A HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced column and an apparatus for measuring the absorbance.

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. HPLC is used to separate components of a mixture by using a variety of chemical interactions between the substance being analyzed (analyte) and the chromatography column. HPLC Modern HPLC systems are highly automated User talk:Emilio8#Image:HPLC.jpg File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... HPLC Modern HPLC systems are highly automated User talk:Emilio8#Image:HPLC.jpg File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... Column chromatography in chemistry is the preparative application of chromatography. ... This article or section does not cite its references or sources. ... Analytical chemistry is the analysis of material samples to gain an understanding of their chemical composition and structure. ... An Analyte is the substance or chemical constituent that is undergoing analysis. ...

Principle

In isocratic HPLC the analyte is forced through a column of the stationary phase by introducing 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, or compounds such as Trifluoroacetic_acid. An Analyte is the substance or chemical constituent that is undergoing analysis. ... Column chromatography in chemistry is the preparative application of chromatography. ... Chromatography is a family of analytical chemistry techniques for the separation of mixtures. ... The use of water pressure - the Captain Cook Memorial Jet in Lake Burley Griffin, Canberra. ... This article or section does not cite its references or sources. ... A chemist is shown using column chromatographic apparatus in the mid-1950s to separate constituents in a coal tar color analysis Pictured is a sophisticated gas chromatography system. ... A solvent is a fluid phase (liquid, gas, or plasma) that dissolves a solid, liquid, or gaseous solute, resulting in a solution. ... Water (H2O, HOH) is the most abundant molecule on Earth, composing 70-75% of the Earths surface as liquid and solid state in addition to being found in the atmosphere as a vapor. ... Methanol, also known as methyl alcohol or wood alcohol, is a chemical compound with chemical formula CH3OH. It is the simplest alcohol, and is a light, volatile, colourless, flammable, poisonous liquid with a distinctive odor that is somewhat sweeter than ethanol (ethyl alcohol). ... Acetonitrile is an organic molecule, often used as a solvent, with the chemical formula of CH3CN. Also known as methyl cyanide, it is the simplest of the organic nitriles. ... Trifluoroacetic acid (TFA) is a strong, hygroscopic, non-oxidizing, organic acid with a molecular formula C2HF3O2. ...

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. Often a series of tests are performed on the analyte and a number of generic runs may be processed in order to find the optimum HPLC method for the analyte - the method which gives the best seperation of peaks. In chemistry, hydrophobic or lipophilic species, or hydrophobes, tend to be electrically neutral and nonpolar, and thus prefer other neutral and nonpolar solvents or molecular environments. ...

Types of HPLC

Normal phase chromatography

Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and separates analytes based on polarity or as structural isomers since it is an adsorptive separation mechanism. This method uses a polar stationary phase and a nonpolar mobile phase, and is used when the analyte of interest differs by polar nature or as geometric isomers. The polar analyte associates with and is retained by the polar stationary phase.

NP-HPLC had fallen out of favor in the 1970's with the development of reversed-phase HPLC because of a lack of reproducibility of retention times as water or protic organic solvents changed the hydration state of the silica or alumina chromatographic media. Recently it has become useful again with the development of HILIC bonded phases which utilize a partition mechanism which provides reproducibility. RPC can refer to: Remote procedure call, a protocol that allows a computer program running on one host to cause code to be executed on another host Revolutionary Policy Committee, a faction within the Independent Labour Party, a United Kingdom political party during the 1930s Rail Passengers Council, a network...

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 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. It has been suggested that strong base be merged into this article or section. ... Columns redirects here. ... In chemistry, a mixture is the product of a mechanical blending or mixing of chemical substances like elements and compounds, without chemical bonding or other chemical change, so that each ingredient substance retains its own chemical properties and makeup. ... Bipyridine is one of the simplest polypyridine compounds. ... Chelation (from Greek, claw like) describes the reversible binding of an organic ligand, the chelator or chelating agent, to a metal ion, forming a metal complex, the chelate. ... Hot metal work from a blacksmith In chemistry, a metal (Greek: Metallon) is an element that readily forms positive ions (cations) and has metallic bonds. ... An ion is an atom, group of atoms, or subatomic particle that normally is electrically neutral and achieve their status as an ion by loss (and addition) of an electron. ... The chemical compound silicon dioxide, also known as silica, is the oxide of silicon, chemical formula SiO2. ...

Size exclusion chromatography

Size exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of size. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, and is the primary technique for determining the average molecular weight of natural and synthetic polymers. Equipment for running size exclusion chromatography. ... Equipment for running size exclusion chromatography. ... Image resolution describes the detail an image holds. ... In biochemistry, the tertiary structure of a protein is its overall shape. ... In biochemistry, many proteins are actually assemblies of more than one protein (polypeptide) molecule, which in the context of the larger assemblage are known as protein subunits. ... A polymer is a long, repeating chain of atoms, formed through the linkage of many molecules called monomers. ...

Ion exchange chromatography

Ion-exchange chromatography allows the separation of ions and polar molecules based on the charge properties of the molecules. It can separate proteins based on their isoelectric points and is often used as a first step in protein purification. Both positively-charged and negatively-charged molecules can be separated based on their charge, meaning that this process is not just restricted to proteins. The ion-exchange chromatography process allows the separation of ions and polar molecules based on the charge properties of the molecules. ...

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.

In physics, spectrophotometry is the quantitative study of spectra. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... Liquid chromatography-mass spectrometry (LC-MS)is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (aka HPLC) with the mass analysis capabilities of mass spectrometry. ... Basic schematic of mass spectrometry Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio of ions. ... Fused quartz is a man-made material manufactured principally from sands. ...

Particle size

Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5μm beads being the most common. Smaller particles 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 particles 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 particles are more often used in non-HPLC applications such as solid-phase extraction. Solid-phase extraction (SPE) is an extraction method that uses a solid phase and a liquid phase to isolate one, or one type, of analyte from a solution. ...

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/in² (~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/in² (~100 MPa or about 1000 atmospheres). (Note that "UPLC" is a trademark of Waters Corporation although it is sometimes used generically.) An electric driven pump of water works nearby the Hengstey See, Germany A pump is a device used to move gases, liquids, or slurries. ... In fluid dynamics, the rate of fluid flow is the volume of fluid which passes through a given area per unit time. ... The use of water pressure - the Captain Cook Memorial Jet in Lake Burley Griffin, Canberra. ... Pounds-force per square inch (lbf/in²) is a non-SI unit of pressure. ...

Manufacturers of HPLC chromatographs

• Agilent Technologies [1] (formerly Hewlett-Packard)
• Eksigent Technologies [2]
• Gilson, Inc. [3]
• Micro-Tech Scientific [4]
• PerkinElmer, Inc. [5]
• Shimadzu Scientific Instruments [6]
• Thermo Electron Corporation [7]
• Varian, Inc.[8]
• Waters Corporation [9]

... PerkinElmer, Inc. ... Headquarters of Shimadzu Corporation in Nakagyo-ku, Kyoto, Japan Shimadzu Corportation ) (TYO: 7701 ) is a manufacturer of precision instruments, measuring instruments and medical equipment, based in Kyoto, Japan. ... Thermo Electron Corporation (Thermo) NYSE: TMO (incorporated 1956) is a major provider of analytical instruments and services for a variety of domains. ... Varian, Inc. ...

Manufacturers of HPLC columns and accessories

• Agilent Technologies
• Gilson, Inc. [10]
• Hitachi [11]
• Micro-Tech Scientific [12]
• Phenomenex [13]
• Shimadzu Scientific Instruments
• Sielc
• Supelco
• Thermo Electron Corporation [14]
• Varian, Inc.
• Waters Corporation

... Hitachi may refer to: Hitachi (train) trains in Melbourne, Australia. ... Phenomenex, Inc. ... Headquarters of Shimadzu Corporation in Nakagyo-ku, Kyoto, Japan Shimadzu Corportation ) (TYO: 7701 ) is a manufacturer of precision instruments, measuring instruments and medical equipment, based in Kyoto, Japan. ... Thermo Electron Corporation (Thermo) NYSE: TMO (incorporated 1956) is a major provider of analytical instruments and services for a variety of domains. ... Varian, Inc. ...

See also

• Chromatography
• Ion exchange chromatography
• Size exclusion chromatography

A chemist is shown using column chromatographic apparatus in the mid-1950s to separate constituents in a coal tar color analysis Pictured is a sophisticated gas chromatography system. ... The ion-exchange chromatography process allows the separation of ions and polar molecules based on the charge properties of the molecules. ... Equipment for running size exclusion chromatography. ...

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
• Novasep - Description of various industrial chromatography technologies