Farnan D, Frey D D, Horváth C
Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520, USA.
Biotechnol Prog. 1997 Jul-Aug;13(4):429-39. doi: 10.1021/bp970044k.
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.
在理论和实验两方面研究了在高速蛋白质色谱当前使用的流速范围内，颗粒内传质阻力对峰形的影响。在这些条件下，蛋白质条带的不对称性通过峰顶点的一阶矩与保留体积之间的差异来量化，这比峰偏度更容易确定。介绍了一种从峰不对称性随折合流速的变化来评估给定色谱吸附剂传质特性的通用方法。当理论塔板数在3至300之间时，该方法显示出最为有用，这正是峰不对称普遍存在的范围。在非保留条件下通过等度洗脱对三种色谱吸附剂进行了测量，每种吸附剂代表一类通用的固定相构型，即大孔、介孔和凝胶填充大孔颗粒。基于峰不对称性随流体流速的变化，使用新方法评估传质参数。为作比较，还从折合塔板高度随折合流速的变化来评估柱传质参数，当峰不对称性较小且在所研究的流速范围内保持恒定时，该方法最为有用。结果表明，这两种方法是互补的，并且在实验误差范围内得出相同的颗粒内扩散参数。使用这些方法证明，对于凝胶填充大孔柱填料，未保留洗脱物的扩散行为和一阶矩对应于一种吸附剂颗粒，其中洗脱物通过含有均匀分布的实心圆柱体的液体填充孔扩散，这些圆柱体代表凝胶材料中的聚合物链。同样，这些方法用于验证，在高流速下，颗粒内对流可对大孔柱填料中颗粒内传质速率有显著贡献。
