HPLC instrument, test

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

Validation of instrument hardware includes testing according to documented specifications. If HPLC instruments consist of several modules, individual modules (modular validation), as well as the entire system (holistic validation), should be validated. However, the latter is preferred, as individual module tests should be performed as part of the diagnosis if the system fails. 

The main qualification tests applied to HPLC instrumentation concern ... 

After the successful completion of these four steps it will be possible to perform reliable analyses. However, it is necessary to perform the instrument test" at regular time intervals, at least once a year. The test for HPLC instramentation is described in detail in Chapter 25. 

A new topic is now included Chapter 20 about quahty assurance. Part of it could be found before in chapter 19 but now the subject is presented much broadly and independent of Analytical HPLC . Two chapters in the appendix were updated and expanded by Bruno E. Lendi, namely the ones about the instrument test (now chapter 25) and troubleshooting (now chapter 26). Some new sections were created 1.7, comparison of HPLC with capillary electrophoresis 2.11, how to obtain peak capacity 8.7, van Deemter curves and other coherences 11.3, hydrophilic interaction chromatography 17.2, method transfer 18.4, comprehensive two-dimensional HPLC 23.3, fast separations at 1000 bar 23.5, HPLC with superheated water. In addition, many details were improved and numerous references added. 

Figure 2. Microbore HPLC of test solutes showing actual and calculated (c) retention times for a 10 minute linear ternary gradient, (conditions listed In Table 4a) using a 155 pi Instrumental gradient delay. Column 250 X 1 mm laboratory bonded Partlsll 10 C3 flow rate 50 pi min Detector Kratos 769, fitted with a 0.5 pi flow cell. Wavelength 254 nm, sensitivity 0.1 aufs. Pump Brownlee MPLC mlcobore gradient pump. Injector Rheodyne 7413 with a 0.5 pi injection loop. Reproduced from Ref. 22. Copyright 1985, American Chemical Society.

Whether you use a standardized test or your assay, it is worthwhile to check column performance on a regular basis and keep a log of it With today s computerized HPLC instruments this is fairly easy to do, and you can ea y generate control charts of the important column characteristics. I recommend monitoring for at least one peak plate count, peak symmetry, and retention time, and relative retention for a critical pair of analytes. Resolution is not as instructive a parameter, since it is affected by both plate count and relative retention. Thus it does not tell you anything about which of the underlying parameters is changing. 

Following completion of the initial experiment, the focus shifts to the separation of the test mixture or organic compounds using the HPLC instrument. The effect of solvent strength on k and the effect of mobile-phase flow rate on will be considered by retrieving previously developed Turbochrom methods and making manual injections. 

The apparatus test includes a protocol about pump, injector and detector functions and includes the requirements listed in Table 19.1 Usually they are easily fulfilled by today s HPLC instrumentation, especially when new. The apparatus test should be performed routinely in adequate time intervals and after each repair. It is a prerequisite for method validation. A proposal can be found in Chapter 24. 

The characteristics of an equipment may change over time, e.g., UV detector lamps lose intensity, or pump piston seals abrade, or short-term noise affecting the LOD is increased because of flow cell contamination. These changes will have a direct impact on the performance of the HPLC instrument. The frequency of performance tests will be determined by experience and is based on need, type, and history of equipment performance. Intervals between the checks should be shorter than the time the instrument drifts outside acceptable limits. New instruments need to be checked more frequently, and, if the instrument meets the performance specifications, the time interval can be increased. 

Figure 2. Result of an automated HPLC hardware test. The user selects test items from a menu and defines acceptance limits. The instrument then performs the tasks and prints the actual results together with the limits, as specified by the user...

The efficiency, or plate count of a column N is often calculated as 5.54 (tr/a)2, where tr is the retention time of a standard and a is the peak width in time units at half-height.1 2 5 This approach assumes that peaks are Gaussian a number of other methods of plate calculation are in common use. Values measured for column efficiency depend on the standard used for measurement, the method of calculation, and the sources of extra-column band broadening in the test instrument. Therefore, efficiency measurements are used principally to compare the performance of a column over time or to compare the performance of different columns mounted on the same HPLC system. 

Most manufacturers of dissolution testing devices offer semi-automated systems that can perform sampling, filtration, and UV reading or data collection. These systems automate only a single test at a time. Fully automated systems typically automate entire processes including media preparation, media dispensing, tablet or capsule drop, sample removal, filtration, sample collection or analysis (via direct connection to spectrophotometers or HPLCs), and wash cycles. A fully automated system allows automatic performance of a series of tests to fully utilize unused night and weekend instrument availability. 

The progress made in interfacingHPLC instruments with mass spectrometry has been a significant development for laboratory analyses in the pharmaceutical industry. The low concentrations of test drugs in extracts of blood, plasmas, serums, and urine are no problem for this highly sensitive HPLC detector. In addition, the analysis is extremely fast. Lots of samples with very low concentrations of the test drugs can thus be analyzed in a very short time. At the MDS Pharma Services facility in Lincoln, Nebraska, for example, a very busy pharmaceutical laboratory houses over 20 LC-MS units, and they are all in heavy use daily. 

HPLC is frequently employed in the analysis of amino acids, peptides, proteins, nucleic acids, and nucleotides. HPLC is also often used to analyze for drugs in biological samples (see Workplace Scene 16.2). Due to the complex nature of the molecules to be analyzed, these techniques tend to be more complex than HPLC applications in other areas of analytical chemistry. For example, separation of nucleotides or amino acids is more difficult than testing for caffeine in beverages, even though the same instrument and same general methods would be employed. A variety of columns and mobile phases are regularly employed. 

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