High performance liquid chromatography
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High performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. It is sometimes referred to as high pressure liquid chromatography but this is primarily a historical term and not widely accepted today. HPLC is used to separate components of a mixture based on a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column.
Principle
In isocratic HPLC the analyte is forced through a column of the stationary phase by a liquid at high pressure. Use of pressure gives the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. 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.A further refinement to HPLC has been to vary the mobile phase composition during the analysis, this is known as gradient elution. A normal gradient for reverse phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes, depending on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of how well the changing solvent mobilizes the analyte. In this example, 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. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte.
Types of HPLC
Normal phase chromatography
Normal phase HPLC (NP-HPLC) was the first kind of HPLC setup used, and retains analyte based on polarity. This method uses a polar stationary phase and a nonpolar mobile phase, and is used when the analyte of interest has a polar nature. The polar analyte associates with and is retained by the polar stationary phase. NP-HPLC has fallen out of favor recently with the development of reversed phase HPLC.Reversed phase chromatography
The reversed phase HPLC (RP-HPLC) consists of a nonpolar stationary phase and a polar mobile phase, and was developed due to the increasing interest in large nonpolar biomolecules. One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. The retention time is therefore longer for molecules which are more non-polar in nature, allowing polar molecules to elute more readily. Today, in an ironic twist, RP-HPLC is by far the most common form of HPLC.Reversed phase columns are quite difficult to damage compared with normal silica columns, however, they should never be used with strong aqueous bases as these will destroy the silica. They can be used with aqueous acid but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine. Because the 2,2'-bipy can chelate the metal it is normal that when a metal ion is present on the surface of the silica the shape of the peak for the 2,2'-bipy will be distorted, tailing will be seen on this distorted peak.
Size exclusion chromatography
- For more details on this topic, see Size exclusion chromatography.
Ion exchange chromatography
- For more details on this topic, see Ion exchange chromatography.
Parameters
Internal diameter
The internal diameter (ID) 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 columns are usually seen in industrial applications such as the purification of a drug product for later use. Low ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.- Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity.
- Analytical scale columns (4.6 mm) 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
- Capillary columns (under 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.
Bead size
Most traditional HPLC is performed with the stationary phase attached to the outside of spherical silica beads. These beads come in a variety of sizes with 5[\mu]m beads being the most common. Smaller beads generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter cubed. This means that changing to beads that are half as big in the same size of column will double the performance, but increase the required pressure by a factor of eight. 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. For example a protein which is only slightly smaller than a pore might enter the pore but not easily leave once inside.Pump pressure
Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure may reach as high as 6000 lbf/in³ (~40 MPa, or about 400 atmospheres). Modern HPLC systems have been improved to work at much higher pressures, and therefore be able to use much smaller particle sizes in the columns (< 2 micrometres). These "Ultra Performance Liquid Chromatography" systems or UPLCs can work at up to 15,000 lbf/in³ (~100 MPa or about 1000 atmospheres).See also
External links
- [HPLC Find] - A directory of HPLC resource sites on the web
- [LC Resources ChromFAQ] - A compilation of frequently asked questions about HPLC
- [Chromatography Forum] - A free on-line chromatography discussion group
- [Overview of HPLC Suppliers] - A HPLC specialized search engine
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