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Interactions between dansyl amino acids and human serum albumin using high-performance liquid chromatography: mobile-phase pH and temperature considerations.

Authors
  • Peyrin, E
  • Guillaume, Y C
  • Guinchard, C
Type
Published Article
Journal
Analytical chemistry
Publication Date
Dec 15, 1997
Volume
69
Issue
24
Pages
4979–4984
Identifiers
PMID: 9414611
Source
Medline
License
Unknown

Abstract

The reversed-phase liquid chromatography (RPLC) retention mechanism of a series of dansyl amino acids was investigated over a wide range of mobile-phase pH and column temperatures using human serum albumin (HSA) as a chiral stationary phase. Thermodynamic constants for the transfer of a solute from the mobile to the HSA stationary phases were determined. Different van't Hoff plot shapes were observed with different mobile-phase pH values, indicating a change in the retention mechanism. Enthalpy-entropy compensation revealed that the solute retention mechanism was independent of the compound molecular structure, the same at four pH values (5.5, 6, 6.5, and 8), but changed at pH = 7 and 7.5. Differential scanning calorimetry was used to show phase transition in the HSA stationary phase at pH = 7 and 7.5. A new theory was presented to explain that the HSA protein structure balance between a disordered and an ordered solid-like state. Variations of column temperature and mobile-phase pH tend to cause this phase transition between these two states, explaining the observed thermodynamic constant variations with pH and temperature.

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