Ion-exchange and hydrophobic-interaction high-performance liquid chromatography of proteins. A practical study

A number of commercially available columns for ion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC) have been tested, ranging from very fast columns with low capacity to preparative columns. The experiments represent a selection of column applications likely to occur in a biochemical laboratory. The use of several ion-exchange columns for separation of serum and membrane proteins has been demonstrated. Columns with a silica gel matrix and with a polymer matrix have proved to be of equal value in experiments with water-soluble proteins. In experiments with hydrophobic membrane proteins the choice of the column depends on the protein to be isolated. A combination of a cation-exchange and an anion-exchange column as a tandem as well as a mixed-bed column, were applied for the separation of serum proteins and for the isolation of a 175,000-dalton membrane glycoprotein. One problem of hydrophobic-interaction HPLC is the poor solubility of some proteins, for example serum proteins at high salt concentrations. This difficulty can be overcome by the use of columns with higher hydrophobicity which require lower initial salt concentrations. Less hydrophobic columns have been shown to separate hydrophobic membrane proteins by a combination of salt and detergent gradients.