HPLC Technique

HPLC stands for high-performance liquid chromatography, a standard qualitative and quantitative analysis procedure used in analytical labs. HPLC is reliable and repeatable, which makes it the preferred technique for separating, identifying and quantifying molecules from complex mixtures of both chemical and biological components.

Four Components

• The HPLC technique contains four major components. First, the solvent delivery system (the mobile phase), which carries the samples through the column, is important, as the polarity affects the system's ability to separate the molecules. The chromatography column is usually a vertical glass column that is filled with an adsorbant. Second, the adsorbant (also known as the stationary phase, since it does not move) is commonly silica gel or alumina. Third, the sample introduction system consists of some sort of injection port to introduce the sample to be analyzed into the solvent delivery system. The fourth component is the detector.

Detectors

• The type of detector used depends upon the type of sample the technicians are running and what molecules they are trying to isolate. The criteria the technician uses when choosing a detector include selectivity, signal-to-noise ratio, detection limit, linear range, time constant, drift and cell volume. Commonly used detectors include ultraviolet-visible detectors, mass spectrometers, refractive indexes, fluorescence detectors or evaporative light scattering devices.

Procedure

• Turn on the detector, the pumps (which are used to add pressure to move the mobile phase through the stationary phase), and the computer that will record and print the chromatogram. Choose the appropriate programs in order to collect the desired data. Inject the sample into the injection port. Keep an eye on the pressure. Each type of column has a maximum pressure; do not exceed this or separation will not occur. Print out the chromatogram and analyze the results. At the end of each run, it's important to flush out the injection port to prevent buildup, which can create blockages.

Types

• Hydrophilic interaction chromatography uses a bonded polar stationary phase and a nonpolar, water-miscible mobile phase to separate molecules based on polarity. This technique has the ability to separate acidic, basic and neutral samples in a single chromatogram.
  Normal phase chromatography uses a polar stationary phase and a nonpolar, nonaqueous mobile phase to separate structural isomers (molecules with the same molecular formula that have atoms bonded together in different ways).
  Reversed phase chromatography is the most widely used type of HPLC. It uses a nonpolar stationary phase and an aqueous somewhat polar mobile phase. This method is commonly used by pharmaceutical companies to qualify drugs before release.
  Size exclusion chromatography separates molecules based on their size and can also determine the structure of proteins.
  Ion exchange chromatography is based on the attraction between the ions in the solute and the charged sites bound on the stationary phase.

Uses

• HPLC is useful in a variety of applications. It can be used to analyze pharmaceutical materials, which is helpful in researching new pharmaceuticals as well as purifying existing ones. It is also used to analyze biological fluids, pesticides, explosives and other materials. Although it is a popular technique for biomedical, biochemical and pharmaceutical research, it is also used in the cosmetics, food, environmental and energy industries.