Gritti Fabrice, Guiochon Georges
Department of Chemistry, University of Tennessee Knoxville, Tennessee 37996-1600, USA.
Anal Chem. 2009 Apr 1;81(7):2723-36. doi: 10.1021/ac8026299.
The mass transfer kinetics of human insulin was investigated on a 50 mm x 2.1 mm column packed with 1.7 microm BEH-C(18) particles, eluted with a water/acetonitrile/trifluoroacetic acid (TFA) (68/32/0.1, v/v/v) solution. The different contributions to the mass transfer kinetics, e.g., those of longitudinal diffusion, eddy dispersion, the film mass transfer resistance, cross-particle diffusivity, adsorption-desorption kinetics, and transcolumn differential sorption, were incorporated into a general mass transfer equation designed to account for the mass transfer kinetics of proteins under high pressure. More specifically, this equation includes the effects of pore size exclusion, pressure, and temperature on the band broadening of a protein. The flow rate was first increased from 0.001 to 0.250 mL/min, the pressure drop increasing from 2 to 298 bar, and the column being placed in stagnant air at 296.5 K, in order to determine the effective diffusivity of insulin through the porous particles, the mass transfer rate constants, and the adsorption equilibrium constant in the low-pressure range. Then, the column inlet pressure was increased by using capillary flow restrictors downstream the column, at the constant flow rate of 0.03 mL/min. The column temperature was kept uniform by immersing the column in a circulating water bath thermostatted at 298.7 and 323.15 K, successively. The results showed that the surface diffusion coefficient of insulin decreases faster than its bulk diffusion coefficient with increasing average column pressure. This is consistent with the adsorption energy of insulin onto the BEH-C(18) surface increasing strongly with increasing pressure. In contrast, given the precision of the height equivalent to a theoretical plate (HETP) measurement (+/-12%), the adsorption kinetics of insulin appears to be rather independent of the pressure. On average, the adsorption rate constant of insulin is doubled from about 40 to 80 s(-1) when the temperature increases from 298.7 to 323.15 K.
在填充有1.7微米BEH-C(18)颗粒的50毫米×2.1毫米色谱柱上，以水/乙腈/三氟乙酸（TFA）（68/32/0.1，v/v/v）溶液作为洗脱剂，研究了人胰岛素的传质动力学。将对传质动力学的不同贡献，例如纵向扩散、涡流扩散、膜传质阻力、跨颗粒扩散率、吸附-解吸动力学和跨柱差异吸附等，纳入一个通用的传质方程中，该方程旨在解释高压下蛋白质的传质动力学。更具体地说，这个方程包括孔径排阻、压力和温度对蛋白质谱带展宽的影响。首先将流速从0.001增加到0.250毫升/分钟，压降从2增加到298巴，色谱柱置于296.5 K的静止空气中，以确定胰岛素在低压范围内通过多孔颗粒的有效扩散率、传质速率常数和吸附平衡常数。然后，通过在色谱柱下游使用毛细管流量限制器，以0.03毫升/分钟的恒定流速增加色谱柱入口压力。将色谱柱依次浸入温度分别为298.7 K和323.15 K的循环水浴中，使柱温保持均匀。结果表明，随着平均柱压的增加，胰岛素的表面扩散系数比其体相扩散系数下降得更快。这与胰岛素在BEH-C(18)表面的吸附能随压力增加而强烈增加是一致的。相比之下，考虑到理论塔板高度（HETP）测量的精度（±12%），胰岛素的吸附动力学似乎与压力相当无关。平均而言，当温度从298.7 K升高到323.15 K时胰岛素的吸附速率常数从约40 s⁻¹增加到80 s⁻¹，翻倍。
