If the HPLC system uses an inappropriate connection method or incorrect capillary application, it may lead to poor peak broadening, and the optimal separation efficiency of the chromatographic column is out of the question. It may even happen that the thinner the column used, the greater the broadening of the elution peak. This article will introduce in detail the influence of capillaries on HPLC separation and how to choose the correct capillary and connection method.
The good separation efficiency of HPLC columns is an important prerequisite for successful HPLC separation. Inappropriate tubing connections or improper use of capillaries may lead to poor peak broadening, so special attention should be paid. When using the “old-fashioned” standard separation column of 4.6×250mm, the role of capillaries and the influence of tubing connections are not very prominent; when using a 2.0×100mm thin-diameter chromatographic column, their influence is very large.
For “Ex-columns”, the main cause of peak broadening is the use of incorrect capillaries and cavities in the tubing connection, which will significantly expand the elution channel. Therefore, in principle, the column diameter should be as narrow as possible and the connecting pipes should be as short as possible.
If the user uses a matching device or a tightly fitted HPLC supplied by the same manufacturer, all pipe connections have been pre-optimized by the supplier. Problems with such equipment only occur when it is necessary to connect a detector from another company or when a HPLC system needs to be assembled from different components.
The peak width through the separation column is proportional to the size of the chromatographic column. In short: a thin column produces a narrow peak. The peak broadening caused by liquid conduction involves the entire system and needs to be coordinated one by one.
Poiseuille’s law
The French physicist Jean-Léonard Poiseuille’s interest in the physiology of the blood circulation system led him to conduct fundamental experimental studies on the fluid behavior of liquids in capillaries in 1840. Instead of a straight interface between the sample and the eluent, a laminar flow is formed. The different flow rates of the fluid in a tube with a diameter of “dt” (HPLC capillary) form a “U” shape (as shown in Figure 2).
Figure 2. Flow behavior of liquids in capillaries as described by Jean Louis Léonard Marie Poiseuilles. dT = inner diameter, U = flow velocity, UMAX = maximum flow velocity, UAVE = average flow velocity.
To minimize the peak width, the smallest possible tube diameter should be chosen. The sample molecules have a tendency to diffuse toward the fluid boundary. Without this diffusion, the elution peak would be infinitely wide, because according to Poiseuille’s theory, the flow velocity is zero at the capillary wall, and the sample molecules would move extremely slowly at this point. The diffusion effect causes the sample molecules to migrate from the wall to the middle of the tube, so that the sample peak can be eluted in a limited time and has a limited peak width.
Post time: Jul-04-2024