Gritti Fabrice, McDonald Thomas, Gilar Martin
Waters Corporation, Milford, MA 01757, USA.
Waters Corporation, Milford, MA 01757, USA.
J Chromatogr A. 2016 Jun 17;1451:107-119. doi: 10.1016/j.chroma.2016.05.035. Epub 2016 May 10.
250μm×100mm fused silica glass capillaries were packed with 1.8μm high-strength silica (HSS) fully porous particles. They were prepared without bulky stainless steel endfittings and metal frits, which both generate significant sample dispersion. The isocratic efficiencies and gradient peak capacities of these prototype capillary columns were measured for small molecules (n-alkanophenones) using a home-made ultra-low dispersive micro-HPLC instrument. Their resolution power was compared to that of standard 2.1mm×100mm very high-pressure liquid chromatography (vHPLC) narrow-bore columns packed with the same particles. The results show that, for the same column efficiency (25000 plates) and gradient steepness (0.04min(-1)), the peak capacity of the 250μm i.d. capillary columns is systematically 15-20% higher than that of the 2.1mm i.d. narrow-bore columns. A validated model of gradient chromatography enabled one to predict accurately the observed peak capacities of the capillary columns for non-linear solvation strength retention behavior and under isothermal conditions. Thermodynamics applied to the eluent quantified the temperature difference for the thermal gradients in both capillary and narrow-bore columns. Experimental data revealed that the gradient peak capacity is more affected by viscous heating than the column efficiency. Unlike across 2.1mm i.d. columns, the changes in eluent composition across the 250μm i.d. columns during the gradient is rapidly relaxed by transverse dispersion. The combination of (1) the absence of viscous heating and (2) the high uniformity of the eluent composition across the diameter of capillary columns explains the intrinsic advantage of capillary over narrow-bore columns in gradient vHPLC.
250μm×100mm的熔融石英玻璃毛细管填充了1.8μm的高强度硅胶（HSS）全多孔颗粒。这些毛细管的制备没有使用会产生显著样品分散的笨重不锈钢终端配件和金属滤片。使用自制的超低分散微型高效液相色谱仪，针对小分子（正链烷苯酮）测量了这些原型毛细管柱的等度效率和梯度峰容量。将它们的分离能力与填充相同颗粒的标准2.1mm×100mm超高压液相色谱（vHPLC）窄内径柱的分离能力进行了比较。结果表明，在相同的柱效（25000塔板）和梯度陡度（0.04min⁻¹）下，内径250μm的毛细管柱的峰容量比内径2.1mm的窄内径柱系统地高15 - 20%。一个经过验证的梯度色谱模型能够准确预测毛细管柱在非线性溶剂化强度保留行为和等温条件下的观察到的峰容量。应用于洗脱液的热力学方法量化了毛细管柱和窄内径柱中热梯度的温差。实验数据表明，梯度峰容量比柱效更容易受到粘性加热的影响。与内径2.1mm的柱子不同，内径250μm的柱子在梯度过程中洗脱液组成的变化通过横向扩散迅速弛豫。（1）不存在粘性加热和（2）毛细管柱直径上洗脱液组成的高度均匀性相结合，解释了在梯度vHPLC中毛细管柱相对于窄内径柱的内在优势。
