Effect of the Spacer Arm in Affinity Chromatography: Determination of Adsorption Characteristics and Flow Rate Effect

Abstract

The effect of the spacer arm in affinity chromatography based on the adsorption equilibrium of l-asparaginase on activated Sepharose 4B with aliphatic diamines (1,4-diaminobutane−1,8-diaminooctane) as the spacer arms and l-(+)-chlorosuccinamic acid as the ligand was studied in a batch reactor and in a packed bed. The adsorption equilibrium constants were determined for all adsorbents. A maximum in the adsorption was observed when 1,6-diaminohexane was used as the spacer arm under optimum adsorption conditions (pH 8.6, 298 K, and 0.05 M NaCl) (Martín del Valle, E. M.; Galán, M. A. Ind. Eng. Chem. Res. 2001, 40 (1), 369−376). Partition coefficients were also obtained in the batch reactor. The values of these coefficients allowed us to quantify the amounts of specifically adsorbed, nonspecifically adsorbed, and occluded enzyme. A pulse−response chromatographic method and statistical moments were used to determine the adsorption equilibrium constants in the packed bed. The values of the adsorption equilibrium constants for different adsorbents were compared with those obtained previously in the batch reactor taking into account the mass-transfer resistance inside the particles of the adsorbent (partition coefficient). The difference in the values obtained was lower than 5−8%. In addition, the effect of the flow rate on adsorption was studied for different adsorbents with spacer arms of different lengths. When the flow rate was increased, the value of the adsorption equilibrium constant increased. This effect was observed when 1,8-diaminooctane was used as the spacer arm