Comparison Techniques for HPLC Column Performance

Jun 01, 2008

When developing a chromatographic method, chromatographers are often required to determine the best column and operating conditions to address the problem at hand. With so many columns and operating conditions to choose from it is often a challenge to choose the optimum column parameters (e.g., particle size, column length, column internal diameter, etc.) and optimum operating conditions (e.g., mobile phase components, buffers, flow-rates, temperature, etc.).

For most chromatographers, analysing HPLC column performance consists of measuring the following four key performance parameters: the column efficiency [height equivalent theoretical plate (HETP) or H], which is a function of the mobile phase flow-rate; the column permeability, K v0 (or pressure drop), the retention capacity (k value); and the column selectivity (α) for important pairs of analytes. Whereas, the former two essentially reflect the packing quality and the mass transfer kinetics of the column, the latter two essentially depend on the dimensions and the chemistry of the stationary phase.1 Although the overall column quality is also determined by other factors, such as lifetime, chemical inertness and mass loadability, this article will focus on the first four parameters (H, K v0 , k and α) because these generally apply to all separations than the latter three, which depend strongly on the chemistry and the economics of the application.

Effective Performance (Absolute Plots)

Figure 1
Traditional column comparison techniques: In a typical performance report or research paper, the main column performance parameters (H, K v0 , k and α) are often treated and discussed separately. Figure 1 shows such an example. The column efficiency is usually represented by the column Van Deemter curve [Figure 1(a)]. To investigate the pure column performance, the reported plate height values should preferably be corrected for the extra-column band broadening because these features only detract from a fair column comparison, but are particularly important for the smaller particle columns that are being used today.

The column permeability is usually assessed from a plot of the column pressure P (preferably also corrected for the extra-column pressure-drop) versus the flow-rate or the interstitial-, the superficial- or the t 0 -marker velocity [Figure 1(b)]. The retention capacity is usually represented as a plot of ln k' versus the fraction of organic modifier (φ) for one or more test components [Figure 1(c)], while the information regarding the column selectivity is usually contained in a table listing the retention factor ratios of close-eluting pairs of components [Figure 1(d)].

The example data shown in Figure 1 have been obtained by comparing two different particle types (similar sub-2 μm size, but bonded with different stationary phases, C8 and C18). Each particle type was evaluated by testing two purchased columns.

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