Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

End-group analysis and stepwise degradation of proteins and peptides
Determination of N-terminal groups by the 2,4-dinitrofluorobenzene method
Identification of DNP-amino acids by thin-layer chromatography

Thin-Layer Chromatography is successfully used to identify DNP-Amino Acids. Compared to paper chromatography, this method is more sensitive and less time-consuming. Mixtures are most commonly separated on silica gel using the following solvent systems [2]: a) toluene–pyridine–Ethylene chlorohydrin–ammonia (0.8 M) (100 : 30 : 60 : 60); this system is heterogeneous, with the upper phase used for chromatography and the lower phase for adsorbent equilibration; b) chloroform–benzyl alcohol–acetic acid (70 : 30 : 3); c) benzene–pyridine–acetic acid (80 : 20 : 2); d) chloroform–methanol–acetic acid (95 : 5 : 1). Approximately 0.2–0.5 µg of DNP-amino acid is sufficient for identification. The Separation of DNP-amino acids by two-dimensional chromatography in various solvent systems is shown in Fig. 61.

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Fig. 61. Identification of DNP-amino acids by thin-layer chromatography on silica gel.

Solvent 1: toluene–pyridine–ethylene chlorohydrin–0.8 M ammonia (100 : 30 : 60 : 60). Solvent 2: chloroform–benzyl alcohol–acetic acid (70 : 30 : 3). Solvent 3: benzene–pyridine–acetic acid (80 : 20 : 2). Solvent 4: chloroform–methanol–acetic acid (95 : 5 : 1). DNA — dinitroaniline, DNP — dinitrophenol.



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