Separation of proteins by high-performance anion-exchange chromatography.
The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000302:isolation & purification; D007782:Lactoglobulins / Q000302:isolation & purification; D009711:Nucleotides / Q000302:isolation & purification; D009961:Orosomucoid / Q000302:isolation & purification; D010047:Ovalbumin / Q000302:isolation & purification; D011094:Polyethyleneimine; D011506:Proteins / Q000302:isolation & purification*
D003574:Cytochrome c Group; D007782:Lactoglobulins; D009711:Nucleotides; D009961:Orosomucoid; D011506:Proteins; D011094:Polyethyleneimine; D010047:Ovalbumin