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Another method, mass spectrometry, has certain advantages over other techniques. Mass spectra could be obtained rapidly; only small amount (sub-μg) of sample is required for analysis, and the data provided by the spectra is very informative of the molecular structure. Mass spectrometry also has strong advantages of specificity and sensitivity compared with other detectors. The combination of HPLC-MS is oriented towards the specific detection and potential identification of chemicals in the presence of other chemicals. However, it is difficult to interface the liquid chromatography to a mass-spectrometer, because all the solvents need to be removed first. The common used interface includes electrospray ionization, atmospheric pressure photoionization, and thermospray ionization.
Flow rate shows how fast the mobile phase travels across the column, and is often used for calculation of the consumption of the mobile phase in a given time interval. There are volumetric flow rate U and linear flow rate u. These two flow rate is related by [link] , where A is the area of the channel for the flow, [link] .
The retention time (t R ) can be defined as the time from the injection of the sample to the time of compound elution, and it is taken at the apex of the peak that belongs to the specific molecular species. The retention time is decided by several factors including the structure of the specific molecule, the flow rate of the mobile phase, column dimension. And the dead time t 0 is defined as the time for a non-retained molecular species to elute from the column.
Retention volume (V R ) is defined as the volume of the mobile phase flowing from the injection time until the corresponding retention time of a molecular species, and are related by [link] . The retention volume related to the dead time is known as dead volume V 0 .
The migration rate can be defined as the velocity at which the species moves through the column. And the migration rate (U R ) is inversely proportional to the retention times. If only a fraction of molecules that are present in the mobile phase are moving. The value of migration rate is then given by [link] .
Capacity factor (k) is the ratio of reduced retention time and the dead time, [link] .
In the separation, the molecules running through the column can also be considered as being in a continuous equilibrium between the mobile phase and the stationary phase. This equilibrium could be governed by an equilibrium constant K, defined as [link] , in which C mo is the molar concentration of the molecules in the mobile phase, and C st is the molar concentration of the molecules in the stationary phase. The equilibrium constant K can also be written as [link] .
The most important aspect of HPLC is the high separation capacity which enables the batch analysis of multiple components. Even if the sample consists of a mixture, HPLC will allows the target components to be separated, detected, and quantified. Also, under appropriate condition, it is possible to attain a high level of reproducibility with a coefficient of variation not exceeding 1%. Also, it has a high sensitivity while a low sample consumption. HPLC has one advantage over GC column that analysis is possible for any sample can be stably dissolved in the eluent and need not to be vaporized.With this reason, HPLC is used much more frequently in the field of biochemistry and pharmaceutical than the GC column.
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