Gritti Fabrice, Felinger Attila, Guiochon Georges
Department of Chemistry, University of Tennessee, 552 Buehler Hall, Knoxville, TN 37996-1600, USA.
J Chromatogr A. 2006 Dec 8;1136(1):57-72. doi: 10.1016/j.chroma.2006.09.074. Epub 2006 Oct 13.
The influences of the errors made in the measurement of the extra-column volume of an instrument on the accuracies of the estimates made of the column efficiency and of the parameters of the mass transfer kinetics were investigated from an experimental point of view. A standard HP1090 apparatus (extra-column volume, approximately 50 micro L) was used to measure the efficiency of a Sunfire-C(18) RPLC column (column hold-up volume, approximately 1.50 mL). The first and second moments of the peaks of phenol (a retained compound) and of thiourea (a practically non-retained compound) were measured at six different temperatures between 22 and 78 degrees C, for flow rates between 0.10 and 4.70 mL/min (i.e., for linear velocities between 0.025 and 1.179 cm/s). Each series of measurements was successively made with the instrument being fitted with and without the column. The experimental HETP data must be corrected for the solute dispersion in the connected tubes in order properly to assess the true column efficiency. Even with a modern, high performance instrument, the dispersion of a non-retained compound is essentially due to the band broadening phenomena that take place in the extra-column volumes, the sum of all these extra-column band broadening contributions accounting for more than 80% of the total band broadening measured. The contribution of the sampling device is particularly deleterious since, for a 2 mu L injection, the maximum solute concentration in the peak that enters into the column is nearly ten-fold lower than that of the sample. Nevertheless, the impact of the extra-column volumes on the estimates of the kinetic parameters (e.g., molecular diffusion coefficient D(m) and effective particle diffusivity D(e)) remains negligible. Obviously, the relative error made on the column efficiency of a retained compound depends much on its retention factor. It decreases from 8 to 1% when the retention factor increases from 5 to 17.
从实验角度研究了仪器柱外体积测量误差对柱效和传质动力学参数估算精度的影响。使用标准的HP1090仪器（柱外体积约50微升）来测量Sunfire - C(18)反相液相色谱柱的柱效（柱死体积约1.50毫升）。在22至78摄氏度之间的六个不同温度下，对于0.10至4.70毫升/分钟的流速（即线性速度为0.025至1.179厘米/秒），测量了苯酚（一种保留化合物）和硫脲（一种几乎不保留的化合物）峰的一阶和二阶矩。每次测量系列都是在仪器安装和未安装色谱柱的情况下依次进行的。为了正确评估真实的柱效，实验得到的理论塔板高度数据必须针对连接管中溶质的分散进行校正。即使使用现代的高性能仪器，未保留化合物的分散主要是由于柱外体积中发生的谱带展宽现象，所有这些柱外谱带展宽贡献的总和占测量的总谱带展宽的80%以上。进样装置的贡献特别有害，因为对于2微升进样，进入色谱柱的峰中溶质的最大浓度比样品中的浓度低近十倍。然而，柱外体积对动力学参数（例如分子扩散系数D(m)和有效颗粒扩散系数D(e)）估算的影响仍然可以忽略不计。显然，保留化合物柱效的相对误差很大程度上取决于其保留因子。当保留因子从5增加到17时，相对误差从8%降至1%。
