Characteristics of Superdex® prep grade media for gel filtration chromatography of proteins and peptides

Abstract

Some chemical and chromatographic properties of Superdex® 30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade, all new composite media for separation of peptides and proteins, were investigated. The selectivity of Superdex 30 prep grade was studied by constructing calibration graphs ( K av vs log M r) of peptides and poly(ethylene glycol) (PEG) standards. The influence of the sodium chloride concentration of the mobile phase on the distribution coefficient of peptides demonstrates that Superdex 30 prep grade contains small amounts of charged groups. Titration results show that the amount of negatively charged groups (carboxyl groups) was about 1 μmol/ml gel for all three investigated Superdex media. A homologous series of n-alphatic (n=4–8) alcohols was used to study the hydrophobic interaction behaviour of Superdex media. The effects of temperature, salt concentration and methanol on K av of the alcohols were used to characterize the elution mechanism. Hydrophobic interactions increased with the amount of dextran coupled to the agarose bead. This means that of the three Superdex media, Superdex 200 prep grade is the least hydrophobic. The functional stability of Superdex 30 prep grade was studied by testing the separation of PEG standards and peptides during repeated cleaning-in-place (CIP) treatments with 1·0 m sodium hydroxide solution and 0·1 m hydrochloric acid. The separation behaviour of the PEG standards and the peptides was unaltered after the medium had been treated for a total contact time of 400 h in 0·1 m hydrochloric acid. However, a slight increase in retention volume was observed after treatment in basic conditions. This effect is a result of hydrolysis of covalently bonded dextran in the agarose pores. In basic conditions, the highest carbohydrate (dextran) leakage was observed for Superdex 30 prep grade and the lowest for Superdex 200 prep grade. In acidic conditions it was found that for all Superdex media, dextran and low amounts of 5-(hydroxymethyl)-2-furaldehyde (5-HMF) were released but the separation characteristics were unaltered after treatment in 0·1 m hydrochloric acid. The highest 5-HMF leakage was observed for Sepharose High Performance (the agarose bead on which Superdex media are based). The lowest amount was released from Superdex 30 prep grade.