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Introduction to HPLC
Chromatography encompasses a diverse group of methods that are utilized for the separation of closely related components of mixtures. In all chromatographic separations, the sample is transported within the mobile phase, which may be a gas (GC), a liquid (LC), or a supercritical fluid (SFC). In column chromatography, the stationary phase is contained within a narrow tube through which the mobile phase is forced by gravity or under pressure. The components of the mixture to be analyzed distribute themselves between the mobile phase and stationary phase in varying proportions. Compounds that interact strongly with the stationary phase migrate very slowly with the mobile phase; in contrast, compounds that are weakly retained by the packing material migrate rapidly with the mobile phase. As a consequence of the differences in mobility between the individual components of a mixture, the sample components are separated into discrete bands (or zones) that emerge from the column at specific ‘retention times’. These bands may be identified qualitatively and/or further analyzed quantitatively using an appropriate detector.
Early liquid chromatography was carried out in glass columns with diameters of 1 to 5 cm and lengths of 50 to 500 cm. The average diameter of the solid stationary phase particles was usually in the 100 to 200 micron range. Recent technology has allowed for the development of packing material with relatively small particle size diameter (3-10 micron). This technology resulted in the development of columns with very high efficiencies, and consequently has involved the use of more sophisticated instrumentation to perform at increased pressures and flows; hence the term High Performance Liquid Chromatography (HPLC).
The typical HPLC separation is based on the selective distribution of analytes between a liquid mobile phase and an immiscible stationary phase. The sample is first introduced by means of an injection port into the mobile phase stream that is delivered by a high-pressure pump. Next, the components of this sample mixture are separated on the column, a process monitored with a flow-through detector as the isolated components emerge from the column.
Today, HPLC is the most widely used analytical separation method. The method is popular because it is non-destructive and may be applied to thermally labile compounds (unlike GC); it is also a very sensitive technique since it incorporates a wide choice of detection methods. With the use of post-column derivatization methods to improve selectivity and detection limits, HPLC can easily be extended to trace determination of compounds that do not usually provide adequate detector response. The wide applicability of HPLC as a separation method makes it a valuable separation tool in many scientific fields.
Analytical
and Preparative Applications of LC
"If you can dissolve it, LC can resolve it."
Liquid chromatography is useful for a multitude of applications in industry and academia. Its use can be broken down into two classifications, analytical LC and preparative LC. In analytical LC the goal is identification and quantification of given components within a sample, usually in the picogram to milligram range. In preparative LC, the objective is to isolate or collect the separated components of the sample in the mg to kg range.
In industry, as well as the sciences analytical LC is employed for:
- Basic Research
- Quality Assurance
- Methods Development
Liquid chromatography is widely used in different types of industry:
- Life Sciences – proteins, nucleic acids, carbohydrates, lipids, metabolites.
- Pharmaceuticals
- Biotechnology
- Industrial Chemicals – fine chemicals, polymers, synthetic mixtures.
- Food and Agricultural Processing – plant products, agrochemicals.
The preparative capabilities of LC are applied at different levels or scales of isolation:
- Small-scale or semi-preparative (mg to g)
- Pilot-scale (g to kg)
- Production or process scale (kg to ton)
In Preparative LC, a partial list of specific needs or reasons to isolate or purify samples is as follows:
- Drug Efficiency Studies
- Full-scale Production of Drugs (e.g., interferon, insulin)
- Spectroscopy/Structure Elucidation
- Biological Screening
- Physical Testing
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