HPLC instrumentation trends

An overview of HPLC instrumentation, operating principles, and recent advances or trends that are pertinent to pharmaceutical analysis is provided in Chapter 3 for the novice and the more experienced analyst. Modern liquid chromatographs have excellent performance and reliability because of the decades of refinements driven by technical advances and competition between manufacturers in a two billion-dollar-plus equipment market. References to HPLC textbooks, reference books, review articles, and training software have been provided in this chapter. Rather than summarizing the current literature, the goal is to provide the reader with a concise overview of HPLC instrumentation, operating principles, and recent advances or trends that lead to better analytical performance. Two often-neglected system parameters—dwell volume and instrumental bandwidth—are discussed in more detail because of their impact on fast LC and small-bore LC applications. 

HPLC INSTRUMENTATION IN PHARMACEUTICAL ANALYSIS STATUS, ADVANCES, AND TRENDS... 

This chapter presents an overview of current trends in high-pressure liquid chromatography (HPLC) instrumentation focusing on recent advances and features relevant to pharmaceutical analysis. Operating principles of HPLC modules (pump, detectors, autosampler) are discussed with future trends. 

This chapter provides the novice and the more experienced analyst with an overview of HPLC instrumentation, operating principles and recent advances or trends that are pertinent to pharmaceutical analysis. 

Column dimensions—length (L) and column inner diameter (dc or i.d.)— control column performance (N, speed, sensitivity, sample capacity) and its operating characteristics (flow rate, back pressure). Designations of various column types based on column inner diameters and their associated characteristics are shown in Table 3.1. Note that void volume, sample capacity, and operating flow rate are proportional to (dc)2, while detection limit, or sensitivity, is inversely proportional to (dc)2. Note also that prep columns (>10mm i.d.), microbore (micro columns (<0.5 mm i.d.) will require specialized HPLC instruments (see Chapter 4). There is a definitive trend toward the increased use of shorter and smaller inner diameter analytical columns due to their higher sensitivity performance and lower solvent usage.9"11 This trend will be explored later. 

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... 

The diameter of HPLC columns may vary from several tens of micrometers to several tens of centimeters. Depending on the size of the separation column - nano-, capillary-, narrow-bore - analytical and preparative separations may be distinguished. A current trend in HPLC instrument development provides the hope that in time it may become feasible to use a column of any size with a single instrument. 

One of the trends in HPLC Instrumentation involves the Incorporation of image detectors In UV spectroscopy and, in particular, that of diode array photometric systems, whose optical scheme departs from the traditional as can be seen from Fig. 12.17. Thus, they have no monochromator the light beam im-... 

The recent development and comparative application of modern separation techniques with regard to determination of alkylphosphonic acids and lewisite derivatives have been demonstrated. This report highlights advantages and shortcomings of GC equipped with mass spectrometry detector and HPLC as well as CE with UV-Vis detector. The comparison was made from the sampling point of view and separation/detection ability. The derivatization procedure for GC of main degradation products of nerve agents to determine in water samples was applied. Direct determination of lewisite derivatives by HPLC-UV was shown. Also optimization of indirect determination of alkylphosphonic acids in CE-UV was developed. Finally, the new instrumental development and future trends will be discussed. 

As long as the health authorities accept 90-110% specification limits on the drug assay, the normalization method presented above will barely suffice for batch release purposes. Since there is a general trend toward tightening the specification limits to 95-105% (this has to do with the availability of improved instrumentation and a world-wide acceptance of GMP-standards), a move toward options 1 (HPLC) and 2 (DA-UV) above is inevitable. 

While typical flow rates for the HPLC analyses of flavonoids lie in the 1.0 to 1.5 ml/min range, the introduction of short columns containing stationary phases with smaller pore sizes (allowing narrower peaks to be obtained in shorter separation times) means that considerably lower flow rates are the trend. Not only is there a decrease in solvent consumption but coupling to mass spectrometers or NMR instruments is facilitated. 

The most common HPLC column diameter is 4.6 mm. There is a trend toward narrower columns (2 mm, 1 mm, and capillary columns down to 25 pm) for several reasons. Narrow columns are more compatible with mass spectrometers, which require low solvent flow. Narrow columns require less sample and produce less waste. Heat generated by friction of solvent flow inside the column is more easily dissipated from a narrow column to maintain isothermal conditions. Instruments must be specially designed to accommodate column diameters <2 mm or else band broadening outside the column becomes significant. 

This chapter provides an overview of modern HPLC equipment, including the operating principles and trends of pumps, injectors, detectors, data systems, and specialized applications systems. System dwell volume and instrumental bandwidth are discussed, with their impacts on shorter and smaller diameter column applications. The most important performance characteristics are flow precision and compositional accuracy for the pump, sampling precision and carryover for the autosampler, and sensitivity for the detector. Manufacturers and selection criteria for HPLC equipment are reviewed. 

Low-dispersion HPLC systems are necessitated by the increasing trend of using shorter and narrower HPLC columns, which are more susceptible to the deleterious effects of extra-column band-broadening. HPLC manufacturers are designing newer analytical HPLC systems with improved instrumental bandwidths compatible with 2-mm i.d. columns by using micro injectors, smaller i.d. connection tubing, and detector flow cells. A new generation of ultra-low dispersion systems dedicated for micro and nano LC is also available. 

Another current trend that is well underway is the use of more specific analytical instrumentation that allows less extensive sample preparation. The development of mass spectrometric techniques, particularly tandem MS linked to a HPLC or flow injection system, has allowed the specific and sensitive analysis of simple extracts of biological samples (68,70-72). A similar HPLC with UV detection would require significantly more extensive sample preparation effort and, importantly, more method development time. Currently, the bulk of the HPLC-MS efforts have been applied to the analysis of drugs and metabolites in biological samples. Kristiansen et al. (73) have also applied flow-injection tandem mass spectrometry to measure sulfonamide antibiotics in meat and blood using a very simple ethyl acetate extraction step. This important technique will surely find many more applications in the future. 

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