Intraparticle mass transfer in high-speed chromatography of proteins.

The effect of intraparticle mass-transfer resistances on the peak shape at high flow velocities in the range currently used in high-speed protein chromatography was investigated both theoretically and experimentally. The asymmetry of the protein bands under these conditions was quantified 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 the mass-transfer characteristics of a given chromatographic sorbent from the variation in peak asymmetry with reduced velocity. The method is shown to be most useful when the number of theoretical plates is between 3 and 300, which is the regime where peak asymmetry is prevalent. Measurements by isocratic elution under nonretained conditions were made on three chromatographic sorbents, each representing a general class of stationary phase configuration, i.e., gigaporous, mesoporous, and gel-filled gigaporous particles. Mass-transfer parameters were evaluated using the new method based upon the variation of the peak asymmetry with the fluid velocity. For the purpose of comparison, column mass transfer parameters were also evaluated from the variation in the reduced plate height with reduced velocity, a method most useful when the peak asymmetry is small and remains constant in the velocity range investigated. It is shown that the two methods are complementary and yield, within experimental error, the same intraparticle diffusion parameters. It was demonstrated using these methods that the diffusional behavior and the first moments of unretained eluites for the gel-filled gigaporous column packing correspond to a sorbent particle where eluites diffuse through liquid-filled pores containing a uniform distribution of solid cylinders, with the cylinders representing the polymer chains in the gel material. Similarly, the methods were used to verify that, at high flow rates, intraparticle convection can contribute substantially to the rate of intraparticle mass transfer in gigaporous column packings.