HPLC systems and modules

High-performance liquid chromatography (HPLC) is a versatile analytical technique using sophisticated equipment refined over several decades. An in-depth understanding of the working principles and trends is useful for more effective application of the technique. This chapter provides the reader with a concise overview of HPLC instrumentation, operating principles, recent advances, and modern trends. The focus is on the analytical scale HPLC systems and modules (pump, injector, and detectors). System dwell volume... 

High Performance Liquid Chromatographic (HPLC) Analysis. A Waters HPLC system (two Waters 501 pumps, automated gradient controller, 712 WISP, and 745 Data module) with a Shimadzu RF-535 fluorescence detector or a Waters 484 UV detector, and a 0.5 pm filter and a Rainin 30 x 4.6 mm Spheri-5 RP-18 guard column followed by a Waters 30 x 3.9 cm (10 pm particle size) p-Bondapak C18 column was used. The mobile phase consisted of a 45% aqueous solution (composed of 0.25% triethylamine, 0.9% phosphoric acid, and 0.01% sodium octyl sulfate) and 55% methanol for prazosin analysis or 40% aqueous solution and 60% methanol for naltrexone. The flow rate was 1.0 mL/min. Prazosin was measured by a fluorescence detector at 384 nm after excitation at 340 nm (8) and in vitro release samples of naltrexone were analyzed by UV detection at 254 nm. 

HPLC systems can be classified as modular or integrated. In a modular system, separate modules are stacked and connected to function as a unit, whereas in an integrated system, modules are built inside a single housing and often share a common controller board. These built-in modules cannot function outside the system solvent lines and electrical wires are inside the housing. Modular systems are considered easily serviceable since internal components are easily accessible, and the malfunctioning module can be swapped. Integrated systems provide... 

A typical HPLC system consists of several modules a pump injector, a detector, and a data-handling device (Figure 1). The simplest... 

This chapter reviews the principles and strategies used for HPLC system calibration that includes the pump, the detector, the autosampler, and the column oven. A case study is used to illustrate the development of the calibration procedures for all system modules and the rationale of setting up acceptance criteria that balance productivity and compliance. 

Most HPLC systems in pharmaceutical laboratories are calibrated every 6-12 months. Periods longer than 12 months are not recommended while periods shorter than 3 months are deemed unnecessary because each HPLC system is also subjected to a daily system suitability check to ensure sufficiency for the application. Ideally, the frequency of calibration should be dictated by the historical data of calibration failures and the manufacturer s recommendation. In practice, 6 months appears to be the norm adopted by most laboratories. Calibration is also required after annual preventive maintenance or major repairs though only the affected modules, and not the entire system, need to be recalibrated. 

In summary, this chapter reviews how to develop expedited calibration procedures and acceptance criteria for all the common modules in an HPLC system. 

Operation Qualification. Operation qualification (OQ) is the process of establishing that the instrument or system modules operate according to the functional requirements in a suitable environment. For an HPLC system, operation of the pump, injector, and detector will be tested at this stage. Typical OQ tests for HPLC modules and a UV-Vis spectrophotometer are as follows ... 

HPLC systems operate on a master-slave arrangement in setting up for automation. One module sets the timing and initiates the process, and the remaining modules accept a signal and follow the leader. In a gradient HPLC system, the master module can be a microprocessor-based controller, a computer software-based controller, an autosampler, or an integrator. 

On-Line Systems Flowing MMLLE systems have been established in different layouts with automation and on-line hyphenation to GC and HPLC analysis. An automated on-line FS-MMLLE-GC system with a loop-type interface compatible with LVI was used for the extraction of pesticides and PAHs in surface waters.86 In another study, pressurized hot water extraction (PH WE) was coupled on-line to a FS-MMLLE-GC-FID system and applied to the analysis of PAHs in soil, where MMLLE was used as a cleanup and concentration step of the PH WE extract prior to final GC analysis.87 In addition, an HF-MMLLE setup was incorporated in PHWE and GC, resulting in an online PHWE-HF-MMLLE-GC system, where the HF membrane module contained 10-100 HFs. The system served for the extraction and analysis of PAHs in soil and sediments ... 

The Prelude" Workstation, which is capable of automating solid sample treatments and includes options such as weighing, mixing, filtration and solid-phase extraction of samples for automatic insertion into HPLC systems, transfer to UV-Vis spectrophotometers and gathering in an EasyFill Sample Collection Module. 

In an HPLC system, problems can arise from many sources. Malfunction can be allocated to various points. Chromatographers should use not only their experience to locate problems but also aU their senses (obviously, except taste) to identify LC problems. For example, a leak can be noticed by smeU before it is actually seen. A strange noise indicates some kind of malfunction and a hot smell indicates an overheating module. Most problems, however, are identified by sight, and they can mainly be observed as changes in the chromatogram. As soon as the problem has been defined, actions should be taken to correct the malfunctioning component. The incident should be recorded, in a log book kept for this purpose, to help with further failure problems at a later time. 

Figure 4.18. Graphical user interface of Agilent ChemStation showing the status of various HPLC modules in the system and a real-time chromatographic plot.

This section summarizes best practices and standard procedures used by experienced practitioners in HPLC system operation. The information is presented as check lists categorized by the module or the column. An additional section... 

McGuire, M. Wong, S.H. Skrinska, V. Fogelman, K. Miles, W. Totally automated sequential analysis of tricychc antidepressants hy SPE module system and reversed-phase HPLC analysis with a hase-deactivated C18 column. J.Anal.Toxicol., 1996, 20, 65 [SPE]... 

---