Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA.
J Chromatogr A. 2010 Apr 30;1217(18):3000-12. doi: 10.1016/j.chroma.2010.02.044. Epub 2010 Mar 6.
A series of experiments and measurements demonstrate the importance of minimizing the extra-column band broadening contribution of the instrument used. The combination of several measures allowed the achievement of the full potential efficiency of three Kinetex-C(18) columns, using a conventional liquid chromatograph. The first measure consists in minimizing the extra-column volume of the instrument, without increasing much its back pressure contribution, by changing the needle seat volume, the inner diameter and length of the capillary connectors, and the volume of the detector cell of a standard instrument (Agilent 1100). The second measure consists in injecting a volume of weak eluent (less than half the elution strength of the mobile phase) right after the sample, before the sample had time to reach the column. Experimental results show that these changes could provide most of the resolution expected from the true column performance. After the changes were made, the resolutions of the 2.1 mm x 50 mm, 4.6 mm x 50 mm, and 4.6 mm x 100 mm Kinetex-C(18) columns for compounds having retention factors close to 1 were increased by about 180, 35, and 30%, respectively. The resolutions obtained are then similar to those measured with advanced instruments like the Agilent 1200, the Agilent 1290 Infinity HPLC, and the Acquity chromatographs.
一系列实验和测量表明，最大限度地减少仪器的柱外带宽展宽贡献非常重要。通过采取多种措施，可以在常规液相色谱仪上实现三个 Kinetex-C(18)色谱柱的全部潜在效率。第一项措施是通过改变针座体积、毛细管件的内径和长度以及标准仪器（安捷伦 1100）的检测池体积，在不增加仪器背压贡献的情况下，最小化仪器的柱外体积。第二项措施是在样品进入色谱柱之前，在样品有时间进入色谱柱之前，立即注入少量弱洗脱液（小于流动相洗脱强度的一半）。实验结果表明，这些变化可以提供从真实柱性能预期的大部分分辨率。进行这些更改后，对于保留因子接近 1 的化合物，2.1mm×50mm、4.6mm×50mm 和 4.6mm×100mm Kinetex-C(18)色谱柱的分辨率分别提高了约 180%、35%和 30%。然后，获得的分辨率与安捷伦 1200、安捷伦 1290 Infinity HPLC 和 Acquity 色谱仪等先进仪器测量的分辨率相似。
