Sugaring out: A new method for removal of acetonitrile from preparative RP-HPLC eluent for protein purification

Pradip B. Dhamole, Prafulla Mahajan, Hao Feng

Research output: Contribution to journalArticle


Reverse phase-high pressure liquid chromatography (RP-HPLC) with an acetonitrile-water mixture as the eluent is widely used for purification of proteins. The separation of acetonitrile (ACN) in RP-HPLC eluent is important for protein recovery. Cooling below subzero temperature and salting out have been used to remove ACN, each with its limitations. In this work we have explored the use of sugaring-out, a new phase separation method developed at University of Illinois for the separation of ACN from a simulated preparative RP-HPLC effluent. The effect of glucose concentration, temperature, and initial amount of ACN in the effluent on phase separation was investigated. Results showed that a good phase separation can be achieved at near room temperature (18 °C). With the optimized conditions, we found that more than 60% (w/w) of ACN was removed and more than 95% (w/w) of water-soluble proteins (bovine serum albumin, trypsin, and pepsin) were recovered.

Original languageEnglish (US)
Pages (from-to)1672-1676
Number of pages5
JournalProcess Biochemistry
Issue number10
StatePublished - Oct 1 2010



  • Acetonitrile
  • Liquid-liquid extraction
  • Proteins
  • Solvent extraction
  • Sugaring-out

ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

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