Adsorption Chromatography
In this chromatography, the protein or enzyme solution suspected to contain other proteinaceous impurities, is passed through a column of inert material packed in a glass or steel tube. Most commonly used column materials include finely divided solids such as charcoal, silica, alumina, calcium phosphate, hydroxyapatite, etc. The effluent solution is continuously collected in small fractions of 1.0 to 2.0 ml. The protein in each fraction is estimated by measuring the absorption at 280 nm using a UV spectrophotometer. The enzyme is also assayed in each fraction. Various spleen enzymes such as basic RNAase, acidic RNAase, acidic DNAase, phosphodiesterase, phosphomonoesterase, etc. are often separated from each other using adsorption chromatography. For large scale chromatographic. separation of enzymes, the process is accelerated by using motors and other mechanical devices for packing the column, for loading the enzyme on the column and for eluting the enzyme.