Ligand exchange chromatography of amino alcohol enantiomers as Schiff bases

Journal of Chromatography, 406 (1987) 343-352 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands CHROMSYMP.

1249

LIGAND EXCHANGE CHROMATOGRAPHY ANTIOMERS AS SCHIFF BASES

OF AMINO ALCOHOL

EN-

C. H. SHIEH*, B. L. KARGER*, L. R. GELBERM, and B. FEIBUSH-** Barn&t Institute and Department of Chemistry, Northeastern University. Boston, MA 02115 (U.S.A.)

SUMMARY

A study was conducted on the ligand exchange high-performance liquid chromatographic separation of enantiomers of a-amino alcohols as their Schiff bases. Using a previously developed L-proline bonded phase diluted with Cl8 alkyl groups, good chromatographic performance and high separation factors, sometimes in excess of 2.0, are achieved. Greater solute retention and enantiomer separation are found by decreasing mobile phase buffer concentration and increasing Cu2+ concentration. For Schiff base formation, use of acetophenone or benzophenone derivatizing agents, particularly with two hydroxyl groups at the 2- and Cpositions of the aromatic ring, enhance enantiomeric separation. These results can be rationalized in terms of a previous chemical model of chiral resolution. A semipreparative scale purification of (R,S)-phenylethanolamine on the basis of the Schiff base derivative is demonstrated.

INTRODUCTION

The separation of enantiomers by ligand exchange high-performance liquid chromatography (HPLC) has been an active area of research over the last few yearslp3. While this method has been demonstrated to be a powerful tool for chiral separations, most applications have been for separation of a-amino acids and a-hydroxycarboxylic acids 4*5. In a previous paper, we reported that solute derivatization can be a simple route to extend this technique to other classes of compound@. In particular, we showed the enantiomer resolution of a-amino alcohols as Schiff bases on a bonded L-proline stationary phase. In the present paper, we examine the chromatographic behaviour of the previously developed system in greater detail. We have observed that mobile phase conditions, such as ionic strength and Cu2 + concentration, as well as Schiff base structure, strongly influence chiral resolution. As a result, substantial control of the system is available for optimization of chiral separations. In addition, the applicability of the Schiff base procedure to semipreparative separations is demonstrated. In this case, the facile reversibility of the Schiff base derivatization is used to advantage. l Present address: Beckman Instruments, San Ramon, CA, U.S.A. ** Present address: Barr Laboratories, Northvale, NJ, U.S.A. Present address: Supelco Inc., Bellefonte, PA, U.S.A.

0021-9673/87/$03.50

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1987 Elsevier Science Publishers B.V.

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EXPERIMENTAL

Materials (R,S)-Phenylethanolamine [(R,S)-Zamino-1-phenylethanol], (R.S)-valinol [(R,S)-2-amino-3-methylbutanol], and (S)-valinol were obtained from Sigma (St. Louis, MO, U.S.A.). 2,4-Dihydroxyacetophenone, 2,4-dihydroxybenzaldehyde, 2hydroxy-4-methoxyacetophenone, 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxyacetophenone, 2-hydroxybenzaldehyde, 2-hydroxybenzophenone, 10% palladium on charcoal and undec-lo-enal were purchased from Aldrich (Milwaukee, WI, U.S.A.) L-Proiine tert.-butyl ester was obtained from Chemical Dynamics (South Plainfield, NJ, US.A.), octadecyltriethoxysilane and triethoxysilane from Petrarch Systems, (Bristol, PA, U.S.A.), and di-p-chlorodichlorobis(ethylene)diplatinum(II) from Strem (Newburyport, MA, U.S.A.). HPLC grade silica gel was Supelcosil, 5 pm, from Supelco (Bellefonte, PA, U.S.A.). Elemental analyses were performed by Multichem Labs. (Lowell, MA, U.S.A.). Instrumentation Infrared spectra were measured with a Perkin Elmer (Norwalk, CT, U.S.A.) Model 1B 467 spectrophotometer, lH NMR spectra with a Varian (Walnut Creek, CA, U.S.A.) T-60 spectrophotometer, and uncorrected melting points with a Thomas-Hoover Uni-melt (Swedesboro, NJ, U.S.A.). The HPLC instrument consisted of a Waters (Milford, MA, U.S.A.) Model 6000A pump, a BioRad (Richmond, CA, U.S.A.) Model AS-48 autosampler, a Waters Model 441 Detector, a Nelson Analytical (Cupertino, CA, U.S.A.) 760 series interface, and an IBM PC with a Nelson Analytical chromatography software package. Synthesis of 11-triethoxysilylundecanal The silane was prepared by a modification of a method described in the literature’. A solution of 20 ml (0.1 mol) of undec-lo-enal with a trace amount of dip-chlorodichlorobis(ethylene) diplatinum(I1) in 25 ml of chloroform was slowly heated to 54°C under a nitrogen atmosphere. A solution of 18.4 ml (0.1 mol) of triethoxysilane in 20 ml of chloroform was slowly added to the first solution over a period of 1 h. The reaction was monitored using IR spectroscopy. After the C = C band (1640 cm-‘) disappeared, the reaction mixture was cooled to room temperature, and the solvent evaporated under reduced pressure. The residue was distilled at 142-143°C at 0.3 Ton, to yield 12.8 g (38%) of product. IR (cm-‘): 3020, 2980, 2935, 1730, 1400, 1110, 1080, 960. ‘H NMR (CZHC13): 6 0.7-1.0 (m, 2H), 1.48 (t, J=6,9H), 1.1-2.1 (m, 16H), 2.45-2.9 (m, 2H), 4.05 (q, J=6,6H), 9.95 (t, J=l, 1H). Synthesis of N-(II-triethoxysilylundecyl)~L-proline tert.-butyl ester A solution of 5.84 mmol of 11-triethoxysilylundecanal, 50 ml of absolute alcohol, 200 mg of 10% palladium on charcoal and 5.84 mmol of L-proline tert.-butyl ester was agitated on a 500 ml hydrogenation apparatus under 39.5 p.s.i. hydrogen atmosphere. After 14 h, the pressure had decreased to 28 p.s.i. The reaction mixture was removed from the hydrogenation apparatus, filtered, and the solvent evaporated on a rotary evaporator. The residue was purified by column chromatography on 90 g of dry silica gel and eluted with 8% ethyl acetate in toluene...The yield was 2.60 g

LIGAND EXCHANGE CHROMATOGRAPHY

OF AMINO ALCOHOL ENANTIOMERS

345

(91.5%). IR (cm-r): 2970, 2920, 2850, 1740, 1160-1070 (broad). NMR (C2HC13): 6 (0.5-0.9 (m, 2H), 1.48 (t, J=6.6, 9H), 1.1-1.7 (m, 29H), 1.8-2.2 (m, 2H), 2.3-2.9 (m, 3H), 3.0-3.4 (m, 2H), 3.96 (q, J=6.6, 6H). Elemental analysis; calculated for C26H53N05Si: C 64.06%, H 10.88%, N 2.87%; found: C 64.29%, H 10.81%, N 2.81%. Preparation of bonded phases and packing of column

A 1:lO diluted and a 1:1 concentrated L-prolineC1s phase were prepared for this study, based on the procedures described previousIy6s*. Bonded phases were packed into 15 cm x 4.6 mm I.D. columns using a slurry solvent containing methanol and carbon tetrachloride, and a Model DSTV 122 air-driven Haskel (Burbank, CA, U.S.A.) pump. Synthesis of Schiff bases

All Schiff bases were made from the amino alcohols and aldehydes or ketones following the previous procedures9 and characterized by melting point. Table I shows elemental analysis data and melting point of Schiff bases derived from phenylethanolamine. Resolution of (R,S)-phenylethanolamine

by recrystallization1Q

A solution of 13.7 g (0.1 mol) of (R,S)-phenylethanolamine in 30 ml of ethanol was added to a solution of 7.5 g (0.05 mol) of D-tartaric acid in 50 ml of ethanol at 65°C. The mixture was stirred for 10 min, after which the white crystalline precipitate was collected by filtration. After four recrystallizations from aqueous acetone, the product was hydrolyzed using a 10% aqueous sodium hydroxide solution. The mixture was extracted twice with 100 ml of chloroform. The chloroform layer was evaporated to dryness and the enriched (R)-phenylethanolamine crystallized from ether. A sample was derivatized to the Schiff base with 2-hydroxy-4-methoxyacetophenone, and HPLC analysis demonstrated that its optical purity was greater than 75%. Semipreparative separation of (R,S)-phenylethanolamine

(R,S)-Phenylethanolamine (0.5 g) and 2-hydroxy-4-methoxyacetophenone (0.6 g) weit: dissolved in 10 ml of methanol and heated to 40°C for 30 min. After cooling to 0°C for 2 h, the solid product was collected and recrystallized from absolute alcohol. A solution of 200 mg of Schiff base in 60 ml of methanol was prepared and sixteen 100-p samples were injected onto the concentrated proline phase in a 15 cm x 4.6 mm column. The mobile phase was 5 . 10m3 M copper(I1) acetate, 0.3 M ammonium acetate, pH 5.0, in 75% methanol. The column temperature was 25”C, and the flow-rate was 1 ml/mm. The fractions corresponding to each peak were collected manually. Each pooled fraction was then evaporated to dryness on a rotary evaporator, after which 10 ml of 50% aqueous acetic acid were added and the solution stirred for 30 min. The solution was then extracted seven times with 10 ml of chloroform to give a combined green chloroform solution. The chloroform solution was washed with water and evaporated to dryness, 10 ml of water and 1 ml of hydrochloric acid were added, and the mixture extracted twice with 100 ml of diethyl ether. The aqueous portion of each fraction was evaporated to dryness to yield pure (R)- or (S)-phenylethanolamine hydrochloride.

346

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TABLE I ELEMENTAL ANALYSIS OF PHENYLETHANOLAMINE SCHIFF BASES X OH

-0

x

R

H

H

OH

H

OCH3

H

H

CH3

OCH3

CH3

H

CsHs

found calculated found calculated found calculated found calculated found calculated found calculated

%C

%H

%N

74.90 74.67 70.11 70.02 70.80 70.83 75.38 75.27 71.58 71.56 79.52 79.47

6.37 6.27 5.92 5.88 6.36 6.32 6.72 6.71 6.74 6.71 6.07 6.03

5.81 5.81 5.92 5.44 5.12 5.16 5.46 5.49 4.88 4.91 4.39 4.41

Melting

poin1(“C) 91- 93 175-177 158-160 115-116.5 194-196 131-134

RESULTS AND DISCUSSION

Bonded phase strategy

As discussed in our previous paper, we employed an L-proline phase diluted with Cis groups attached to the silica gel surface?. In this study, we prepared N(1 I-triethoxysilylundecyl)~L-proline tert.-butyl ester, which was well characterized by spectral and elemental analysis. This silane was diluted with octadecyltriethoxysilane (l:lO), and the mixture of silanes co-bonded to the silica surface. After bonding, the rert.-butyl group was hydrolyzed with trifluoroacetic acid, and the amount of active ligand was determined by gas chromatographic analysis of the isobutylene produced”. The active ligand concentration was found to be 0.37 pmol/m2. The total coverage of the phase was calculated from carbon analysis, using the equation previously described’ l, and found to be 3.78 pmol/m* which is consistent with the original composition of the silane mixture used for bonding. The L-proline-Cla (1: 10) phase was used continuously over a period of four months (50 1 of solvent) with less than 5% change in retention and no change in selectivity. All the peaks were identified by injection of Schiff bases derived from mixtures enriched with the (R)-phenylethanolamine or (S)-valinol isomers. Chromatographic characteristics

The structure of the Schiff bases used in this study are presented in Table II. All the Schiff bases were prepared from the a-amino alcohols, phenylethanolamine or valinol, using the corresponding derivatives of benzaldehydes, aceto- or benzophenones. In agreement with previous results, the diluted L-proline phase provides good separation of the enantiomers of Schiff base derivatized amino alcohols, as

LIGAND EXCHANGE CHROMATOGRAPHY

OF AMINO ALCOHOL ENANTIOMERS

347

TABLE II STRUCTURES OF SCHIFF BASES STUDIED R

x

CH3 H CHg H CHg H

OH OH OCHo OCH3 H H

(35

14

CHB H CHo

OH OH OCHB

11 12 13

H CHB H

OCHO H H

14

CsHs

H

Compound 1 2 3 4

Structure Old

‘i

H20H

yx,

5 6

=N-CH-CH(CH& d

I 8 9 10

0

X

X

illustrated in Fig. 1 for compounds 9 and 11. Note the use of the 350 nm detection wavelength, in a region relatively free of UV absorbing interferences. This detection wavelength represents a particular advantage in the selection of Schiff base derivatives. Note also that no peak for salicylaldehyde is observed. If the sample is not freshly made up, a peak for this species would be seen. Ammonium acetate concentration Table III summarizes the retentions and enmantioselectivities of the Schiff bases as a function of the concentration of ammonium acetate. It can be seen that enantioselectivity and retention decrease markedly with increase in the concentration of the buffer leading to a total loss of enantiomer resolution for six solutes at 0.4 A4 ammonium acetate. These trends are due to competition for the limited amount of Ct.?+ ions present. Since ammonium acetate can complex with Cuz+, increasing the concentration of the buffer effectively decreases the formation of the Schiff base solute L-prolinato-copper(I1) complex, leading to reduced retention, and a loss of enantioselectivity. Cu2 + concentration The results obtained when the Cu2+ concentration of the mobile phase was varied from 5 . lo-’ A4 to 5 . 10e5 M are presented in Table IV. Retention and enantioselectivity are generally greater the higher the Cu2+ concentration. It should be noted that inversion of elution order is observed for several of the Schiff bases derived from phenylethanolamine at low Cu2+ concentrations. In addition, retention increases with decreasing Cu2 + concentration when inversion in elution order occurs. On the other hand, none of the valinol derived Schiff bases exhibit inversion of elution order in the range of Cu2+ concentrations tested. The equilibrium theory of ligand exchange chromatography, developed by

C. H. SHIEH et al.

TIME, min Fig. 1. Chiral separation of compounds 9 and ‘11 (see Table II) by ligand exchange chromatography. Conditions: stationary phase, dilute L-proline bonded phase; mobile phase, 5 . 10m3 A4 Cu2+, 0.2 M ammonium acetate, pH 5.0 in 75% (v/v) methanol; temperature, 3X, flow-rate, 1 ml/mine UV detection at 350 nm. Solutes: (a) (R)-9, (b) (s)-9, (c) (R)-11 and (d) (3-11.

HelfferichlzJ3, predicts that changing the concentration of the metal ion should alter the equilibrium distribution between all complex species present in solution and on the bonded phase surface, and this change could in principle affect the separation factors. The behavior observed, including inversions of elution order, suggests the possibility that multiple complex species are involved in the separation. Since the relative concentration of these species could be influenced by the Cuz+ concentration in different ways, species in which one enantiomer is more stable could be favored at high Cu2 + concentration, while other species in which the other enantiomer is more stable could be favored at low Cu2+ concentration. It is not possible to specify these different complexes at the present time. Schlj- base structure

Tables III and IV further indicate that the Schiff base structure has a marked effect on enantiomer separation. It may be observed that the substitution of the benzyl hydrogen of the Schiff bases with a methyl or phenyl group has a significant effect on enantioselectivity (see Table II). Compounds 2,4, 6,9, 11 and 13 are benzaldehyde derivatives; compounds 1, 3, 5,8, 10 and 12 are acetophenone derivatives, while compounds 7 and 14 are benzophenone derivatives. Under optimized chromatographic conditions, the separation factors, a, of the benzophenone derivatives are larger than those of similar acetophenone derivatives, and those of the benzaldehyde derivatives are the smallest. Methyl or phenyl substitution on the benzyl carbon not only increases steric hindrance but also drives the Schiff base to its ke-

LIGAND EXCHANGE CHROMATOGRAPHY

349

OF AMINO ALCOHOL ENANTIOMERS

TABLE III ENANTIOMER RESOLUTION TIONARY PHASE

OF AMINO ALCOHOL SCHIFF BASES ON DILUTED L-PROLINE STA-

Mobile phase, 5 . 10e3 A4 Cu2+, pH 5.0 in 75% (v/v) methanol with different concentrations of ammonium acetate; temperature, 30°C; bonded chelate, 0.37 pmole/mz. Compound number

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0.1 M Ammonium acetate

0.2 M Ammonium acetate

0.3 M Ammonium acelate

0.4 M Ammonium acetate

kh

KS

a

kk

ks

a

kin

KS

a

kk

KS

a

0.63 0.64 1.53 1.52 1.34 1.44 3.55 1.68 1.29 4.07 3.51 4.07 3.57 7.98

1.14 0.98 2.36 2.03 2.19 1.94 5.99 5.42 3.20 10.10 6.38 9.95 6.45 21.82

1.80 1.48 1.54 1.34 1.64 1.35 1.69 3.22 2.48 2.48 1.82 2.44 1.81 2.73

0.28 0.34 0.65 1.39 0.68 0.71 1.74 0.92 0.92 2.07 2.53 2.20 2.66 4.98

0.48 0.47 0.92 1.65 1.00 0.93 2.76 2.53 1.66 4.65 3.48 4.82 3.61 11.17

1.76 1.38 1.41 1.17 1.48 1.31 1.59 2.75 1.81 2.25 1.37 2.19 1.36 2.24

0.22 0.30 0.55 0.72 0.58 0.69 1.62 0.64 0.81 1.56 2.47 1.67 2.58 4.31

0.29 0.38 0.66 0.84 0.77 0.81 2.24 1.57 1.19 3.08 2.79 3.20 2.84 8.00

1.29 1.26 1.19 1.16 1.31 1.18 1.38 2.44 1.46 1.97 1.13 1.92 1.10 1.86

0.23 0.32 0.50 0.69 0.47 0.66 1.31 0.53 0.77 1.21 2.08 1.43 2.49 3.79

0.57 1.69 1.17 0.96 2.16 2.31 2.40 6.54

1 1 1 1 1.20 1 1.27 2.2 1.29 1.78 1.11 1.64 1 1.73

toamine form through conjugation or hyperconjugation (see Fig. 2). The ketoamine form provides increased stability for the mixed Cu’ + complex. Similarly, the presence of a hydroxyl group at the 4-position of the aromatic ring of the SchilI bases also enhances the ketoamine form; compare compound 1 with compounds 3 and 5; compound 2 with compounds 4 and 6; compound 8 with compounds 10 and 12; and compound 9 with compounds 11 and 13. In all four cases the first member of each set has a hydroxyl group in the Cposition, the second a methoxy, and the third a hydrogen. Compounds 1, 2, 8 and 9 always exhibit higher d! values than their 4methoxy or 4-hydrogen analogues.

HO

Fig. 2, Tautomeric equilibrium between enolimine and ketoamine forms of amino alcohol Schiff bases derived from 2-hydroxybenxaldehyde and

C. H. SHIEH et al.

3.50 TABLE IV ENANTIOMER RESOLUTION TIONARY PHASE

OF AMINO

ALCOHOL

SCHIFF BASES ON DILUTED

L-PROLINE

STA-

Mobile phase, 0.2 M ammonium acetate (pH 5.0) in 75% (v/v) methanol, with different concentrations of Cu2+; temperature 3o’C; bonded chelate, 0.37 pmole/mz. Compound number

1 2 3 4 5 6 I 8 9 10 11 12 13 14 l

5. 1o-2 h4 c2+

5.10-a

M cl2+

5

lo+

A4 cu2+

5. 1o-5 lucu2+

vi

4

a

&

ks

a

kk

ks

a

kZ

ks

a

0.34 0.32 0.48 0.69 0.75 0.64 1.55 0.95 0.72 2.09 1.52 2.05 1.45 3.77

0.63 0.47 0.79 0.95 1.15 0.90 2.52 2.77 1.71 4.92 3.12 4.78 2.94 8.49

1.83 1.49 1.64 1.38 1.54 1.41 1.63 2.90 2.38 2.35 2.06 2.34 2.03 2.26

0.28 0.34 0.57 0.75 0.68 0.71 1.74 0.92 0.92 2.07 2.53 2.20 2.66 4.98

0.48 0.47 0.74 0.90 1.00 0.93 2.76 2.53 1.66 4.65 3.48 4.82 3.61 11.17

1.76 1.38 1.30 1.20 1.48 1.31 1.59 2.75 1.81 2.25 1.37 2.19 1.36 2.24

0.19 0.29 0.40 0.92 0.45 0.76 1.12 0.59 1.86 1.56 7.58 2.16 8.34 8.32

0.43 0.88 1.44 1.12 1.97 2.37 6.07 3.08 6.66 9.14

1 1.20 1 1 1 1.15 1.28 1.90 1.06 1.51 0.80* 1.43 0.80* 1.17

0.19 0.32 0.36 0.93 0.35 0.97 1.05 0.36 4.64 0.96 19.22 1.71 21.85 13.49

-

1 1 1 1 1 1

3.26 13.25 15.32 10.10

0.70* 1 0.69* 1 0.70* 0.75*

1 1

Inversion in elution order.

Let us further examine the tautomeric equilibrium between the enolimine and ketoamine forms, demonstrated in Fig. 2 i4v15. The possible structures of the mixed complex necessary for separation were described in our previous paper (see Fig. 2a of ref. 6). The Ct.?+ complex has two ligands, an N-alkylated-L-proline bidentate ligand and a tridentate Schiff base ligand. The ketoamine structure of the Schiff base is more favored in such a complex l*. As illustrated in Fig. 2, when there is an additional hydroxyl group in the 4-position of the aromatic ring, the ketoamino form does not involve the free electrons of the 2-hydroxyl group in the resonance structure. The 4-hydroxyl group increases electron density of the oxygen and nitrogen atoms which participate in complex formation in the ketoamine form. The 4-hydroxyl group should thus enhance ketoamine formation, resulting in increased coordination and greater enantioselectivity. It may also be noted that the Schiff bases derived from phenylethanolamine exhibit greater enantiomer resolution than those derived from valinol. Compare, for example, the a values of compounds 1-7, derived from valinol, with those for compounds 8-14, derived from phenylethanolamine. As discussed previously6, phenylethanolamine possesses on the asymmetric carbon the hydroxyl group involved in the coordination. In contrast, valinol has the hydroxyl on a carbon adjacent to the chiral center and is less influenced by the L-prolinato ligand in the mixed Cu* + complex.

LIGAND EXCHANGE CHROMATOGRAPHY

OF AMINO ALCOHOL ENANTIOMERS

351

Semipreparative scale resolution of phenylethanolamine

A semipreparative scale resolution of (R,S)-phenylethanolamine, as the SchilI base of 2-hydroxy4methoxyacetophenone, was performed on a concentrated L-proline phase, a phase with greater loading capacity than that of the diluted phase. The chromatographic conditions are listed in the experimental section. In this study, we resolved 0.3 mg of Schiff base per injection without difficulty on a 15 cm x 4.6 mm I.D. column. After chromatographic purification, the phenylethanolamine was regenerated by the procedure described in the experimental section. Each enantiomer was then rederivatized to the Schiff base with 2-hydroxy4methoxyacetophenone. Reinjection of each enantiomer revealed that the (R)-phenylethanolamine was 100% pure and the (,5’)-phenylethanolamine was 90% pure. This result demonstrates that the two peaks observed in the chromatogram correspond to the two enantiomers of phenylethanolamine and that the starting alcohols can be easily recovered from the derivative. This purification strategy takes advantage of the easy recovery of the amino alcohols from the Schiff base by hydrolysis and the stabilization of the Schiff bases under the chromatographic conditions with Cu2+ (ref. 6). CONCLUSION’S

The results described in this paper indicate that facile and rapid derivatization of racemic mixtures for enantiomer separation by ligand exchange HPLC has great potential. Schiff bases of cl-amino alcohols appear particularly promising in this regard. The observed dependence of chromatographic behavior on mobile phase conditions such as ionic strength and Cu2+ concentration suggests that mobile phase composition must be carefully controlled for good chromatographic reproducibility. However, this also presents the chromatographer with a useful means to influence and optimize chromatography to suit the particular circumstances of the separation. Choice of derivatizing agent can also be used effectively to modify resolution as desired, as well as to control detection levels. The feasibility of semipreparative scale enantiomer resolution using labile derivatives in chiral ligand exchange chromatography has also been demonstrated. ACKNOWLEDGEMENTS

The authors gratefully acknowledge the National Science Foundation for support of this work. This is contribution No. 291 from the Barnett Institute of Chemical Analysis and Materials Science. REFERENCES I V. A. Davankov, A. S. Kurganov and A. S. Buchkov, Adv. Chromatogr., 22 (1983) 71. 2 Y. Tap&i, N. Miller and B. L. Karger, J. Chromatogr., 205 (1981) 325. 3 E. Gil-Av and S. Weinstein, in W. S. Hancock (Editor), Handbook of HPLC for the Separation of Amino Acid, Peptides and Proteins, Vol. I, C.R.C. Press, Boca Raton, FL, 1982, p. 429. 4 B. Lefebre, R. Audebert and C. Quivoron, J. Liq. Chromatogr., 1 (1978) 761. 5 W. Kelmisch, A. Von Hodenberg and K. 0. Volimer, J. High Resolut. Chromatogr. Chromatogr. Commun., 4 (1981) 535. 6 L. R. Ge.lbe.r, B. L. Karger, J. L. Neumeyer and B. Feibush, J. Am. Chem. Sot., 106 (1984) 7729.

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