High-performance liquid chromatography (sometimes referred to as 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 to provide a characteristic retention time for the analyte. The detector may also provide additional information related to the analyte, (i.e. UV/Visspectroscopic 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. It is a form of liquid chromatography that utilizes smaller column size, smaller media inside the column, and higher mobile phase pressures.
With HPLC, a pump (rather than gravity) provides the higher pressure required to move 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.
Operation
The sample to be analyzed is introduced, in small volumes, into the stream of mobile phase. The solution moved through the column is slowed by specific chemical or physical interactions with the stationary phase present within the column. The velocity of the solution 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 given sample. The use of smaller particle size column packing (which creates higher back pressure) 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.
A further refinement of HPLC is to vary the mobile phase composition during the analysis; gradient elution. A normal gradient for reversed phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes; the gradient depends on how hydrophobic the sample is. The gradient separates the sample mixtures as a function of the affinity. This partitioning process is similar to that which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, more hydrophobic components will elute(come off the column) when the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase).
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.
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