Separation of the valence intermediates of human haemoglobin by high-performance chromatofocusing

Ezio Bolzacchini, Isabella Fermo, Ermanna Rovida, Roberto Colombo, Michele Samaja

Research output: Contribution to journalArticlepeer-review


Investigations into the properties of haemoglobin often require the isolation of the valence intermediates (αo)2β+)2 and (α+ βo 2)2. Chromatofocusing with an anion-exchange gel (Mono PTM; Pharmacia, particle size 10 μm) in an HR5/20 column at various temperatures (10-25°C) provides an excellent method for this task. A linearly decreasing pH gradient (8 to 7, generated by Polybuffer 96, Pharmacia) eluted sequentially the species methaemoglobin, (αo 2β+)2, (α+ βo 2)2 and oxygenated haemoglobin. Calibration graphs help in quantitative analyses. This method is simpler and less time consuming and provides a similar or even better resolution than the traditional ion-exchange or isoelectric focusing methods.

Original languageEnglish
Pages (from-to)233-237
Number of pages5
JournalJournal of Chromatography A
Issue numberC
Publication statusPublished - Jun 26 1987

ASJC Scopus subject areas

  • Analytical Chemistry
  • Clinical Biochemistry
  • Molecular Medicine


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