Cross-linked crystals of glucose isomerase (CLGI) were characterized as a liquid chromatographic separation material. The experiments were done with crystals having an average diameter of 83 mu m. Porosity (epsilon(p)) and pore size distribution of the CLGI crystals were measured with size exclusion chromatography using D2O and polyethylene glycols as probes. CLGI material was capable of separating (1000 g/mol polyethylene glycols. Fifty two percent (epsilon(p) = 0.47) of the total crystal volume was in pores. Pore size measurement showed that CLGI crystals were microporous material, having an average apparent pore diameter of 29 +/- 0.08 Angstrom. CLGI material separated n-alcohols C-1 to C-8 based on the hydrophobic interaction between the protein material and the carbon chain of the alcohols. Height equivalent to a theoretical plate (HETP, in millimeters) ranged from 1.6 to 0.89 for the C-1 to C-7 n-alcohol series. Despite the large crystal size, CLGI as a chirally active phase effectively separated D- and L-arabitol (R-s = 0.58) and showed potential for chiral separation of amino acids.