High Performance Liquid Chromatography : Module 2

Liquid Chromatography Evolution

     “Learn something new. Try something different. Convince yourself that you have no limits “

                                                                                                                                   — Brian Tracy

Column Chromatography


High Performance Liquid Chromatography

Liquid Chromatography originated in early 1900 when Russian botanist Mikhail S. Tswett separated plant pigments using calcium carbonate packed glass columns. It was not until mid century that the technique was applied to develop Paper Chromatography, HPLC and GC.

HPLC was originally referred to as High Pressure Liquid Chromatography as high pressure was required to allow liquid to flow through packed columns. However, with continued advances in instrumentation and packing materials the name was changed to High Performance Liquid Chromatography leading to improvements in separation, identification, purification and quantification of complex molecules over previously known techniques.

Advances in pump technology contributed to greater control and flexibility of mobile phase composition. Isocratic operation maintains same mobile phase composition throughout the analytical run whereas gradient elution mode permits composition programming as per analysis requirements.

Column efficiency was known to increase with reduction in particle size. However, non-availability of technology for manufacture of small size particles held back progress in this direction. In 1990’s particle sizes in the range of 3-5 µ m were in use. The sub – 2 µ m barrier was broken in 2003 and at Pittcon 2005, columns packed with sub – 2µ m particles were demonstrated. This development launched the era of UPLC (Ultra Pressure Liquid Chromatography) or Fast LC. It became possible to reduce column lengths and diameters to give high separation efficiencies. The advantages could be realized only after technological advancements in instrumentation which permitted operations at high pressures as well as high speed detectors and electronics for capturing fast signals and narrow peaks.

In the recent years there has been increasing interest in the synthesis and separation of Enantiomers due to their importance in biochemistry and pharmaceutical industry. Conventional chromatography could not separate Enantiomers but Chiral Chromatography offered this option for both analytical and preparative scale applications.

In the following years the growth in applications has been phenomenal in areas such as Pharmaceuticals, Life sciences, Foods, Polymers and Forensics. Pharmaceuticals industry alone accounts for 35% of all HPLC instruments and is poised to continue as the biggest end use market. Major advances in  LC-MS and fast LC have further expanded the scope of applications in addition to advantages of speed, sensitivity and cost saving on expensive solvents. HPLC is set to dominate the analytical instruments market in future as well.  The driving force has been the large increase in number of Quality Control, Method Development and drug development laboratories. Demand has been further fuelled by emergence of CRO’s and Bioequivalence studies laboratories.

We hope that so far we have captivated your interest and now you would be looking forward to learn about the basic components of HPLC system. The next module will help you understand the individual parts of a HPLC system.

Vice President - Training & Development




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