Common HPLC Problems

A clogged column frit is another common HPLC problem. To minimize this problem from the start, the... 

Common HPLC problems are caused by component malfunctions (pump, degasser, injector, detector, data system, column), and faulty preparation of the mobile phase or sample preparation. Problems can be categorized into several areas ... 

Table 10.4. Summary of Symptoms of Common HPLC Problems and Probable Causes...

Table 10.4 summarizes the symptoms of common HPLC problems and the most probable causes. Developing HPLC troubleshooting skills often takes many years of operating experience and a working understanding of the principles of the instrument as well as considerable patience to eliminate all the typical causative factors. 

Ion-exchange columns can be substituted into the general HPLC instrument shown in Eigure 12.26. The most common detector measures the conductivity of the mobile phase as it elutes from the column. The high concentration of electrolyte in the mobile phase is a problem, however, because the mobile-phase ions dominate the conductivity, for example, if a dilute solution of HCl is used as the mobile phase, the presence of large concentrations of H3O+ and Ck produces a background conductivity that may prevent the detection of analytes eluting from the column. 

Oligomeric additives with broad MWD tend to be a problem in conventional HPLC conditions. In cases where no interest exists in the oligomer distribution it is common practice to solve the problem by creating a uniform structural unit useful for analysis. For example, isocratic (or gradient) LC-UV was used for the determination of the polymeric light stabiliser Tinuvin 622 in polyolefins using dissolution (toluene)/derivatisation (TBAH)-precipitation (alcohol) the diol formed was quantitatively determined by NPLC [653]. 

Matrix effect is a phrase normally used to describe the effect of some portion of a sample matrix that causes erroneous assay results if care is not taken to avoid the problem or correct for it by some mechanism. The most common matrix effects are those that result in ion suppression and subsequent false negative results. Ion enhancement may lead to false positive results.126 127 Several reports about matrix effects include suggestions on what can cause them and how to avoid them.126-147 While various ways to detect matrix effects have been reported, Matuszewski et al.140 described a clear way to measure the matrix effect (ME) for an analyte, recovery (RE) from the extraction procedure, and overall process efficiency (PE) of a procedure. Their method is to prepare three sets of samples and assay them using the planned HPLC/MS/MS method. The first set is the neat solution standards diluted into the mobile phase before injection to obtain the A results. The second set is the analyte spiked into the blank plasma extract (after extraction) to obtain the B results. The third set is the analyte spiked into the blank plasma before the extraction step (C results) these samples are extracted and assayed along with the two other sets. The three data sets allow for the following calculations ... 

As mentioned previously, introducing the sample to the flowing mobile phase at the head of the column is a special problem in HPLC due to the high pressure of the system and the fact that the liquid mobile phase may chemically attack a rubber septum. For these reasons, the use of the so-called loop injector is the most common method for sample introduction. 

Problems that arise with HPLC experiments are usually associated with abnormally high or low pressures, system leaks, worn injectors parts, air bubbles, or blocked in-line filters. Sometimes these manifest themselves on the chromatogram and sometimes they do not. In the following subsections, we address some of the most common problems encountered, pinpoint possible causes, and suggest methods of solving the problems. You can also refer to the troubleshooting guide in Chapter 12 for possible solutions. 

What is the most common cause of an unusually high pressure in an HPLC flow stream and how is the problem solved ... 

Particulate sorbents are available almost exclusively in the shape of micrometersized beads. These beads are packed in columns and represent currently the most common stationary phases for high-performance liquid chromatography (HPLC). Despite their immense popularity, slow diffusional mass transfer of macromolecular solutes into the stagnant pool of the mobile phase present in the pores of the separation medium and the large void volume between the packed particles are considered to be major problems in the HPLC of macromolecules, frequently impairing their rapid and efficient separation [1]. 

When compared to fluorescence detectors for HPLC, the design of a fluorescence detector for CE presents some technical problems. In order to obtain acceptable sensitivity, it is necessary to focus sufficient excitation light on the capillary lumen. This is difficult to achieve with a conventional light source but is easily accomplished using a laser. The most popular source for laser-induced fluorescence (LIF) detection is the argon ion laser, which is stable and relatively inexpensive. The 488-nm argon ion laser line is close to the desired excitation wavelength for several common fluorophores. The CLOD for a laser-based fluorescence detector can be as low as 10 12 M. 

With capillary electrophoresis (CE), another modern primarily analytically oriented separation methodology has recently found its way into routine and research laboratories of the pharmaceutical industries. As the most beneficial characteristics over HPLC separations the extremely high efficiency leading to enhanced peak capacities and often better detectability of minor impurities, complementary selectivity profiles to HPLC due to a different separation mechanism as well as the capability to perform separations faster than by HPLC are frequently encountered as the most prominent advantages. On the negative side, there have to be mentioned detection sensitivity limitations due to the short path length of on-capillary UV detection, less robust methods, and occasionally problems with run-to-run repeatability. Nevertheless, CE assays have now been adopted by industrial labs as well and this holds in particular for enantiomer separations of chiral pharmaceuticals. While native cyclodextrins and their derivatives, respectively, are commonly employed as chiral additives to the BGEs to create mobility differences for the distinct enantiomers in the electric field, it could be demonstrated that cinchona alkaloids [128-130] and in particular their derivatives are applicable selectors for CE enantiomer separation of chiral acids [19,66,119,131-136]. 

An area of study related to this topic is the use of subcritical, but superheated water as a mobile phase for chromatographic separations [78], These separations use water heated to 100-220°C and pressures up to 50 bar, avoiding problems due to hydrolysis and oxidation, which is common when supercritical water is used. Although this is a new area of investigation, several reports on the hyphenation of HPLC using... 

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. 

Analytical Method Development for TRIS. The detection of brominated compounds of very low volatility such as TRIS posed special analytical problems. Since TRIS has no recognizable chromophore, the detection systems which are commonly used with high performance liquid chromatography (hplc), such as refractive index or short wavelength (<220 nm) uv detectors, are too non-specific to be of much practical use for the analysis of environmental samples. Furthermore, the sensitivities available with these detection methods are generally inadequate. 

---