A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research
A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research
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DOI：
10.1016/j.chroma.2005.12.050
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发表时间：
2006-02-24
影响因子：
4.1
通讯作者：
Carr, PW
中科院分区：
文献类型：
--
作者：
Wang, XL;Barber, WE;Carr, PW
峰容量最大化是开发复杂样品的一维分离的一个非常重要的步骤。最近的工作表明，使用小颗粒与超高压液相色谱 (UHPLC) 新技术相结合能够产生非常高的峰容量。在这里，我们展示了传统 HPLC 仪器能够在肽复杂混合物的重要情况下提供与 UHPLC 中获得的峰容量相当的峰容量，但通过使用 60 厘米长的一组填充有 5 μm 薄膜（表面多孔）颗粒的色谱柱，压力要低得多。我们首先表明，与等度分离理论的众所周知的结果完全一致，当时间不受限制时，梯度洗脱色谱中的最佳峰容量是通过使用大颗粒和可以在泵的压力极限下操作的最长的柱来获得的。比较了两种不同类型的 5 μm 颗粒（表面多孔和完全多孔）在肽梯度色谱中的效率。我们发现，与类似的多孔材料相比，薄膜材料的峰值容量高出约 50%。将短柱串联而成60cm的填充薄膜颗粒的柱组；室温下4小时内获得约460的峰容量。将柱温提高到70℃，分析时间缩短到2It，峰容量进一步提高到500以上。牛血清白蛋白胰蛋白酶肽分离中观察到的峰数量大大增加，分离质量显着提高。 (c) 2005 Elsevier B.V. 保留所有权利。
Maximization of peak capacity is a very important step in developing one-dimensional separations of complex samples. In recent work, it was shown that the use of small particles in combination with the new technique of ultrahigh pressure liquid chromatography (UHPLC) was able to generate very high peak capacities. Here we show the ability of conventional HPLC instrumentation to give comparable peak capacities to those obtained in UHPLC for the important case of complex mixtures of peptides but at much lower pressures by using a 60cm long set of columns packed with 5 mu m pellicular (superficially porous) particles. We first show, in complete agreement with the well known results of the theory of isocratic separations that, when time is not limiting, the best peak capacities in gradient elution chromatography are obtained by using large particles and the longest column that can be operated at the pump's pressure limit. Two different types of 5 mu m particles (superficially porous and totally porous) were compared for their efficiency in gradient chromatography of peptides. We find that the pellicular material gave about 50% higher peak capacity compared to the analogous porous material. A 60 cm column set packed with pellicular particles was made by connecting short columns in series; a peak capacity of about 460 was obtained in 4 h at room temperature. Increasing the column temperature to 70 degrees C reduced the analysis time to 2 It and further increased the peak capacity to more than 500. The number of peaks observed in the separation of bovine serum albumin tryptic peptides was greatly increased and the separation quality was significantly improved. (c) 2005 Elsevier B.V. All rights reserved.