HPLC problems case studies

Case study 4 shows several examples of problems caused by equipment malfunctions and their subsequent diagnosis and solution. The first one involved a situation of poor retention time reproducibility of a gradient assay. It involved the analysis of a complex natural product, using a narrowbore column (2-mm i.d.) at 0.5 mL/min. System suitability test showed retention times to be erratic and could vary by 1-2 minutes without any obvious trends. Flow rate accuracy was found to be acceptable, however, the compositional accuracy test failed (see Chapter 9 on HPLC calibration). The tentative diagnosis was that of a malfunctioning of the proportioning valve. After its replacement, the retention time precision performance was re-established. 

In many cases, both a number of factors are varied in experiments and many response variables are measured. In such situations, there are often correlations between response variables. This suggests that it may be useful to try to find out to what extent the response variables can be independently affected by the factors and interactions between the factors. One way to analyse this problem is to use PCA or PLS and determine how many significant dimensions that can be extracted in designed studies. As an example, the influence of various mobile phase factors in a HPLC system for separating catecholamines and some catechol- and indolamine metabolites was investigated measuring the retention... 

For pharmacokinetic and metabolic studies it is essential that thiols and disulfides are not measured in isolation. Pre-column reduction procedures partially overcome this problem, but qualitative data on the disulfides are lacking. Postcolumn reduction can be restrictive since sensitivity depends on the ability of the reactor to reduce disulfides in some cases this is impossible. HPLC-ED is proving in some cases to be the method of choice for this dual application. 

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