Preparative separation of proteins and enzymes in the mean molecular-weight range of 10,000–100,000 LiChrosorb diol® packing by high-performance size-exclusion chromatography

Abstract

LiChrossorb Diol® packing has been to be well-suited for the separation of proteins and enzymes according to a size-exclusion mechanism in a mean molecular-weight (MW) range between 10,000 and 100,000. Loadability of a small bore column of 6 mm I.D. (A) and a large-bore column of 23.5 mm I.D. (B), both of 250 mm in length, were examined. Defining a 20% decrease of the number of theoretical plates as loadability limit the volume load at constant mass of chymotrypsinogen as representative test solute was ≈ 100 μl for column (A) and 1500 μl for column B at 0.78 · 10 -5 g/g of packing for column A and 0.78 · 10 -6 g/g of packing for column B, respectively. Mass load to constant injection volume (20 μl for column A and 200 μl for column B, respetively) was ≈ 30 mg/ml or ≈ 1.10 -4 g/g of packing for both columns. As demonstrated in one instance, 75 mg of a mixture of 3 proteins which differ in MW by a factor of 2, could be separated in one run.