Vs. Gradient Analysis

Traditionally, most pharmaceutical assays are isocratic analysis employing the same mobile phase throughout the elution of the sample. Isocratic analyses are particnlarly common in quality control applications since they nse simpler HPLC eqnipment and premixed mobile phases. Notable disadvantages of isocratic analysis are limited peak capacity (the maximnm nnmber of peaks that can be accommodated in the chromatogram), and problems with samples containing analytes of diverse polarities. Also, late eluters (such as dimers) are particularly difficult to quantitate in isocratic analysis due to excessive band broadening with long retention times. 

Key Gradient Parameters (Flow rate, Gradient time [tj, 

FIGURE 18 HPLC separation of a stabiiity sampie under isocratic conditions showing iow ieveis of various reiated substances (API active pharmaceutical ingredient). Reprinted with permission from Reference 19. 

The concept of the capacity factor k is also considerably more complex in gradient chromatography and is best represented by an average k or k where 

FIGURE 20 Chromatogram illustrating the concept of peak capacity (P) which is the maximum number of peaks that can be accommodated in a chromatogram. 

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An excellent and comprehensive review has covered HPLC analysis of AOs and light stabilisers up to 1990 [576]. Normal vs. reversed-phase and isocratic vs. gradient-elution HPLC separation of synthetic mixtures of additives and of solvent extracts from polymers were discussed. 

Analytical methods for the determination of one antidepressant and/or its metabolite(s) were usually performed in isocratic mode, with total run times from seconds to a few minutes. However, as previously mentioned, multianalyte procedures are preferable, particularly if the method is intended for clinical or forensic analysis. Gradient separation was usually applied when the most common antidepressants were included in the methodology however, total chromatographic run times varied widely, from 5 to 40 min [57, 76], depending on column length, extraction technique (offline vs. online techniques), biological matrix or the specific application of the method. 

If the retention vs. composition plots of all solutes are known, then it is in principle possible to calculate the optimum program parameters for a simple, continuous gradient (figure 6.2a-d). In such a procedure an appropriate optimization criterion can be selected such that the distribution of all the peaks over the chromatogram, as well as the required analysis time, can be taken into account (see chapter 4). 

This expression highlights both factors whose gradients affect Rf the liquid velocity ratio v/vf and the phase ratio Vm/Vs. Analysis shows that vlvf is always less than unity, decreasing as one moves back from the liquid front [31]. In paper strips, this ratio can vary from unity at the front to somewhere around 0.6 near the liquid source. Factor VJV5 varies even more, increasing twofold upon retreating from 90% of the distance to the front to 10%. 

Figure 5.13. A good vs. a bad blank chromatogram from a gradient trace analysis for impurity testing of pharmaceuticals. The ghost peaks from the blank injection are derived mostly from the trace contaminants in the weaker mobile phase, which are concentrated during column equilibration. Reprint with permission from reference 16.

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