In the development of a chromatographic method, chromatographers often are 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 sometimes a
challenging task to choose the optimum column parameters (for example, particle size, column length, and column internal diameter)
and optimum operating conditions (for example, mobile phase components, buffers, flow rates, and temperature). Often, a given
is the high performance liquid chromatography (HPLC) instrumentation at hand, with its maximum pump pressure and flow rate
capability and a column that can be installed in the instrument or in the drawer.
For most chromatographers, analyzing HPLC column performance consists of measuring the following four key performance parameters:
the column efficiency (H or HETP) (as a function of the mobile phase flow rate), the column permeability, Kv0 (or pressure drop), the retention capacity (k value), and the column selectivity (α) for important pairs of analytes. Whereas the former two essentially are reflecting
the packing quality and the mass transfer kinetics of the column, the latter two depend upon the dimensions and the chemistry
of the stationary phase (1). Although the overall column quality also is determined by other factors, such as lifetime, chemical
inertness, and mass loadability, the present contribution will focus on the first four parameters (H, Kv0, k, and α) because these are of a more general nature than the latter three, which depend strongly upon 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) often are treated and discussed separately. Figure 1 shows such an example. The column efficiency usually is represented
by the column Van Deemter curve (Figure 1a). To investigate the pure column performance, the reported plate height values
preferably should be corrected for the extracolumn 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
usually is assessed from a plot of the column pressure P (preferably also corrected for the extracolumn pressure drop) versus the flow rate or the interstitial-, the superficial-,
or the t0-marker velocity (Figure 1b). The retention capacity usually is represented as a plot of ln k' versus the fraction of organic
modifier (φ) for one or more given test components (Figure 1c), while the information regarding the column selectivity usually
is contained in a table giving the retention factor ratios of close-eluted pairs of components (Figure 1d).
The example data shown in Figure 1 were obtained by comparing two 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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