High-performance liquid chromatography (HPLC) is one of the most powerful tools in analytical chemistry. It has become a very common laboratory instrument that in fact it is the third most common instrument found in laboratories, trailing behind the balance and pH meter. HPLC has the ability to separate, identify, and quantitate compounds over a wide range of polarity that are present in any sample that can be dissolved in a liquid. Today, compounds in trace concentrations as low as parts per trillion (ppt) may easily be identified with the appropriate detector. HPLC can be applied to just about any sample, such as pharmaceuticals, environmental, forensic, food, nutraceuticals, and industrial chemicals.
In HPLC, separation is achieved through the distribution of the analytes between a stationary phase packed in a column and a liquid mobile phase that is pumped through the column. Traditionally, the stationary phase was polar, e.g., silica, and the mobile phases used were relatively non-polar. This HPLC mode is called normal phase. Today though, reversed phase HPLC is more common, accounting for over 75% of separations being done. In reversed phase HPLC, the column is packed with hydrophobic particles, for example a C-18 bonded phase. The mobile phase is any miscible combination of water (or water modified with acid or base, or buffers) and an organic solvent like methanol and acetonitrile. Other HPLC modes include:
- Ion-exchange chromatography
The stationary phase is cation-exchange or anion-exchange resins that have fixed charges which attract the ions of opposite charge. Separation is effected by the strength of attraction of the analyte to the resin, which in turn is based on the size and charge on the analyte.
- Size exclusion chromatography
The column contains particles with defined pore size that allow only small molecules to penetrate them. The larger molecules in the sample will then reach the detector first, followed by the smaller ones, then the solvent molecules.
- Affinity chromatography
The column contains analyte-specific or group-specific affinity ligand. The analyte recognizes and binds to the ligand in a very specific manner. Another type of HPLC is called denaturing HPLC (DHPLC), which is used in determining single nucleotide polymorphisms (SNPs), the most common type of genetic variation. In DHPLC, the stationary phase has differential affinity for single and double-stranded DNA. At present, there are two competing columns available for DHPLC analysis: The original column is packed with alkylkated poly(styrene-divinylbenzene) particles, and the other one is alkylated silica.
An HPLC system is composed of six basic modules that are connected by appropriate tubing and fittings: reservoir, pump, injector, column, detector, and data system (Figure 1).
Figure 1: Block diagram of an HPLC system
A reservoir holds the solvent (also called the “mobile phase” because it moves). A high-pressure pump generates a specified flow rate of mobile phase. The injector introduces (injects) the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column is the heart of HPLC, since it contains the chromatographic packing material (stationary phase) needed for the separation to take place. A detector “sees” the separated compound bands as they elute from the HPLC column. Since sample compound characteristics can be very different, several types of detectors are available. The UV-absorbance detector is very common, and is useful for compounds that can absorb ultraviolet light. If the compound fluoresces, a fluorescence detector is used. If the compound does not have either of these characteristics, a more universal type of detector is used, such as an evaporative-light-scattering detector (ELSD). These days, it is common to couple a mass spectrometer to an HPLC system (a technique called LC/MS). The mobile phase exits the detector and goes to waste, or can also be collected.
In modern HPLC systems, the operation of the HPLC is controlled via a computer (data system), which also records the electrical signal needed to generate the chromatogram. A chromatogram is a representation of the separation that has taken place in the column. It consists of a series of peaks rising from a baseline drawn on a time axis. Each peak represents the detector response for a different compound.
There are two basic elution modes in HPLC: isocratic and gradient. In the isocratic mode, the mobile phase composition remains the same throughout the run. In gradient elution, the mobile phase composition changes during the separation. This mode is useful for samples that contain compounds with wide range of polarity. As the separation proceeds, the elution strength of the mobile phase is increased (that is, increasing the amount of organic solvent) in order to elute the more strongly retained sample components.
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