High Performance Liquid Chromatography (HPLC Instrument)

High Performance Liquid Chromatography (HPLC) which was once called High Pressure Liquid Chromatography is a form of liquid chromatography used to separate compounds that are dissolved in solution with the help of HPLC instrument. HPLC technique is used in biochemistry which is a branch of biotechnology dealing with the study of chemicals used in biological processes. Thus High Performance Liquid Chromatography (HPLC) Instrument is an important biotechnology lab equipment.

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What is High Performance/ Pressure Liquid Chromatography

High Performance Liquid Chromatography or HPLC, is a chromatographic technique- in fact, the most widely used analytical separations technique- that can separate a mixture of compounds using a liquid mobile phase to separate components of mixture with the help of high pressure that pushes solvent through the column. There are many reasons for which HPLC is preferred by the researchers. These include:

• High Performance Liquid Chromatography has ready adaptability to accurate quantitative determination.
• HPLC is also suitable for separating nonvolatile species or thermally fragile species.
• HPLC technique can be widely applied to substances that are of interest to industry, various fields of science, as well as to the public.
• It is ideally suited for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and many other inorganic substances.

High Performance Liquid Chromatography Applications

High Performance Liquid Chromatography or HPLC is ideal for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and many other inorganic substances. Some of the HPLC applications include the following fields.

• Drug analysis
• Toxicology
• Explosives analysis
• Ink analysis
• Forensic applications involving fibers and plastics etc.

HPLC Instrument Components

The components of HPLC instrument include a reservoir of mobile phase or solvent delivery system, a pump, an injector, a separation column, and a detector. The compounds are separated by injecting a plug of the sample mixture onto the column. Different components in the mixture pass through the column at various rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase.

HPLC Solvent Delivery System- A mobile phase is pumped under pressure from one or multiple reservoir which flows through the column at a constant rate.

HPLC Pump- A pump is a significant component of high performance liquid chromatography instrument as its performance directly affects retention time, reproducibility and detector sensitivity. Three types of pumps are generally used in HPLC equipment to propel the liquid mobile phase through the system. These are - displacement pump, reciprocating pump, and pneumatic or constant pressure pump.

HPLC Injection System- Quality HPLC instruments have a narrow plug for inserting sample onto the pressurized column. The sample is inserted into the injection port of HPLC instrument through three ways- loop injection, valve injection and on column injection. In "loop injection," a fixed amount of volume is injected through fixed volume loop injector. In "valve injection," a variable volume is injected through an injection valve and in the "On column injection," a variable volume is injected by a syringe through a septum.

HPLC Chromatographic Column- The column of HPLC lab instrument is made up of heavy glass or stainless steel tubing so that it may withstand the high pressure. These columns are generally 10-30 cm long and 4-10 mm inside diameter containing stationary phase at particle diameter of 25 µm or less.

HPLC Detectors- The detectors in HPLC instrument monitor the mobile phase as it merges from the column. There are two general types of detectors used in high performance liquid chromatography instruments- bulk property detectors and solute property detectors. The bulk property detectors compare overall changes in the physical property of the mobile phase with and without an eluting solute such as refractive index, dielectric constant or density. The solute property detectors respond to the physical property of the solute which is not exhibited by the pure mobile phase such as UV absorbance, fluorescence or diffusion current. These detectors are more sensitive when giving a detectable signal for a few nanograms of sample.

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