Gusev I, Huang X, Horváth C
Department of Chemical Engineering, Yale University, New Haven, CT 06520-8286, USA.
J Chromatogr A. 1999 Sep 3;855(1):273-90. doi: 10.1016/s0021-9673(99)00697-4.
Capillary columns with monolithic stationary phase were prepared from silanized fused-silica capillaries of 75 microns I.D. by in situ copolymerization of divinylbenzene either with styrene or vinylbenzyl chloride in the presence of a suitable porogen. The porous monolithic support in this study was used either directly or upon functionalization of the surface to obtain a stationary phase that was appropriate for the separation of peptides by capillary electrochromatography (CEC). The main advantages of monolithic columns are as follows. They do not need retaining frits, they do not have charged particles that can get dislodged in high electric field, and they have relatively high permeability and stability. Whereas such columns are designed especially for CEC, they find application in micro high-performance liquid chromatography (mu-HPLC) as well. Five different porogens were employed to prepare the monolithic columns that were examined for permeability and porosity. The flexibility of fused-silica capillaries was not adversely affected by the monolithic packing and the longevity of the columns was satisfactory. This may also be due to the polymerization technique, which resulted in a fluid-impervious outer layer of the monolith that precluded contact between the fused-silica surface and the liquid mobile phase. For the most promising columns, the conductivity ratios and the parameters of the simplified van Deemter equation, both in mu-HPLC and CEC, were evaluated. It was found that the efficiency of the monolithic columns in CEC was significantly higher than in mu-HPLC in the same way as observed with capillary columns having conventional particulate packing. This is attributed to the relaxation of band-broadening with electroosmotic flow (EOF) with respect to that with viscous flow. It follows then that the requirement of high packing uniformity to obtain high efficiency may also be relaxed in CEC. Angiotensin-type peptides were separated by CEC with columns packed with a monolithic stationary phase having fixed n-octyl chains and quaternary ammonium groups at the surface. Plate heights of about 8 microns were routinely obtained. The mechanism of the separation is based on the interplay between EOF, chromatographic retention and electrophoretic migration of the positively charged peptides. The results of the complex migration process, with highly nonlinear dependence of the migration times on the organic modifier and the salt concentration, cannot be interpreted within the framework of classical chromatography or electrophoresis.
具有整体固定相的毛细管柱由内径75微米的硅烷化熔融石英毛细管通过在合适的致孔剂存在下使二乙烯基苯与苯乙烯或乙烯基苄基氯原位共聚制备而成。本研究中的多孔整体载体可直接使用，也可在表面功能化后使用，以获得适合通过毛细管电色谱（CEC）分离肽的固定相。整体柱的主要优点如下。它们不需要保留筛板，没有会在高电场中脱落的带电颗粒，并且具有相对较高的渗透率和稳定性。虽然此类柱是专门为CEC设计的，但它们也可应用于微高效液相色谱（μ-HPLC）。使用了五种不同的致孔剂来制备整体柱，并对其渗透率和孔隙率进行了检测。整体填充对熔融石英毛细管的柔韧性没有不利影响，并且柱的寿命令人满意。这也可能归因于聚合技术，该技术导致整体柱形成了不透液的外层，从而避免了熔融石英表面与流动相液体之间的接触。对于最有前景的柱，评估了μ-HPLC和CEC中的电导率比以及简化的范德姆特方程的参数。结果发现，与具有传统颗粒填料的毛细管柱一样，整体柱在CEC中的效率明显高于在μ-HPLC中的效率。这归因于电渗流（EOF）相对于粘性流的谱带展宽的松弛。因此，在CEC中获得高效率对高填充均匀性的要求也可能放宽。通过CEC使用填充有表面带有固定正辛基链和季铵基团的整体固定相的柱分离了血管紧张素型肽。通常可获得约8微米的板高。分离机制基于带正电肽的EOF、色谱保留和电泳迁移之间的相互作用。迁移时间对有机改性剂和盐浓度具有高度非线性依赖性的复杂迁移过程的结果无法在经典色谱或电泳的框架内进行解释。
