Amino acids separation phase

The first chiral phases introduced for gas chromatography were either amino acid esters, dipeptide, diamide or carbonyl-bis(amino acid ester) phases [721,724,756-758]. In general, these phases exhitdted poor thermal stability and are infrequently used today. Real interest and progress in chiral separations resulted from the preparation of diamide phases grafted onto a polysiloxane backbone. These phases were thermally stable and could be used to prepare efficient open tubular columns [734,756,758-762]. These phases are prepared from commercially available poly(cyano-propylmethyldimethylsiloxanes) or poly (cyanopropylmethylphenyl-... 

The best correlation equation for racemic a-amino acids separated on a-cyclodextrin column using 1% triethyl-amine acetate (pH=5.1) as a mobile phase is ... 

The process of separation and quantitation of amino acids has been automated. In one automated method, a single cation exchange resin column separates all the amino acids in the protein hydrolysate. The analyzer is capable of detecting as little as 1-2 nmol of an amino acid and a complete analysis can be obtained in about 4 hours. In newer procedures, the complete analysis can be performed in about Ihour and permit detection of as little as 1-2 nmol of an amino acid. Picomole amounts of amino acids can be determined when the separated amino acids are coupled to fluorescent reagents such as o-phthalaldehyde. Amino acid separation and quantitation can also be accomplished by reverse-phase high-pressure liquid chromatography of amino acid derivatives—a rapid and sensitive procedure. 

Selective Crystal Dissolution. An effective method for distinguishing bulk (lattice trapped) versus surface impurities involves the selective dissolution of a crystal sample while testing the liquid and/or crystalline phases for relative purity. In this technique, a small sample of crystals of a narrow size fraction is washed with successive small amounts of clean solvent until most of the crystalline phase is dissolved. The filtrate and/or crystalline phase are analyzed after each washing to discern whether impurities reside predominantly at the surface, or are more evenly distributed throughout the crystalline phase. The general approach is described by Narang and Sherwood (1978) for quantifying caproic acid incorporation in adipic crystals, and by Addadi et al. (1982) for amino acid separations. 

Peptide chromatography can also be carried out on a reverse-phase column by the ion-pairing technique. The ion pair has increased affinity for the support and allows small hydrophilic peptides to be retained. It also offers a different selectivity and has been reported to improve resolution. Heptane-sulfonic acid and tetrabutylammonium phosphate were used with large peptides (Hancock et al, 1978b), and trifluoroacetic acid was used for both peptides and proteins (Bennett et al, 1979). Amino acid separations have also been carried out on a reverse-phase column using anionic surfactants for ion pairing (Kraak et al, 1977). 

The model chiral phases, iV-(tert-butylaminocarbonyl)-(5 )-valylaminobutane (Phase 1) and (J )-l-(a-naphthyl)ethylaminocarbonyl-glycylaminobutane (Phase 2) are shown in Figure 8.1. Phase 1 was used for the enantioseparation of N-acetylamino acid methylesters and [R]- and (5)-4-nitrobenzoyl amino acids, but Phase 2 could not separate these enantiomers. The enantiomer selectivities of N-(5 )-l-(a-naphthyl)ethylaminocarbonyl-(5)-valylaminobutane (Phase 3), N-(5 )-l-(a-naphthyl)ethylaminocarbonyl-(P)-valylaminobutane (Phase 4), N-[R]-1-(oc-naphthyl)ethylaminocarbonyl-(R)-valylaminobutane (Phase 5), and N-[R)-l- a-naphthyl)ethylaminocarbonyl-(5 )-valylaminobutane (Phase 6), which all have two chiral centers, were examined by computational chemical analysis. The structures of model Phases 3-6 are also shown in Figure 8.1. 

Reversed-phase LC is becoming increasingly popular for the separation of amino acids. Reversed-phase columns are readily available commercially and exhibit higher efficiencies than most commercially available ion-exchange columns. They are also compatible with aqueous samples, since water is generally a major component of the mobile phase. Therefore, it is not necessary to employ additional sample preparation steps in order to produce a sample in a nonaqueous environment. 

The problem of amino acid separation was the starting point for the development of PC. Separation was considered first to be based on partition of the substances between water, bound to cellulose by imbition causing swelling, and a mobile phase, immiscible with water (e. g., phenol saturated with water). This attitude has changed somewhat since it was found that single-phase solvents could also be used with these, the less mobile liquid involved in the cellulose swelling can be regarded as a type of phase, separate from the more mobile solvent-hquid. A partial transition to Freundhch adsorption on the cellulose surface may even occur sometimes. 

We have carried out preliminary experiments with Alox for TLC (Firm 60) (a suspension of 20 g Alox in 60 ml water was spread with the usual spreader and the layer thus obtained was dried as for silica gel) they were unsatisfactory because of the slow rate of flow of the mobile phase, a mixture of n-butanol-acetic acid-water (80 + 20 + 20) 4 hours were required for a 10 cm run The amino acid separation was, however, comparable to that achieved on silica gel. 

Bhushan and Ali (1987) tested amino acid separations on silica gel layers impregnated with various metal salts. Bhushan and Reddy (1989) reported the separation of phenylthiohydantoin (PTH) amino acids on silica gel with new mobile phases. Laskar and Basak (1988) de.scribed a new ninhydrin-based procedure that produced different colors and good sensitivity for amino acid detection. Bhushan and Reddy (1987) reviewed the TLC of PTH amino acids. Gankina et al. (1989) described a unidimensional multistep silica gel HPTLC method for the separation and identification of PTH and dansylamino acids. Bhushan et al. (1987) developed numerous solvent systems for effective separations of 2,4-dini-trophenyl-(DNP) amino acids. Bhushan (1988) reviewed the TLC resolution of enantiomeric amino acids and their derivatives. Kuhn et al. (1989) reported the amino acid enantiomer separation by TLC on cellulose of d- and L-tryptophan and methyltryptophan. Guenther (1988) determined TLC-separated enantiomers by densitometry. 

The purpose of this experiment is to demonstrate TLC amino acid separations on a reversed-phase plate as described in Sherma et al. (1983). 

Sherma et al. (1983) reported that their amino acid separations on C g reversed-phase thin layers were similar to those previously aehieved on conventional silica gel and cellulose by Sleckman and Sherma (1982). Sherma et al. (1983) concluded that it may be difficult to predict the relative separation behavior of numerous compounds including amino acids on the basis of chemical notions of reversed - or normal -phase chromatography. 

Figure 12 Chromatographic resolution and analysis time for mobile phase gradients designed to produce different average capacity factor. Amino acid separations using an average capacity factor (k" of 23 (A), 11.6 (B), and 5.8 (C). The gradient amplitude (35-63%B), flow rate (0.3 pL/min), and injection volume (250 nL) for all the separations were held constant while the gradient slope was varied. Anal5d e and retention order the same as in Figure 9.

Paper chromatography has largely been replaced by thin-layer chromat< raphy (TLC), which differs from paper chromatography in that TLC uses a plate with a coating of solid material instead of filter paper. How the amino acids separate depend on the solid material and the solvent chosen for the mobile phase. 

FIGURE 6.3 Reversed-phase LC separation of AQC-labeled amino acids. Separation... 

The hydrophobic macroporous polystyrene beads are exceptionally chemically stable and are used for organic phase gel permeation chromatography products, Styragel (Waters) and PLgel (Polymer Laboratories) and reversed phase HPLC separations, PRP (Hamilton) and PLRP-S (Polymer Laboratories). It is possible to surface modify the particle to mask the hydrophobicity of the base polymer and produce ion exchange and hydrophilic materials, PI SAX, PL-GFC (Polymer Laboratories). In the microporous form after derivatisation to form strong cation exchangers these materials are commonly used for carbohydrate and amino acid separations, Aminex (BioRad). 

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