High Performance Liquid Chromatography, also known as
High Pressure Liquid Chromatography and usually abbreviated as
HPLC, is a form of column chromatography used frequently in biochemistry and analytical chemistry. The analyte is forced through a column of the stationary phase in a liquid (mobile phase) at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to diffuse within the column. This leads to narrower peaks in the resulting chromatogram and thence to better resolution (it's easier to differentiate one peak from another) and sensitivity (tall, narrow peaks can be easier to discriminate from noise than shorter, broader peaks). Solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components. Advances in the HPLC technology have brought about the use of gradients in the mobile phase composition. A normal gradient might be 5 - 50% methanol (depending on how hydrophobic the analyte is) over 25 minutes. The gradient separates the analyte mixtures as a function of how well the changing solvent mobilizes the analyte. For instance, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) under conditions of relatively high methanol; whereas the more hydrophilic will elute under conditions of relatively low methanol. Whether one starts with high organic or low organic depends on the nature of the stationary phase and the analyte.
Stationary Phases -
Reverse phase HPLC - Traditionally High Peformence Liquid Chromatography stationary phases were polar, whereas so-called "reversed" phase (RP-HPLC) stationary phases are hydrophobic. On an RP-HPLC column, then, hydrophobic analytes would tend to be retained on the column, eluting more readily as the proportion of the hydrophobic component of the mobile phase is increased. Today RP-HPLC is by far the most common form of HPLC. It's a funny twist of history for the "reversed" mode to become the usual (normal) mode and the "normal" mode to become almost obscure such that many practitioners have never even seen a normal mode column. Standard-bore RP-HPLC has lower resolution than Gas Chromatography.
Normal Phase Chromatography - Normal phase chromatography retains analyte based on hydrophilicity. That is the more polar (~larger electric dipole) the analyte the more it will be retained. Often a gradient from a highly non-polar (hydrophobic) solvent like hexane to a moderately hydrophilic solvent like isopropanol is used in the analysis.
Size Exclusion Chromatography - In size exclusion chromatography smaller analytes are allowed to enter into small pores in the stationary phase while larger analytes are excluded. This results in a much shorter path length for the larger analytes that then elute early in the so called exclusion volume. The resolution of SEC is relatively low and careful column selection is important for each application since the SEC columns have a limited size range in which good separation is achieved.
Other Parameters -
Internal Diameter - The internal diameter (I.D.) of an HPLC column is a critical aspect that determines quantity of analyte that can be loaded onto the column and also influences sensitivity. Larger I.D. columns (>10 mm) are used to prepare (purify) usable amounts of material because of their large loading capacity. They are usually seen in industrial applications such as the purification of a drug product for later use. The next smaller column size are the analytical scale columns (4.6 mm). These have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector. Narrow-bore columns (1-2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, fluorescence detection or with other detection methods like liquid chromatography-mass spectrometry. The smallest size are the capillary columns (<0.3 mm) which are used almost exclusively with alternative detection means such as mass spectrometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ. The advantage of low I.D. columns is improved sensitivity (with the right detector) and lower solvent consumption at the expense of loading capacity.
Bead Size - Most tradiational HPLC stationary phases are attached to the outside of silica beads. These beads come in a variety of sizes. The most common bead size is the 5
m beads. Smaller beads generally provide more surface area and better chromatography but with signifcantly increased pressures. Larger beads are more often used in non-HPLC applications such as solid-phase extraction.
Pore Size - Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics especially for larger analytes.
See also -
Chromatography, an overview article covering different chromatography techniques
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