HPLC
High-performance liquid chromatography
(or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.
HPLC typically utilizes different types of stationary phases, a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase.
With HPLC, a pump (rather than gravity) provides the higher pressure required to propel the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography.
(1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.
Operation
The sample to be analyzed is introduced in small volume to the stream of mobile phase. The solution movement through the column is slowed by specific chemical or physical interactions with the stationary phase present within the column. The velocity of the solution moves depends on the nature of the sample and on the compositions of the stationary (column) phase. The time at which a specific sample elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered an identifying characteristic of a the given sample. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity giving the components less time to diffuse within the column, improving the chromatogram resolution. Common 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 sample components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.
The choice of solvents, additives and gradient depend on the nature of the column and sample. Often a series of tests are performed on the sample together with a number of trial runs in order to find the HPLC method which gives the best peak separation.
Throughout the molecular cluster a variety of HPLC systems is available. Please conctact the person responsible if you want to make use of a system. For general questions and repairs please contact Helene Amatdjais-Groenen.
Maybe a good start at Reverse Phase HPLC Basics