Isocratic using short columns

Fig. 3. Plot of plate count I s. analysis time for fast isocratic separations using short columns.

Donovan SF, Pescatore MC (2002) Method for measuring the logarithm of the octanol-water partition coefficient by using short octadecyl-poly(vinyl alcohol) high performance liquid chromatography columns. J Chrom A 952 47-61 Du CM, Valko K, Bevan C et al. (1998) Rapid gradient RP-HPLC method for lipophilicity determination s solvation equation based comparison with isocratic methods. Anal Chem 70 4228-4234... 

For isocratic LC, the solute does not need to fully elute from the second-dimension column prior to the next sampling period. This is illustrated in Fig. 6.4, where it is shown that more than one sample can be resident in the column at one time. Using the chromatograms shown in Fig. 6.5, which show the effect of various injection volumes that will be discussed later, it is not necessary to wait for the full 2 min of sampling time. This significantly helps to speed up the sampling process and is most useful for SEC, where short elution time ranges are typically found and short times between the injection and nonretained peaks are typical of column operation. 

First, we look at isocratic separations. Let us assume that the analysis can be accomplished within a retention factor of 10. We also suppose that the analysis is carried out with a typical reversed-phase solvent and a sample with a typical molecular weight of a pharmaceutical entity. In order to manipulate the analysis time, we will keep the mobile phase composition the same and vary the flow rate. The maximum backpressure that we will be able to apply is 25MPa (250 bar, 4000psi). In Figure 1, we have plotted the plate count as a function of the analysis time for a 5 J,m 15-cm column. We see that the column plate count is low at short analysis times and reaches a maximum at an analysis time of about 1 h. A further increase in analysis time is not useful, since the column plate count declines again. This is the point where longitudinal diffusion limits the column performance. The graph also stops at an analysis time of just under 5 min. This is the point when the maximum allowable pressure drop has been reached. 

El-Fizga (108) developed a simple, rapid method for the detection of oils high in linoleic acid in olive oil by RP-HPLC and a simple authenticity factor and a derived equation to determine the extent of adulteration with a one short chromatographic step, completed in less than 15 min. They used two 150 X 4.5-mm ID stainless steel columns packed with an octyl-bonded silica stationary phase (Supelcosil-LC 8) (Supelco. Bellefonte, PA, USA) and a differential reffactometric detector. The isocratic mobile phase was acetone-acetonitrile (70 30, v/v) (Table 5). 

Analyses of 30 wine samples by the proposed method and the official EEC spectrophoto-metric method show that there is a good correlation between the results of the two methods, although the HPLC analyses are clearly more precise and accurate. The use of a (DVB-H) HPLC column, which allows isocratic elution, together with the short analysis time (about 15 minutes) makes this method rapid and readily amenable to automation (80). 

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