HPLC analysis (high performance liquid chromatography) is a variety of chromatography which is often used in biochemistry and analytical chemistry laboratories whose purpose is to identify, separate and quantify compounds in a sample.
The method is a type of column chromatography using a column which contains a chromatographic packing known as the stationary phase, a pump whose purpose is to move the mobile phase or phases through the column and a detector which is used to determine and display the retention time of the compounds in the column. The retention time (which is the time elapsed from introduction to elution) will vary based on the interactions of the analyte, the solvents used and the stationary phase in the column.
In an HPLC, GC, GCMS analysis labs in Chennai procedure, a small volume of the sample is introduced to the mobile phase stream; the pace of progress of the analyte through the column will be quicker or slower depending on the physical and/or chemical interactions between the analyte and the stationary phase as the sample travels through the column. As stated above, the time between introduction of the sample and elution is referred to as the retention time; this time provides a relatively unique characteristic of the analyte which is useful in identification of the compounds contained in the sample.
In order to create a higher back pressure and a higher linear velocity of the analyte through the column, the stationary phase used in columns for HPLC analysis applications tends to be in small particle sizes. This gives the compounds in the sample being analyzed less time to diffuse; meaning that the resulting chromatogram will be of higher resolution.
The solvents commonly used in HPLC procedures are water combined with methanol or acetonitrile, though any miscible combination of water and/or organic solvents may be used depending on the specific requirements of the application. The water used as a solvent often contains salts or chemical buffers added to assist in separating the constituent components of the analyte. Ion pairing agents like trifluoroacetic acid are also commonly used.
The mobile phase composition may be varied during the HPLC analysis, a practice known as gradient elution. In a reversed phase liquid chromatography process, a typical gradient may be 5% methanol in water at the beginning of the procedure, with a linear progression to 50% methanol in water over the space of 25 minutes. The exact gradient used is dependent on how hydrophobic (or hydrophilic) the analyte is. The analyte mixtures are separated by the gradient as a function of how strong of an affinity the analytes has for the mobile phase composition relative to the composition of the stationary phase.
The process of portioning in a gradient elution HPLC analysis procedure is similar to what is seen in liquid-liquid extraction, but rather than being sequential, the gradient elution method results in a continuous partitioning. With a water/methanol gradient, the components of an analyte which are more hydrophobic will elute when the gradient of the mobile phase is higher in ethanol; the more hydrophilic compounds elute when the gradient of the mobile phase is at a point of lower methanol and higher water content.
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