Band broadening caused by the multiple labeling of proteins in micellar electrokinetic chromatography with diode laser-induced fluorescence detection

Peng Jing, Takashi Kaneta, Totaro Imasaka

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

When a labeling reagent is used, in the determination of proteins by capillary electrophoresis with laser-induced fluorescence detection, the multiple labeling of proteins frequently occurs, which can degrade the separation efficiency. In order to understand the influence of the multiple labeling of proteins on separation efficiency, the band broadening caused by a labeling reaction between bovine serum albumin (BSA) and a cyanine fluorescent dye (Cy5) was investigated using micellar electrokinetic chromatography in conjunction with diode laser-induced fluorometry. With the aid of an internal standard, methylene blue, the height equivalent to the theoretical plate (HETP) ratio of BSA to methylene blue was used as an indicator for band broadening under optimum separation conditions. Labeling conditions, including reaction buffer pH, reaction time, and initial concentration of Cy5 to bovine serum albumin, were found to influence the HETP ratio. The separation efficiency for the labeled protein was degraded by experimental conditions employed in the labeling, which indicates an increase in the heterogeneity of the final products.

Original languageEnglish
Pages (from-to)281-287
Number of pages7
JournalJournal of Chromatography A
Volume959
Issue number1-2
DOIs
Publication statusPublished - Jun 14 2002
Externally publishedYes

Keywords

  • Band broadening
  • Capillary electrophoresis
  • Column efficiency
  • Derivatization
  • Proteins

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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