Effect of Stationary Phase-Support Ratio on the Gas Chromatographic Separation of Trifluoroacetylamino Acid Butyl Esters M. Stefanovic and B. L. Walker Department of Nutrition, University of Guelph, Guelph, Ontario, Canada

Trifluoroacetylamino acid butyl esters were frac- tionated by gas chromatography on ethylene glycol adipate polyester columns containing different levels of liquid phase. For certain amino acids, the elution pattern obtained was a function of the amount of liquid phase on the column packing. Relative retention times of glycine, methionine, phenylalanine, proline, and to a lesser extent alanine, increased with increas- ing levels of liquid phase, affecting the ability of the column packing to separate the amino acid derivatives. This appeared to be due to interaction of the liquid phase and polar moieties on these compounds.

FRACTIONATION OF AMINO ACID DERIVATIVES by a gas chromatographic technique would provide the protein chemist with a valuable research tool. The speed of the analysis, re- quirement for microgram quantities, and relatively low initial cost of equipment are among a few of the advantages of this technique over ion-exchange procedures. Problems have arisen in the development of a gas-liquid chromatographic procedure, however. The usual derivatives employed are the acetyl or trifluoroacetyl esters of the acids with lower alcohols. Cruickshank and Sheehan (I) separated the methyl esters of the trifluoroacetylamino acids on a neopentyl glycol succinate polyester column. Hagen and Black (2) separated these same derivatives on Carbowax using two different columns. Un- fortunately, the methyl ester derivatives of glycine and alanine are volatile and easily lost during preparation and isolation (3, 4). The butyl esters are more readily handled and have been successfully fractionated by Gehrke ef u!. (5) on a mixed polyester phase consisting of diethylene giyco! succinate and ethylene glycol succinate-silicone copolymer (EGSS-X). They were unable to separate all of the amino acids as this derivative on a single phase (6).

Problems arise in the separation of the amino acid deriva- tives because the diversity of functional groups present leads to differences in polarity and boiling point. Glycine fre- quently overlaps isoleucine or leucine. Proline and threonine may overlap and so may phenylalanine and aspartic acid. In the course of our investigations, we have discovered that the stationary phase-support ratio can play a major role in determining the elution patterns of these derivatives and, if carefully selected, can enable complete separation to be achieved on a single stationary phase.

EXPERIMENTAL Reagents. Individual amino acids of high purity were

purchased from the California Biochemical Co., Santa Monica, Calif. An amino acid standard calibration mixture was -~

(1) P. A. Cruickshank and J. C. Sheehan, ANAL. CHEM., 36, 1191

(2) P. B. Hagen and W. Black, Can. J. Biochem., 43, 309 (1965). (3) A. Darbre and K. Blau, J . Chromatog., 17, 31 (1965). (4) W. M. Lamkin and C. W. Gehrke. ANAL. &EM., 37,383 (1965). ( 5 ) C. W. Gehrke, W. M. Lamlun, D. L. Stalling, and F. Shahrokhi,

:6! C. VI. Geiirke and F. Snahrokhi, -4nU.'. Srocnt.n;., IS, 97 (1966)

( 1964).

Biochem. Biophys. Res Commun.. 19, 328 (1965).

obtained from Beckman Instruments, Spinco Division, Palo Alto, Calif. This mixture contained equimolar amounts of the amino acids commonly occurring in protein hydrolyzates. Dry, analytical grade reagents were employed when available.

Procedure. The trifluoroacetylamino acid butyl esters were prepared by the method of Lamkin and Gehrke (4). Methyl esters were first prepared by stirring 10 to 20 mg of the amino acid or mixture of acids with 10 ml of 1.5N an- hydrous methanolic HCl at room temperature for 30 minutes. The solvent was removed under vacuum at room temperature and 10 ml of 1.5N anhydrous butanolic HC1 added. This mixture was stirred at 100" for 3 hours. After cooling, the solvent was evaporated under vacuum at 60" C. The butyl esters were then heated in a sealed tube with 2 ml of methylene chloride and 0.5 ml of trifluoroacetic anhydride for 5 minutes at 150" C. The solvents were removed under a stream of nitrogen and the sample was dissolved in chloroform for gas chromatographic analysis. The acetylation procedure was that proposed by Stalling and Gehrke (7).

Chromatography was carried out in a Barber-Colman 5000 gas chromatograph equipped with dual stainless steel columns, 100 cm long and 4 mm in internal diameter, and dual hydrogen flame ionization detectors. Column packings consisted of 0.5, 0.7, 0.8, 1.0, or 2.0% (wlw) ethylene glycol adipate (Analabs, Inc., Hamden, Conn.) on 80- to 100-mesh acid- washed Chromosorb W. Columns were preconditioned overnight at a temperature of 230" C. Samples were injected as a solution in 1 to 2 p1 of chloroform. The instrument was temperature-programmed from 80" to 230" C at 4" C per minute. Injector and detector bath temperatures were 200" and 230", respectively, and nitrogen (carrier gas), hydrogen, and air flow rates were 60, 30, and 400 ml per minute.

RESULTS AND DISCUSSION

Figure 1 demonstrates the effect of stationary phase-support ratio on the elution pattern of the amino acid derivatives. The sample consisted of the calibration standard, containing equimolar amounts of 17 acids, to which were added cysteine, ornithine, and tryptophan. No effort was made to maintain the quantitative relationship of the three acids added to those in the mixture, as we were primarily interested in qualita- tive rather than quantitative data.

When 0.5 % ethylene glycol adipate (EGA) was employed, sharp well defined peaks were obtained and all of the acids except cysteine and methionine were completely separated. Only partial separation of cysteine and methionine was ob- served. As the level of the stationary phase was increased, the cysteine-methionine separation improved and complete separation was achieved on columns containing 0.7% or higher levels of the stationary phase.

Changing the level of the stationary phase from 0.5% to 0.7z resulted in a shift of the glycine peak toward the iso- leucine peak. Separation of proline and threonine was also

(7) t?. L. Stalling and C. W. Gehrke, Biochem. Biophys. Re:. Commun., 22, 329 (1966).

PHE . GLU

ILEU LEU

A L A vALl.d h PUE +

GLYc P R O + 1

10 20 30 40 TIME (MIN)

Figure 1. pattern of trifluoroacel ylamino acid butyl esters

Effect of stationary phase-support ratio on elution

reduced, as was the separation of phenylalanine and aspartic acid. These trends uere continued as the level of stationary phase was still further increased to 0.8 and 1.0%. -4t the latter level, glycine and isoleucine were eluted together, as were the proline-threonine and phenylalanine-aspartic acid pairs. The peaks were broader with the higher levels of liquid phase. When 2 % EGA was employed (Figure 2), there were actual changes in the order of elution of the acids. Glycine was

b Figure 2. Elution pattern of trifluoroacetylamino acid butyl esters on 2% EGA

1 I

-

Alanine Valine Glycine Isoleucine Leucine Proline Threonine Serine Cysteine Methionine Phenylalanine Aspartic acid Glutarmc acid Tyrosine Ornithine Lysine Tryptophan

0.285 0.347 0.379 0.420 0.470 0.499 0,348 0.627 0.733 0.751 0.814 0.856 1 o(Kl 1,102 1 . i91 1,264 1.316

0.311 0 359 0.430 0.430 0.481 0.539 0.539 0.621 0.732 0.781 0.849 0.849 1 .ooo 1.088 1.199 1.268 1.316

26 12 51 10 I1 40

-9 -6 -1 36 35

.- I

- 14 8 4 0

u Relative retention time on 1.0% EGA-relative retention time on 0.5 7; EGA.

eluted between isoleucine and leucine, and threonine was eluted before proline. Phenylalanine and aspartic acid were eluted as a single broad peak, with the phenylalanine tending to be eluted after aspartic acid. Cysteine, ornithine, and tryptophan were not included in the mixture used for the analysis on 2 % EGA.

Additional experiments were carried out to determine the position at which hydroxyproline was eluted. This again de- pended on the level of stationary phase employed (Figure 3). On the 0.5 % EGA column hydroxyproline was eluted between methionine and phenylalanine, on 1 .O % EGA it did not sepa- rate from the methionine, and on 2 % EGA it was eluted ahead of methionine. The 0.7 and 0.8 EGA columns behaved in a manner similar to the 1.0% EGA column.

i n order to ascertain just which compounds of the various critical pairs were susceptible to changes in the level. of the stationary phase, relative retention times were determined. .%though the relarive retention time concept is usually re-

2.0% E G A

PHE + ASP

LYS 1 ,I

I

,

PHE

~

P

MET

I

I -

0 . 5 % E G A - Figure 3. Effect of stationary phase-support ratio on location of hydroxyproline in elution sequence

stricted to isothermal gas chromatography, it was felt that this concept would be useful in indicating the compounds involved in the changes in elution pattern under the conditions of linear temperature programming employed in this study. Table I lists the relative retention data for analyses carried out on 0.5 and 1.0% EGA columns. The data for the 0.7 and 0.8% EGA columns were intermediate between these values and were similar to each other. As may be seen from the final column in this table, appreciable changes in relative retention time with level of stationary phase occurred with four acids: glycine, proline, methionine, and phenylalanine. There was a moderate increase in the relative retention time of alanine. The first four of these acids were involved in changes in the elution pattern with change in level of stationary phase.

The changes in elution retention data and hence in the elution pattern observed in this experiment probably resulted from changes in the interaction of the polar stationary phase with polar groups on the amino acid derivatives, thus changing the partition coefficients of these compounds. Increasing the level of the stationary phase in effect increased the polarity of the column packing. Glycine behaved as a polar compound by virtue of the absence of a hydrocarbon chain. Alanine, which has a relatively short hydrocarbon chain, also showed evidence of polarity. The free electron pairs on the sulfur in

LEU ILEY 1

methionine and the ring nitrogen in proline, and the pi-electron system of phenylalanine, could account for the interaction of these compounds with the polar stationary phase.

The other amino acids with polar substituents, such as threonine, serine, and cysteine, did not exhibit this enhanced polarity. In these cases, the polar function was blocked during derivative formation. Tyrosine did not show any enhanced interaction with the polyester in spite of the presence of the aromatic ring. The presence of the 0-trifluoroacetyl group on the ring may be responsible for reducing the inter- action of the aromatic ring with the stationary phase. The inductive effect of this group would reduce the availability of the benzene ring electrons for polar interactions. A similar mechanism may account for the behavior of hydroxyproline, because the changes in the elution of this acid relative to meth- ionine and phenylalanine appeared to be attributable to the increasing retention times of the latter two acids.

We were able to separate arginine, histidine, and cystine on ethylene glycol adipate columns. However, very small peaks were obtained for these three acids under the above conditions, indicating either a failure to produce appropriate derivatives or destruction of the derivatives during chromatography. Stal- ling and Gehrke (7) demonstrated the partial conversion of a disubstituted-arginine trifluoroacetate salt to the trisubstituted derivative in the hot metal flash heater of the chromatograph. They recommended the acylation of amino acids at 150 O and use of on-column injection and an all-glass system to avoid these problems. However, we have been unable to reproduce their results using this procedure and all-glass system. Work is continuing on this problem. Preliminary quantitative data on the acids other than those referred to above were similar to those of Gehrke et al. (5).

In all probability this variation in elution pattern applies to most polar packings. We have tested only one other poly- ester phase, ethylene glycol succinate, and found this to be true. Should this prove to be generally applicable, the frac- tionation of trifluoroacetylamino acid butyl esters on a single stationary phase may not be too difficult. Problems could arise when high temperatures are employed with polar phases or with mixed phases of differing thermal stability. The con- tinuous bleeding of the stationary phase or one component of a mixed phase may lead to changes in elution pattern with loss in effectiveness in separating certain acids. That this can be a serious problem was demonstrated by the use of 0.7 and 0 . 8 x EGA columns. There were notable differences in the elution patterns with these two columns in spite of the fact that the

PHE

b Figire 4. Separation of trifluoro- acetylamino acid butyl esters on IO

P S P d 0.65Y.EGA

20 io 0.65% EGA

712 ANALYTICAL CHEMISTRY

TIME ( M I N I

weights of stationary phase present differed by only about 12z. In actual practize we have encountered this problem with several polyester phases, when initially promising results were hard to duplicate i l s columns aged. Fortunately, ethyl- ene glycol adipate appesrs to have sufficient thermal stability to overcome this problim. The columns employed in these experiments were conditioned at 230” C for 24 to 48 hours and have been in constant use for periods in excess of 8 weeks without noticeable deterioration.

On the basis of our results, we feel that the fractionation of N-trifluoroacetylamino iicid butyl esters on a single stationary phase is possible. A c,olumn consisting of 0.65% ethylene glycol adipate will accomplish this and we are currently em- ploying such a column (Figure 4). Problems still exist with arginine, histidine, and (cystine. The ability of the column to separate these compounds from any other amino acids is not in question. However, the low responses obtained for these acids indicate that the correct derivatives were not prepared

or that the derivatives were decomposed in the column or reacted chemically with the polar stationary phase. This problem is still under investigation. By varying the liquid- phase content of a column packing it is possible to tailor the packing to a specific need. Thus, whereas a column packed with 0.65 EGA is the best compromise for the separation of all the amino acids considered in this paper, in the absence of cysteine, a situation frequently encountered with acid hydroly- zates of proteins, a column packing containing 0.5% EGA would be the better choice, as the separation of phenyl- alanine and aspartic acid is more efficient. Undoubtedly other instances exist where this principle can be applied.

RECEIVED for review February 6, 1967. Accepted March 27, 1967. Study carried out with financial assistance from the National Research Council of Canada and the Ontario De- partment of Agriculture and Food.

Fast Analysis of Phenol, Methylphenols, and Polymethylphenols by Gas Chromatography Using Packed Capillary Columns Catherine Landault and Georges Guiochon

Laboratoire du Professe#vr L. Jacque, Ecole Polytechnique, Paris, France

Packed capillary columns, using silanized Chromosorb P coated with a mixture of di (3,3,5-trimethylcyclohexyl) o-phthalate and tri 2,4-xylenyl phosphate, allow rapid analysis of the mixtures of phenol, methylphenols, and polymethyl phenols resulting from the pyrolysis of phenol-formol polycclndensates. Although the practi- cal upper temperature limit is 115”C, the high perme- ability and efficiency of the packed capillary columns allow a complete analysis of the 10 compounds (phenol cresols and xylenols)i in less than 15 minutes and the analysis of 19 of thtr 20 compounds (phenol and all methyl- and polymethylphenols) in 55 minutes. The performance of the packed capillary columns is de- scribed, and retention data of the various phenols on the pure liquid phases are given.

THE COMPLETE ANALYSIS of a mixture of phenol, the three methylphenols, and the 16 polymethylphenols is very diffi- cult. These compouncls are highly polar and have a low vapor pressure at modctrate temperatures. In addition, cer- tain pairs of these cornpounds have nearly the same vapor pressure-e.g., 3-methylphenol and 4-methylphenol, 2,4-di- methylphenol and 2,5-dimethyIphenol (see Table I).

High efficiency colunins using specific selective stationary phases are therefore necessary to obtain a good resolution of these compounds. Thi,s leads to long analysis times because such columns are long and the best selective phases are not stable at high temperatupe.

In the present state o l the art much better analyses are ob- tained with derivatives than with the free phenols (3-6).

(1) J. Franc, CoNection C.:ech. Cliem. Commun., 25, 1573 (1960). (2) R. R. Dreisbach, “Physical Properties of Chemical Com-

pounds,” American Chemical Society., Washington, D. C. (3) W. Carruthers, R. A. W. Johnstone, and J. R. Plimrner,

Clzem. Ind. (London), 1958, 331. (4) R. W. Freedman and G. D. Charlier, ANAL. CHEM., 39, 1880

(1964). (5) D. W. Grant and Cr. A. Vaughan, “Gas Chromatography

1962,” M. Van Swaay, ed., Butterworths, London, 1962, p. 305. (6) E. Pillion, J. Gas Chromatog., 3, 238 (1965).

However, because our primary interest was in the pyrolysis gas chromatography of phenol-formaldehyde polyconden- sates (7, 8), we could not use this approach but had to analyze the phenols themselves. Furthermore, because pyrolysis of phenol-formaldehyde resins gives a mixture of phenol, methyl phenols, and polymethylphenols, the composition of which is related to the composition of the raw materials used to prepare the resin (8), we have to resolve most of these phenols .

A comprehensive review of the work on phenol analysis has been recently published, and it covers the literature from 1956 to 1965 (9). Suffice it to say that the nonpolar liquid phases (Apiezon, silicone oils) and most of the polar phases are un- able to give sufficiently large relative retention of some pairs, so that their resolution would need impracticably long columns. Only two phases appear to be convenient. The first one was selected by Brooks (10): tri 2,4-xylenylphosphate (TXP) gives a good resolution of phenol, methylphenols (MP), and 2,6-dimethylphenol (DMP) (8, 10). This phase is the most frequently used. The second phase, recommended by Sassenberg and Wrabetz (11, 12), is di[3,3,5-trimethyl cyclo- hexyllo-phthalate (TMCP); this gives a good resolution of the MP’s and of 24- and 2,5-DMP.

However, both phases have a low maximum working temperature (125” C), leading to long analysis times (more than 1 hour) for the complete analysis of DMP. Further- more, on both phases there is a pair which is quite difficult to resolve. This pair is made of 2,4- and 2,5-DMP on TXP and of 2-MP and 2,6-DMP on TMCP.

(7) J. Martinez and G. Guiochon, Paris, unpublished data, 1966. (8) J. Zulaica and G. Guiochon, J . Polymer. Sci., 4, 567 (1966). (9) S. T. Preston, “A Guide to the Analysis of Phenols by Gas

(10) V. T. Brooks, Chem. Ind. (London), 1960,1090. (11) W. Sassenberg and K. Wrabetz, 2. Anal. Chem., 184, 423

(12) K. Wrabetz and W. Sassenberg, Ibid., 179, 333 (1961).

Chromatography,” Polyscience, Evanston, Ill., 1966.

(1961).

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