Peptides underivatized separation

Although the majority of reports of macrocycles in analytical chromatography have involved ligand association with the stationary phase, their use as mobile phase constituents has also been investigated. Lamb and Drake [11] showed that addition of water-soluble crown ethers to the mobile phase altered the retention of alkali metal cations on an underivatized reversed phase column. Nakagawa et al. [63-66] also used crown ether-containing mobile phases in the separation of protonated amines, amino acids and peptides, and [1-lactam antibiotics. 

Adoubel, A. et al. Separation of underivatized small peptides on a porous graphitic carbon column by ion-pair chromatography and evaporative light scattering detection. J. Liq. Chromatogr. Rel. Technol. 2000, 23, 2433-2446. 

Figure 6 (A) On-line enzyme digestion of propyl 4-hydroxylase subunit by S. aureus V8. Ce separation of underivatized peptides monitored at 214 nm, (B) Same as 6A except peptides derivatized on-line with FITC.

The first example of alkylation of underivatized xanthine with chloroacetic acid to yield a separable mixture of N-7- and N-9-(methylenecarboxyl)xanthme and conversion to a peptide nucleic acid monomer have been described <05SL1442>. 

Underivatized peptides separation with quantitative yields... 

The CSPs can be operated in normal as well as reversed-phase mode and additionally in SFC mode. Their main field of application is the separation of underivatized amino acids (Berthod et al., 1996) and small peptides (Ilisz, Berkecz, and Peter, 2006). As especially underivatized amino acids and small chiral acids are difficult to be separated there has been some effort put into an integrated separation approach by first using SMB-chromatography to obtain an enantiomerically enriched fraction and later obtaining the final purity by fractionated crystallization. 

A method for interfacing the peptides from the chromatographic separation to some form of spectroscopic detection (primarily MS, due to its superior sensitivity to underivatized peptide chains or amino acids, and the high information content of mass specta, especially when employed as multistage MS ). 

Chirobiotic T exhibits a unique chiral selectivity for a number of classes of solutes. It is particularly useful for resolving the enantiomers of underivatized amino acids, N-derivatized amino acids and small peptides but is widely applicable to many classes of compounds. Chirobiotic T can also be used with a very wide range of solvents, and can be used with the polar organic system, in the reversed phase mode and in the normal phase mode. In addition, the chiral selectivity changes greatly from one development mode to another. The optimization protocol as seen in the chart Chirobiotic T is very similar to that used for Chirobiotic V and the same adjustments are made to optimize the separation. 

The extent of HPLC separation between derivatized and underivatized peptides depends on the length of the polypeptide chain, the type of stationary phase used for separation (e.g. C4-C18), and the elution conditions. When using C18 columns the difference in retention time between the two species varies from about 10 min for peptides of 40-60 residues to about 5 min in the case of larger sequences. A typical elution profile for a 100-residue polypeptide is shown in Figure 1. As the length of the chain increases, a progressive reduction in the separation between labelled and unlabelled chains is observed. Unpublished results showed that beyond about 150 residues, the retention times of a probe-peptide adduct and underivatized chains are very similar. However, for sequences up to 120-130 residues, the method is sequence independent and can therefore be standardized. 

Due to the very limited solubility of underivatized peptides and proteins in nonpolar solvents, it is unlikely that NP separations for peptides and proteins will ever become an important technique. However, derivatized amino acids and short-chain peptides (seven amino acid residues or fewer) have been successfully chromatographed. Dia-stereomeric separations are done easily on the many chiral stationary phases currently available. In these cases, IPA is frequently used as the low-level mobile phase... 

---