Separation of double-stranded DNA in conventional and isoelectric buffers: Studies on stability and separation performance

Soffia Magnusdottir, Cecilia Gelfi, Mahmoud Hamdan, Pier Giorgio Righetti

Research output: Contribution to journalArticle

Abstract

In the capillary electrophoresis of double-stranded DNA in isoelectric buffers, worsening of resolution was observed in electropherograms as a function of time passed from the preparation of the separation solution, which consisted of 0.7% hydroxypropylcellulose, HPC, M(r) 106, diluted in 150 mM histidine buffer. The DNA standards used were: kilobase pair-ladder, Marker V and Marker VI. In order to understand what happens in the histidine-HPC solution with ageing, the absorbance spectrum (200-500 nm), the conductivity and the pH of the solutions as a function of time were monitored. Fresh His gave a distinct peak at 206 nm. For all the solutions a significant diminution in the maximum absorbance value at 206 nm was observed as a function of ageing, with the concomitant appearance of a peak at 278 nm as the solutions became older. Also the conductivity increases dramatically with the ageing of the solutions and seemed to reach a plateau after ca. 40 days. In concomitance with the conductivity increments with time, the pH of the His solution (isoelectric point, pI=7.6) grew slowly up to pH 7.9; these combined data indicated that a new species contributing to the conductivity and altering the pH was formed from the His molecule, suggesting that His degraded in time. When the dipeptide His-Gly was used instead, a similar ageing phenomenon was observed, but with much reduced kinetics. Mass spectrometry, coupled to RP-HPLC, detected, in aged His solutions, in addition to intact His, two main degradation products: a 110.1 u species and a 93.2 u compound. The mass of the former coincides with the protonated species derived from the formation of a Schiff base on the α-amino group of His and subsequent decarboxylation without transformation of the final Schiff base into a chetonic group (a histamine-like molecule terminating with an imino, rather than with an amino group). Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)87-98
Number of pages12
JournalJournal of Chromatography A
Volume859
Issue number1
DOIs
Publication statusPublished - Oct 22 1999

Keywords

  • Buffer composition
  • DNA
  • Isoelectric buffers

ASJC Scopus subject areas

  • Analytical Chemistry

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