Operation
The sample to be separated and analyzed is introduced, in a discrete small volume, into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are functions of specific physical or chemical interactions with the stationary phase. The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called the retention time. The retention time measured under particular conditions is considered an identifying characteristic of a given analyte. The use of smaller particle size packing materials requires the use of higher operational pressure ("backpressure") and typically improves chromatographic resolution (i.e. the degree of separation between consecutive analytes emerging from the column). Common mobile phases used include any miscible combination of water with various organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see Normal-phase chromatography below). The aqueous component of the mobile phase may contain buffers, acids (such as formic, phosphoric or trifluoroacetic acid) or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant ("isocratic elution mode") or varied ("gradient elution mode") during the chromatographic analysis. Isocratic elution is typically effective in the separation of sample components that are not very dissimilar in their affinity for the stationary phase.
In gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times with high eluting strength producing fast elution (=short retention times). A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile (in water or aqueous buffer) and progress linearly to 95% acetonitrile over 5–25 minutes. Period of constant mobile phase composition may be part of any gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.
The composition of the mobile phase depends on the intensity of interactions between analytes and stationary phase (e.g. hydrophobic interactions in reversed-phase HPLC). Depending on their affinity for the stationary and mobile phases analytes partition between the two during the separation process taking place in the column. 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/acetonitrile gradient, more hydrophobic components will elute (come off the column) late, once the mobile phase gets more concentrated in acetonitrile (i.e. in a mobile phase of higher eluting strength).
The choice of mobile phase components, additives (such as salts or acids) and gradient conditions depend on the nature of the column and sample components. Often a series of trial runs are performed with the sample in order to find the HPLC method which gives the best separation.
Read more about this topic: High-performance Liquid Chromatography
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