High Performance Liquid Chromatography (HPLC) is an analytical method used to identify the components of complex mixtures or compounds. The technology was developed in the 1980s with the development of software and different types of columns used for complex analysis. HPLC is predominantly used in industries such as pharmaceutical, environmental, food, drink and energy, and for research and development. The HPLC instrument consists of a solvent reservoir, degasser (pump), injector, detector, integrator and column, the last of which is a temperature-control oven. The principal manufacturers of HPLC instrumentation are Waters, Argilent, Shimadzu and Hewlett Packard. All HPLC instruments can function with different detectors and software.
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Ultraviolet (UV) detectors are cost-effective and popular and are widely used in industry. This type of detector responds to substances that absorb light. The UV detector is mainly used in biomedical and pharmaceutical science to separate and identify the principal active components of a mixture. UV detectors are the most versatile, having the best sensitivity and linearity. UV detectors cannot be used for testing substances that are low in chromophores (colourless or virtually colourless) as they cannot absorb light at low range.
The fluorescence is the most sensitive HPLC detector, used almost exclusively in liquid chromatography (LC). This is a specific detector that senses only those substances that emit light. This detector is popular for trace analysis in environmental science. As it is very sensitive, its response is only linear over a relatively limited concentration range. As there are not many elements that fluoresce (undergo fluorescence or become fluorescent), scientists need to synthesise samples to make them detectable.
The mass spectrometry detector coupled with HPLC is called HPLC-MS and forms one of the best analytical tandems. HPLC-MS is the most powerful detector, particularly relevant for medical science and widely used in pharmaceutical laboratories and research and development. The principal benefit of HPLC-MS is that it is capable of analysing and providing molecular identity of a wide range of components.
Refractive Index (RI) Detection
The refractive index (RI) detector uses a monochromator and is one of the least sensitive LC detectors. This detector is extremely useful for detecting those compounds that are non-ionic, do not absorb ultraviolet light and do not fluoresce.
Samples examined with this detector are sugar, alcohol, fatty acid and polymers.
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