INTRAPARTICLE MASS-TRANSFER IN HIGH-SPEED CHROMATOGRAPHY OF PROTEINS

The effect of intraparticle mass-transfer resistances on the peak shap e at high flow velocities in the range currently used in high-speed pr otein chromatography was investigated both theoretically and experimen tally. The asymmetry of the protein bands under these conditions was q uantified by the difference between the first moment and the retention volume of the peak apex, this being much easier to determine than the peak skewness. A general method is introduced for the evaluation of t he mass-transfer characteristics of a given chromatographic sorbent fr om the variation in peak asymmetry with reduced velocity. The method i s shown to be most useful when the number of theoretical plates is bet ween 3 and 300, which is the regime where peak asymmetry is prevalent. Measurements by isocratic elution under nonretained conditions were m ade on three chromatographic sorbents, each representing a general cla ss of stationary phase configuration, i.e., gigaporous, mesoporous, an d gel-filled gigaporous particles. Mass-transfer parameters were evalu ated using the new method based upon the variation of the peak asymmet ry with the fluid velocity. For the purpose of comparison, column mass transfer parameters were also evaluated from the variation in the red uced plate height with reduced velocity, a method most useful when the peak asymmetry is small and remains constant in the velocity range in vestigated. It is shown that the two methods are complementary and yie ld, within experimental error, the same intraparticle diffusion parame ters. It was demonstrated using these methods that the diffusional beh avior and the first moments of unretained eluites for the gel-filled g igaporous column packing correspond to a sorbent particle where eluite s diffuse through liquid-filled pores containing a uniform distributio n of solid cylinders, with the cylinders representing the polymer chai ns in the gel material. Similarly, the methods were used to verify tha t, at high flow rates, intraparticle convection can contribute substan tially to the rate of intraparticle mass transfer in gigaporous column packings.