Replacement of acetonitrile by ethanol as solvent in reversed phase chromatography of biomolecules.
Acetonitrile, which is a by-product of acrylonitrile synthesis, is the commonly used solvent in ion-pair reversed phase chromatography. In consequence of the decreasing demand for acrylonitrile due to the financial crisis, a worldwide shortage of acetonitrile is observed. Therefore, the aim of this study was to establish ion-pair reversed phase chromatographic assays using alternative eluents for acetonitrile and to decrease costs incurred hereby. We compared the performance of ion-pair reversed phase chromatography using acetonitrile with the alternative eluents methanol, ethanol and n-propanol, using monolithic reversed phase C5 as well as C18 chromatography columns. We used triethylammonium acetate (TEAA) and tetrabutylammonium sulfate (TBA) as representative cationic ion-pair reagents and trifluoroacetic acid (TFA) as representative anionic ion-pair reagent. For covering a large field of applications, we fractionated representative low, middle and high-molecular weight biomolecules, in particular dinucleoside polyphosphates, peptides, proteins and tryptic digested human serum albumin. Whereas the chromatographic characteristics of both methanol and n-propanol were partly insufficient, ethanol was characterised equally or partly even better in the matter of elution strength and separation quality compared to the eluent water-acetonitrile. In conclusion, ethanol is an appropriate alternative for acetonitrile in ion-pair reversed phase chromatography of biomolecules.
Charité-CBF, Medizinische Klinik IV, Hindenburgdamm 30, D-12200, Berlin, Germany.
F
F
Brettschneider
V
V
Jankowski
T
T
Günthner
S
S
Salem
M
M
Nierhaus
A
A
Schulz
W
W
Zidek
J
J
Jankowski
D000097:Acetonitriles; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation*; D000431:Ethanol; D006801:Humans; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D012997:Solvents
D000097:Acetonitriles; D010455:Peptides; D011506:Proteins; D012997:Solvents; D000431:Ethanol; C032159:acetonitrile