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Study of retention and separation of peptides in HPLC
Boudová, Hana ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
This diploma thesis deals with the study of retention and separation of structurally different peptides and digest products of cytochrome c using reverse phase and mixed modes of liquid chromatography. As the model peptides, five unmodified dipeptides, nine dipeptides containing a protective benzoyl group on the N-terminal amino acid and eight therapeutic peptides containing five to ten amino acids were selected. The chromatographic behaviour of the model peptides was tested on three different columns: XBridge C18, XSelect CSH C18 and Atlantis Premier BEH C18 AX under different conditions. The influence of the pH of the aqueous part of the mobile phase, i.e. 2,1; 3,0; 4,7 and 6,8, and the amount of acetonitrile in the mobile phase on retention and separation of model peptides was tested. The effect of the concentration of ammonium acetate buffer, pH = 6,8 in the mobile phase on the retention and peak symmetry in the concentration range of 10-50 mmol L-1 was assessed. The chromatographic conditions for the baseline separation of 20 different model peptides were optimized. The conditions were as follows: XSelect CSH C18 column, mobile phase composed of acetonitrile/10mM ammonium acetate, pH = 6,8 (v/v) under gradient elution. The suitable chromatographic conditions for the analysis and identification...
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