0.73 >5 3 ’ 0.95 30-60 Leucine 15-30 1 0.62 0.71 > 5 3 0.95 Lysine 2 0.04 0.31 10 4 0.94 30-60 Methionine 15-30 1 0.63 0.81 > 5 3 0.95 Ornithine 2 0.07 0.30 10 4 0.90 15-30 Phenylalanine 1 0.66 0.82 >5 3 0.95 >15 Proline 1 0.38 ... ... .. . . .d >15 Serine 15-30 1 0.31 0.72 >5 3 0.39 Threonine 3Ck60 1 0.27 0.69 > 5 3 0 56 Tryptophan 1 0 59 > 5 3 0 81 15-30 0 81 Tyrosine 15-30 1 0 66 0 78 >5 3 0 59 Valine 30-60 1 0 54 0 69 >5 3 0 95 a Reaction time required for disaopearance of starting material. Solvent System 1. Ethanol-water AO:40 3. Chloroform-acetone 90:10 2. Phenol-water 7.5:25 4. Chloroform-methanol 90:10 c Two components observed on TLC plate; both probably are decomposition products of original derivative (see text). Not detected by hypochlorite/starch iodide stain.
trifluoroacetylamino acids was dissolved in methanol and esterified with diazomethane (Equation 1). Although this procedure HzN-CHR-COzH
(CFaC0)rO CFsCOzH
agent (12) has afforded rapid and nearly quantitative conversion of amino acid hydrochlorides to the corresponding methyl esters. This esterification reCFICO-XH-CHR-CO~H
(1) CF~CO-NH-CHR-CO2H
E A +
has the advantage of extreme rapidity for derivative preparation (about 15 minutes), it is not a suitable general method since no volatile derivatives were obtained from arginine and histidine. The second approach investigated involved preparation of the amino acid methyl ester hydrochlorides followed by trifluoroacetylation (Equation 2). Introduction of dimethyl sulfite as a methylating
Table 111.
Columna length, ft,. 2 2
CFaCO--NH-CHR-COICH3 action has been followed for 21 aminn acids by means of thin layer chromatography (Table I). The majority of the amino acids were completely esterified after heating under reflux for 30 minutes with dimethyl sulfite in methanolic hydrogen chloride; in only four instances were traces of unesterified material present (valine, isoleucine, threonine, and lysine). Acylation of the amino acid ester hydrochlorides with trifluoroacetic anhydride also was
Separation Factors for Derivatives of Leucine, Threonine, and Glycine T- h... r
retention time, Separation factors ml./min. min. Leu-Gly Leu-Thr Thr-Gly 1.120 1.060 1.059 18.0 21.3 1.039 1.046 26.3b 1.091 7.0 6*5 1.080 1,042 1.035 18.0 21 .o 2 55 1.098 1,068 1.029 18.0 21.6 3 70 1.A 1.062 1.062 LOO0 18.0 23.5 4 95 1.6 All columns I .5-mm. i.d.; parked with 570 w./w. neopentyl glycol succinate on 80- to 100-mesh Gas Chrom P. Broad peak.
1 194
Initial temp., C. 65
Temp. program, C./min. 1.5 1.5 2
ANALYTICAL CHEMISTRY
Argon flow,
examined by thin layer chromatography (Table I). All compounds escept lysine and ornithine methyl ester hydrochlorides were completely acylated after 5 minutes of heating under reflux; these compounds were completely converted after 10 minutes.
Table II. Infrared Study of Hydroxy Amino Acid Derivatives
Infrared absorption frequences Products Products from from reaction 3a reaction 3b 3700 cm.-I 3450 cm.-1 1755 Cm.? 1740 crn.-’
3450 cm.-l 1795 ern:-' 1755 cm.? 1740 cm.?
Assignment Hydroxy Amide N-H CF3CO-0-C02CH3 CFaCO--IL”
The reaction of arginine methyl ester dihydrochloride with trifluoroacetic anhydride afforded as the main product a derivative with a retention time of 60 minutes under the conditions described in this paper. This material was accompanied by small amounts of derivatives having retention times of 47.4 and 56.0 minutes; the latter was probably bis(trifluoroacety1)ornithine methyl ester, as the retention time was identical to that obtained with the authentic ornithine derivative. There was no interference in the determination of ornithine since the amount of the derivative from arginine was proportional to the main arginine peak, and thus could be crllculated and deducted from the total ornithine. If a solvent other than trifluoroacetic anhydride was used to inject the amino acid derivatives, the 60-minute retention time derivative of arginine disappeared, to be replaced by a substance with a retention time of 52 minutes; thin layer chromatography indicates that a t least two major substances are formed, but only one has been observed by gas chromatography. With the hydroxy amino acids, distinctly different gas chromatographic results were obtained by each of the derivatization schemes. When the amino acids were first acylated, then esterified with diazomethane, much greater retention times were observed for the derivatives than when the amino acid ester hydrochlorides were acylated. These results are illustrated in Figure 1. Products prepared from pure amino acids by each reaction scheme were esaminpd by infrared spectrometry (1). This study established that the products with greater retention times contained free hydroxyl groups, whereas the products with lower retention times were the N.0-bistrifluoroacetyl derivatives (Table I1 and Equation 3).
R-CH-OH
R-CH-OH
I
(CHsC0)zO
H,S-CH-C02EI
\
I
lengthening of this column resulted in poorer separation of 4,O-bis-trifluoroacetyl threonine methyl ester and N-trifluoroacetyl glycine methyl ester (Table I11 and Figure 2c). Initial column temperature, temperature program rate, and argon flow rate also had to be carefully adjusted to effect best resolution of this same pair of derivatives (Table 111 and Figures 2a and 26); values selected for these parameters were respectively, 65" C., 1.5" C./ minute, and 18 ml./minute. From the separation factors given in Table 111, it is apparent that the resolution of the leucine and threonine derivatives was least effected by a change in column length, while the glycine derivative gradually fused with the threonine derivative with increasing column length. Use of longer columns required higher initial temperatures with resultant higher elution tctmperatures for the substance; changes in other parameters which afforded higher elution temperatures also resulted in lower separation factors for the glycine and threonine derivatives. Apparently,
1
CHzNz CHaOH-
CF3CO--h"-CH--CO2CH3
I
(CH30)gSO
(3a)
(CF&O),O
HC1,'CHsOH
OH
R
\
/'
CH
'R
0
/
II
CH-0-C-CFI
I Cl-HJ
+-CH-CO&H,
(CFsCO)e+ C F & O - - N H - - ~ H - C 0 2 C H ~
,\lthough the cystine derivative could bo observed directly by gas chromatography. an alternate procedure for its detrrrnination as cysteine was also developed. Treating cystine, alone or mixed with other amino acids, with ethanethiol in dilute aqueous sodium bicarbonate solution results in complete reduction to cysteine. The amino acids were recovered b,r acidification, evaporation of solvent, and extraction of the residue with metlianolic hydrogen chloride.
(3b)
Column and Operating Parameters.
Of t h e many different solid supports and stationary phases tried for the separation of trifluoroacetylated amino acid methyl esters, Gas Chrom P, 80- to 100-mesh (Applied Science Laboratories, College Park, Pa.), coated with 5% w./w. neopentyl glycol succinate proved most satisfactory. Column length proved to be extremely critical. Contrary to espectation, optimum resolution of all components was achieved with a 2-foot column; any DEGREES CENTIGRADE
210
I70
I35
115
95
80
I
I
I
I
,
i isothermal I
P
80
I
e
3 70
1
I
I
1
1
I
inject ion I.5V m in
2.0°/min
4.0°/min
65
B
J L U
70
I
I
60
I
I
50
I
I
I
40
I
30
I
I
I
20
I IO
I
I
0
MINUrEs Figiure 3.
Separation of a mixture of 20 trifluoroacetylated amino acid methyl esters
Conditions os described in Figure 1 .
Concentration of each amino acid adjusted to keep oll peaks on scale without attenuation
VOL. 3 6 , NO. 7,JUNE 1964
1195
I70
I
I
isothermal
100 -
DEGREES CEMf GRADE 135 115 I
I
4.00/min
95
80
65
I
I
I
2.0Ymin
iniectior I .5*/min
I
I
1
-
90
210
80 -
8
P
7060-
e
2Q
50-
u
5
40-
Q
30
-
20 IO
-
w v u rEs Figure 4.
Determination of amino acids in a ribonuclease hydrolysate as trifluoracetylamino acid methyl esters
Conditions as described in Figure 1 . 1 X IO-' ampere
0.2 MI. of a solution containing amino acid derivatives obtained from 1 .O mg. of ribonuclease.
Table IV. Retention Times for Trifluoroacetylated Amino Acid Methyl Esters
Trifluoroacetylated Retention time, methyl ester minutes Alanine 12 5 Valine 14 0 Isoleucine ii 4 Glycine 20 1 Threonine 21 3a, 35.9b Leucine 22 4 Proline 26 3 Serine 28 3", 40 8 b Aspartic acid 33 6 Cysteine 35 0 Hydroxyproline 36.7", 52 2 b Methionine 41 0 Glutamic acid 42 6 Phenvlalanine 43 4 Tyrosine 5 1 Oa, 62 6 b Ornithine 66 9 Lvsinp ,57 .i Tryptophan 58 8 A4rgin~ne 60 O(47 3, 55 9)' Histidine 64 0-67 0 Cystine 70 8-71 3 a S,O-Bis(trifluoroaretyl) derivative. S-Monn( trifluoroacetyl) derivative. Minor components at 47.3 and 55.9 minutes retention time.
1 196 *
ANALYTICAL CHEMISTRY
the distribution coefficient for trifluoroacetylglycine methyl ester decreases a t a more rapid rate with increasing temperature than the coefficients for the other amino acid derivatives. Separation of the glutamic acid and phenylalanine derivatives was not complete when uqing optimum conditions for separation of the qlycine and threonine derivatives. However, the resolution was satisfactory for unambiguous identification and ' for an approximation of the relative peak areas. Complete resolution of these two trifluoroacetylamino acid esters could be achieved by lengthening the column, but this was always accompanied by fusion of the peaks for the threonine and glycine derivatives. Chromatographic Results. The result of the chromatography of trifluoroacetylated amino acid methyl esters obtained from a standard mixture of 20 amino acids is shown in Figure 3. A mixture of dry amino acid hydrochlorides was dwivatized by the esterification/acylation procedure;
Detector response
the chromatography was carried out under the optimum conditions described above, except that the temperature program rate was increased to 2' C./ minute after 20 minutes and to 4' C./ minute after 42.5 minutes. These increased rates of heating enabled the run to be completed in 75 minutes with no loss of resolution of any components. Retention times for each amino acid derivative were very reproducible, and are given in Table IV for the 21 amino acids studied. Variations in the retention times for the histidine and cystine derivatives prequmably were due to slight variations in the final temperature and in the time a t which this temperature was attained. Vtility for this method of amino acid analysis was demonstrated with a hydrolysate of the protein ribonuclease. The results are shown in Figure 4. All amino acids known to be present in this protein were detected by the gas chromatographic procedure. The significant losses observed with serine and threonine were due to the con-
~
ditions of hydrolysis. The procedure . has been used routinvly to check the amino acid content of peptides prepared in a program devoted to synthesis of hormone and e nzyme-active-site analogs. Quantitative Aspects. T h e relative peak areas for most of the amino acid derivatives remained remarkably constant during six repetitions of the procedure with a standard mixture of amino acids. A11 but two of the 20 amino acids analyzed gave reproducible peak area ratios (based on trifluoroacetylalanine methyl ester = 1.00) within =t 10% a t a 95% confidence limit; of these, 10 were within + 5yo (Table V). Poor reproducibility with cystine and his1 idine was probably due to decomposition of the sensitive side chain functional groups (sulfhydryl and Imidazolyl, respectively). Through utilization of these peak area ratios, determination of amino acid ratiob in hydrolysates of peptides of moderate complexity is possible. By including as an internal standard a known amount of an amino acid not present in the sample under investigation, a true quantitative analysis could be carried out. LITERATURE CITED
( 1 ) Bellamy, L. J., “The Infrared Spectra of Complex Molecules,” 2nd ed., Wiley, Sew York, 1958. ( 2 ) Beyer, E., “Gas Chromatography,”
Table V.
Peak Area Ratios for Trifluoroacetylated Amino Acid Methyl Esters“
Amino acid
Peak area ratiob.c
Alanine J’aline Isoleucine Glycine Threonine Leucine Proline Serine Aspartic acid Cysteine Hydroxyproline Methionine Glutamic acid Phenylalanine Tyrosine Lysine Tryptophan
1.00 1.07 1.14 0.96 0.63 2.12 1.85 0.49 1.57 0.26 1.32 3.69 1.45 7.39 3.38 3.70 3.74
95VG
Confidence limits 2 f 3 f 2 f 8 f 2 f 3 f 6 f 3 f30 4 f 3 f 8 f 2 f 4 f 8 f 6 f
*
370 6% 0% 1% 9% 5% 0% 8% 0% 6% 3% 4% 370 470 7% 6%
D. H. Desty, ed., p. 333, Academic Press, Sew York, 1958. ( 3 ) Black, R. J., Durrum, E. L., Zweig, G., “Paper Chromatography and Paper Electrophoresis,” 2nd ed., Academic Press. New York. 19,58. ( 4 ) Gross, D.. Grodsky, G., J . Am. Chem. Soc. 77, 1678 (1955). ( 5 ) Johnson, D. E., Scott, S. J., Meister, A., ANAL.CHEM.33, 669 (1961). ( 6 ) Kvrides. L. P., J . .Am. Chem. SOC.66. 1006 (1944). ( 7 ) Levy, A. L., Chung, D., ANAL.CHEM. 25, 396 (1953). (8) Peiz, K. A., Moiris, L., Anal. Biochem. 1, 187 (1960). ( 9 ) Saraff, H. A., Karmen, A., Ibzd., 1,344 (1960).
95 70
Amino acid
Peak area rati0b.C
Confidence limits
Arginine Histidine Cystine
0 55 0 48 0 46
8 1% f 1 5 797, f 9 7%
*
a Characteristic for Jarrell-Ash Argon Diode Detector, Model 26-752, Serial no. 126262, with 20 mc. of Sr90. Chromatography conditions were described in Figure 1, except 2 pl. of a 0.01M solution containing derivatives obtained from a 0.01M amino acid standard solution was used. Relative to trifluoroacetylalanine methyl ester. e Peak areas were determined automatically with a Texas Instruments Servo/ Riter integrating recorder.
(10) Schwartz, D. P., Pallansch, M. J., ANAL.CHEM.30, 219 (1958). (11) Spackman, D. H., Stein, W. H., Moore, S., Ibid., 30, 1190 (1958). (12) J’oss, W., Blanke, E., Ann. 485, 258 (1931 ). (13) Wagner, J., U‘inkler, G., Z. Anal. Chem. 183, 1 (1961). (14) Weygand, F., Kolb, B., Prox, A., Tilak, M. A,. Tomida. I.. Z. Phwsiol. Chem: 322. 38 (19601. ’ ’ (15) Zomzely, C., Marco, G., Emery, E., ANAL. CHEM.34, 1414 (1962).
RECEIVED for review June 12, 1963. Resubmitted March 9. 1964. AcceDted March 9, 1964.
Composition of Straight Chain Alkylbenzenes by Gas Chromatography W. J. CARNES Procter & Gamble Co., Ivorydale Technical Center, Cincinnati, Ohio 452 17
b Apiezon-L, SE-30, and DC-550 were evaluated as liquid coatings on 15o-foot, 0.01-inch capillary columns. The best separation of components was achieved with a DC-550 liquid substrate and temperature increased uniformly at 1.5” per minute between 120” and 170” C. Linear alkylbenzenes with side chain ranging from CS to C14 were completely separated in 65 minutes. The isomers arising from the position of phenyl attachment were resolved, except for 7- and 6-phenyl subiitituted alkanes. A criterion for judging column performance is included.
T
widely usesd detergent raw material has been alkylbenzene prepared by alkylation of benzene with propylene tetramer. The alkylbenzenes produced are primarily dodecylbenzene HE MOST
isomers but also contain varying amounts of alkylbenzene with chain lengths ranging from Clo to CIS. C p to 80,000 isomers are possible in the final mixture (S), and the complete characterization of a typical ‘alkylbenzene detergent alkylate has not been accomplished. Gas chromatography can be applied, but the samples a r e so complex that separation and identification of components is not possible. The best that has been achieved is a general profile chromatogram indicating the molecular weight range. The chromatogram of a typical tetrapropylenederived detergent alkylate is shown in Figure 1. Since tetrapropylbenzene sulfonate is relatively resistant to biodegradation, manufacturers of detergent alkylate have sought a detergent alkylate that is readily biodegradable. Alkylbenzene produced by reaction of 1-olefins with benzene is now available.
This material is primarily straight sidechain isomers having chain lengths of Clo to Cla. Length of side chain and position of attachment of the phenyl group mean 26 compounds are possible. Under these more favorable circumstances efforts to establish a gas chromatographic method of analysis for straight chain alkylbenzene raw materials were worthwhile. Both chain length and position of phenyl substitution must be specified because they affect detergency characteristics. Packed column gas chromatographic methods for analyzing detergent alkylates ( I , 2, 6 , 8 , IO) resulted in partial or no resolution of the higher chain length isomers when applied to typical straight chain alkylbenzenes. Analysis times were also excesqive for a convenient routine analysis qchedule applicable to a large number of camples. Capillary columna have also ticen uqed for the VOL. 36, N O . 7, JUNE 1964
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