Resolution of Enantiomers on Optically Active Columns

In 1966, Gil-Av demonstrated the first resolution of amino acid enantiomers on an optically active stationary phase. The N-TFA-2-butyl esters of alanine, valine and leucine were resolved on an N-TFA-l-isoleucine lauryl ester phase coated on a capillary column. However, phases of this type quickly gave way to dipeptide phases such as N-acyl-l,l-dipeptide alkyl esters which were first introduced by Feibush and Gil-Av in 1967 and which produced better resolution.

In 1970 Nakaparskin and colleagues separated 17 amino acid enantiomers on an N-TFA-l-val-l-val-cyclohexyl ester phase (val-val). In earlier studies, stainless-steel columns up to 500 ft long were used.

Consequently, analysis times were prolonged and the cystine, serine and threonine derivatives were degraded. In addition, dipeptide stationary phases such as val-val were functional over a limited temperature range or a limited maximum operating temperature. Columns were usually operated in isothermal mode.

Konig addressed the problem of temperature stability by introducing the N-TFA-l-phenylalanyl-l-leucine cyclohexyl ester which could be operated at 140°C. A later modification, the N-TFA-l-phenylalanyl-l-aspartic acid fo's-(cyclohexyl) ester, was stable over the range 96-165°C and allowed the use of temperature programming. In addition, the introduction of glass capillary columns reduced degradation of the amino acid derivatives. The high boiling N-PFP isopropyl esters of aspartic acid, methionine, phenylalanine, glutamic acid, tyrosine, ornithine and lysine were eluted using a 20 m column. However, the diamide phases still left room for improvement in thermal stability and in peak resolution.

Another generation of phases was introduced by Frank, Nicholson and Bayer who linked the diamide moiety, l-valine tert-butylamide, to a polysiloxane backbone. Later termed Chirasil Val®, phases of this general type became predominant and are still in use. Early versions of this phase resulted in the overlap of d- and l-proline, d-isoleucine and l-o//o-isoleucine, and l-threonine and d-o//o-isoleucine. Nevertheless, the enantiomers of all the other proteic amino acids were resolved as the N-PFP «- or isopropyl esters in about 30 min by temperature programming from 90 to 190°C (Figure 5). Acid treatment of the glass capillary followed by methanol washing was necessary rigorously to exclude basic sites and thus to obtain satisfactory elution of cysteine, serine, threonine and tyrosine and to obtain a sharp peak for arginine. The relative retention times of the amino acids can be manipulated by including polar modifiers such as cyanopropyl and phenyl groups but the effect varies with specific amino acids. The l-valine tert-butyl moiety was subsequently grafted to chloropropionyl-methyl phenylmethyl silicone, a modified OV-225, and to Silar 10C, but no overall improvement was achieved.

Chirasil-Val® was further improved by the incorporation of 15% phenyl groups substituted for methyl groups in the dimethylsiloxane units and the introduction of fused silica capillary columns. Thermal stability, ease of handling and separation efficiency were improved. The product is commercially marketed as Heliflex™ Chirasil-Val®.

Later improvements included the enhancement of enantioselectivity and thermal stability by immobilization of the Chirasil-Val® by radical or thermal i: o

I 85 iso i" g'o 100 ilo 120 130 140 150 160 170 180 190195 °C

I 85 iso i" g'o 100 ilo 120 130 140 150 160 170 180 190195 °C

Figure 5 Resolution of a racemic mixture of proteic amino acids as the N-(O, S)-pentafluoropropionyl n-propyl esters. (Reproduced with permission from Bayer E, Nicholson G and Frank H (1987) Separation of amino acid enantiomers using chiral polysiloxanes: quantitative analysis by enantiomer labeling. In Gehrke CC, Kuo KCT and Zumwalt RW (eds) Amino Acid Analysis by Gas Chromatography, Volume II, pp. 35-53. Boca Raton, FL: CRC Press.)

reactions. Chiral polysiloxanes with regular repeat units, e.g. trifiuoroethyl ester-functionalized polysiloxanes supporting l-val-tert-butylamide or l-a-naphthylethylamine liquid phases, have shown improved enantioselectivity. Backbone modification achieved by replacing one methyl group per dialkyl-siloxy unit with a pentyl or hexyl group improved resolution of arginine and tryptophan N-TFA n-propyl esters but the overall separation of the other amino acids was not significantly affected. However, satisfactory results have been obtained by varying the proportion of l-val-tert-butylamide on the poly-siloxane backbone. A ratio of about 6-7 dimethyl-siloxane units per chirally modified dialkyl siloxane unit is effective for the complete resolution of all components present in a chiral mixture of the 20 proteic amino acids in about 35 min on a 20 m x 0.3 mm glass capillary column.

Most studies on amino acid enantiomer resolution on Chirasil-Val® type columns have used N-per-fluoroacyl alkyl ester derivatives. However, other derivatives may present advantages in specific contexts. For example, the N-alkyloxycarbonyl alkylam-ide derivatives of proline are completely resolved on a Chirasil-Val® column. Similarly, Konig demonstrated the utility of isocyanate derivatives for resolving the enantiomers of N-methyl and ^-hydroxy amino acids.

A radically different approach to enantiomer resolution has become possible with the development of cyclodextrins as stationary phases. Although suitable for liquid chromatography, the high melting point of cyclodextrins rendered them unsuitable for GC without further modification. Konig reduced the melting point and increased stability by introducing hydrophobic moieties by both partial and complete alkylation and acylation of the hydroxy groups. y-Cyclodextrin substituted with 3-O-butyl and 2,6-di-O-pentyl residues was found to resolve most of the common amino acid enantiomers as the N-TFA methyl esters. Histidine enantiomers were only partially separated and arginine did not elute from the column. However, proline, 3,4-dihydroxyproline and pipecolic acid enantiomers were resolved, strongly suggesting that hydrogen bonding is not involved in the separatory mechanism. Atypical amino acids such as N-methyl and ^-amino acids were also resolved.

More recently (1994), Abe explored capillary columns coated with four types of cyclodextrin derivatives of 6-O-tert-butyldimethylsilyl-2,3-di-O-acetyl or n-butyl-^- and y-cyclodextrin. Depending on the phase, all proteic amino acid enantiomers except for those of tryptophan were resolved as the N-TFA isopropyl esters. Variants such as 2,6-di-O-pentyl-3-O-propionyl-y-cyclodextrin have also been used to separate a number of amino acid enantiomers. Molecular modelling has positively correlated the GC elution order of proline derivatives on 2,6-di-O-methyl-3-O-trifluoroacetyl-^-cyclodextrin with the energies of the host-guest complexes.

Several satisfactory methods now exist for the resolution of amino acid enantiomers. Typically, 0.1% of a minor enantiomer can be precisely determined and, depending on the context and the specific method used, it is possible to assay as little as 0.01% or less.

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