Peptides enantioseparation

As previously observed, for most free amino acids and small peptides unbuffered hydroorganic mixtures are enough to yield good enantioseparations however, for some bifunctional amino acids and most other compounds, an aqueous buffer is usually necessary to enhance resolution. TEAA and ammonium nitrate are the most effective buffer systems, while sodium citrate was also effective for the separation of 2-arylpropionic acids (profens) on vancomycin CSPs [78], and ammonium acetate is the most widely used and appropriate in view of LC-MS applications (see Section 2.3.1.4). Small changes in ammonium acetate concentration of MeOH-water (90 10) mobile phases scarcely affected retention and— to a lesser extent—enantioselectivity of carnitine derivatives [61]. 

The major area of applications of cinchona 9-O-carbamate type CSPs embraces enantioseparation of diverse N-protected amino acids and peptides, carboxylic, phosphonic, and phosphoric acids as well as other acidic compounds. 

The most extensive studies on molecular-imprinted polymers as stationary phases for enantioseparations were conducted using amino acids and their derivatives as the template, but many other classes of compounds have been studied, including peptides, p-blockers, and nonsteroidal anti-inflammatory drugs (see Table 3). Examples of separations are reported in Figs. 6-8. 

As a result, the baseline enantioseparation was obtained, as shown in Figure 19.1a. Monomeric Pro is one of these rare amino acids, which develop yellow (and not bluish) color when visualized with ninhydrin. l-Pto used as an external standard (Figure 19.1b) confirmed the identity of the lower yellow spot number 2 as enantiomer L and the upper yellow spot number 3 as enantiomer d, as shown in Figure 19.1a. The respective Rp values were 0.57 0.02 and 0.74 0.02. Brownish-purple spot number 1 (Rp = 0.32 0.02 Figure 19.1) apparently originates from the Pro-derived peptides and it is also fully separated from the monomeric l-Pto spot number 2. The presence of peptides in the two freshly prepared Pro samples wimesses to rapid peptidization of this amino acid (although contamination of the commercial monomeric DL-Pro and L-Pro samples with peptides cannot be excluded). 

Sajewicz, M., Matlengiewicz, M., Juziuk, M., Penkala, M., Weloe, M., Schulz, M., and Kowalska, T. 2013. Thin-layer chromatographic evidence of proline peptidization in solution and its thin-layer chromatographic enantioseparation, J. Liq. Chromatogr. Relat. TechnoL, 36 2497-2511. 

Berthod A, Liu Y, Bagwill C, Armstrong DW (1996) Facile liquid chromatography enantioseparations of native amino acids and peptides using a teicoplanin chiral stationary phase. J Chromatogr A 731 123-137... 

The first cellulose enantioseparation was of a racemic amino acid by paper chromatography [8]. Early cellulose TLC smdies were aimed at repeating paper chromatography enantiomer separations more quickly and with better resolution. Cellulose TLC has mostly been used for amino acids, their derivatives, and peptides and the following are examples of successful enantioseparations. Aromatic amino acids were separated on polyester-backed 20 x 20 cm plates by elution with methanolic aqueous 0.1 M HCl (A) or ethanol/pyridine/water (1 1 1) (B). Typical respective R values were 0.75/0.81 for l- and o-tyrosine in mobile phase A and 0.37/0.46 for L- and D-5-methyl tryptophan in B [9,10]. 

The type of structure of chiral compounds, as well as that of the stationary phase, could strongly affect the separation. LeFevre et al. [28] investigated the effect of racemic dansyl-amino acids structure on the resolution using p-CD as a CMPA in the RP-TLC system. The same authors confirmed stereochemistry of the peptide-derived amino acids and detected a partial racemization of several amino acids occurring during hydrolysis of enkephalin, gramicidin, achatin-I, and cyclosporin by using /3-CD as a CMPA and RP-TLC [25]. In some cases, /3-CD as a CMPA can improve chiral resolution of cellulose and can be used for enantioseparation of some flavanones [16], amino acids, and oxazolidinone [15], as well as of budesonide in pharmaceuticals [29], and some aromatic amino acids, and aromatic amino alcohols [19]. 

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