Peptides reverse-phase chromatograph

Various applications of PITC in analyses of acid hydrolysates of purified proteins and peptides (91) and of hydrolysates of feeds (92) have been described. Bidlingmeyer et al. (93) showed reversed-phase chromatographic analysis of free amino acids and hydrolysates of foods (soybean flour, mozzarella cheese, beer, and soy sauce) with formation of PITC derivatives to be a fast, reproducible method that presented a very good correlation with the results obtained by ion-exchange chromatography. 

In this context, a major impact has been the development of high-resolution reversed phase chromatographic (RP-HPLC) techniques for analytical and preparative purposes.t l In fact, with the discovery of reversed stationary phases and with applications to chromatographic resolution of amino acid and peptide derivatives,HPLC very soon emerged as the most powerful technique for synthetic peptides.P l... 

Fluorescamine (Fluram). Fluorescamine was developed from the study of ninhydrin chemistry as a reagent for detecting amino groups (Udenfriend et al., 1972) and has been successfully used in the reversed phase chromatographic analysis of proteins and peptides using an on-line post-column detection system. This reagent requires two separate pumps for the delivery of the mobile phase buffer and the reagent and this method may therefore be considered to be less... 

Reversed-phase high performance Hquid chromatography has come into use for estimating the purity of proteins and peptides as weU. However, before employed, a high performance Hquid chromatographic (hplc) profile of a given protein must be completely vaHdated (43). 

McKern, N. M., Edskes, H. K., and Shukla, D. D., Purificatiion of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic system, Biomed. Chromatogr., 7, 15, 1993. 

Mohammad, J, Jaderlund, B., and Lindblom, K., New polymer-based prepacked column for the reversed-phase liquid chromatographic separation of peptides over the pH range 2-12, J. Chromatogr. A, 852, 255, 1999. 

Analysis using reversed-phase chromatography at low pH was carried out under conditions typically used for 2DLC-MS/MS analysis of peptides. The mobile phases for other chromatographic conditions were also chosen to be compatible with MS detection, including SCX LC, where the peptides were eluted with volatile ammonium formate buffer. 

Chloupek, R.C., Hancock, W.S., Marchylo, B.A., Kirkland, J.J., Boyes, B.E., Snyder, L.R. (1994). Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples, n. Selectivity effects observed in the separation of several peptide and protein mixtures. J. Chromatogr. A 686, 45-59. 

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