Effect of spacer arm length on protein retention on a strong cation exchange adsorbent

The retention of five proteins was compared on a set of three strong cation exchange adsorbents that differed in spacer arm chemical structure and length. The adsorbents included a commercial product, Amersham Biosciences SP Sepharose Fast Flow, containing a six-carbon spacer between the agarose matrix and the anionic ligand, and two custom-prepared materials. One of the custom adsorbents contained a spacer of about half the length of the SP Sepharose Fast Flow, and the other contained no spacer arm. The adsorbent with no spacer arm was found to be significantly more retentive for all of the test proteins examined, in both isocratic and gradient elution tests. Reducing the spacer arm length by half resulted in increased retention for four of the five proteins, but this increase was less than what was observed when the spacer arm was eliminated. Retention increases were obtained without increasing the density of the anionic charge groups and appear to result from an enhancement of electrostatic or secondary nonelectrostatic interactions, or both. The results indicate that spacer arm length may be a useful variable in manipulating stationary-phase retention properties.