Recent Developments in Analytical Science - High Pressure Liquid Chromatography

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  • Published: Jul 18, 2016
  • Channels: Electrophoresis / Gas Chromatography / Proteomics & Genomics / Ion Chromatography / HPLC / NMR Knowledge Base / MRI Spectroscopy / X-ray Spectrometry / Raman / Base Peak / Proteomics / Atomic / Infrared Spectroscopy

High Pressure Liquid Chromatography

Scientists have just celebrated the 50th anniversary of HPLC but new innovations continue to be announced regularly. Novel stationary phases are being developed to give better performance in HPLC and ultrahigh pressure liquid chromatography and columns packed with superficially porous particles are becoming more popular, due to their lower operating pressures and higher efficiencies compared with the conventional totally porous particles.

Monolithic columns can be tailored for particular applications by tuning the sizes of the macro- and mesopores of the silica or polymeric material during production. With the ability to separate small and large molecules and operating over a relatively wide pH range at low pressures, the polymeric type has much potential, which has yet to be realised. It is more suited than silica for use in lab-on-a-chip and other micro-HPLC systems because the polymers can be produced directly in the separation channels.

For separations of polar and hydrophilic compounds, hydrophilic interaction liquid chromatography (HILIC) columns can be employed. They are regularly used for pharmaceuticals, which often have polar functional groups like amines. The polar stationary phase is produced from materials such as silica gel, zwitterionic compounds, amides and polyethyleneimine. Each of these types gives different selectivity, affording a degree of choice to the analyst.

One growing area is affinity chromatography which uses biological interactions as the basis of the trapping mechanism. Affinity ligands such as nucleic acids, lectins, antibodies and metal ion chelates are held on inert solid supports in the column. They are chosen to trap the analytes in a highly selective procedure. Bioaffinity chromatography, in which the ligand is a biomolecule such as a protein, is used to purify the modern type of pharmaceutical based on antibodies, peptides and proteins. It has been helped by the production of libraries of ligands to help researchers select the right one.45

Ion chromatography separates and measures ions, generally using modified polymeric resins as the mobile phase. One of the principle detectors measures electrical conductivity but atomic spectroscopy, mass spectrometry, fluorescence, luminescence and potentiometric methods are also suitable. The technique has been applied to the analysis of air, water and soil quality, the forensic detection of explosives, ions in foodstuffs, and the authentication of pharmaceuticals. The development of new stationary phases and the exploitation of hyphenated methods will be essential to reach ever lower detection limits.

The growth of 2D HPLC, in which two columns of different selectivity are connected in series, has continued in recent years. In comprehensive 2D LC, complex samples like plant extracts are analysed by sequentially transferring all of the fractions from the first column onto the second. Alternatively, heartcutting transfers a particular fraction to the second column and directs the other fractions to waste. This has been used to measure the contents of active ingredients and impurities in pharmaceuticals. In addition, an advanced mode allows multiple heartcutting from numerous peaks by parking additional cuts while their predecessors are being analysed in the second dimension.46

A number of conventional HPLC detectors have been employed routinely for many years but new ones continue to be developed. The corona charged aerosol detector is a universal detector based on the particle size and mobility of the analyte, rather than any physical property, generating equal responses for the same amounts of different analytes. So, chemists can quantify all of the analytes eluting from a column with just one detector. It has been demonstrated for many classes of compounds, including carbohydrates in edible plants, dissolved silicon and boron in seawater and pharmaceuticals.

Not all HPLC systems use a single detector. Splitting the effluent to different detectors allows analytes with different properties to be identified and measured. In HPLC-SPE-NMR, the fractions are trapped by SPE then eluted for NMR analysis, providing increased amounts of analytes compared with direct HPLC-NMR. Commercial LC-NMR-MS systems have been employed in drug discovery and metabolism and natural products analysis.

HPLC will continue to progress with the development of more columns using improved particle technology, improvements in UPLC systems, and miniaturisation, through the use of microfluidics and chip-based systems.

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