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In chromatography, the retardation factor (R) is the fraction of an analyte in the mobile phase of a chromatographic system. [1] In planar chromatography in particular, the retardation factor R F is defined as the ratio of the distance traveled by the center of a spot to the distance traveled by the solvent front. [2]
The CRFs in thin layer chromatography characterize the equal-spreading of the spots. The ideal case, when the RF of the spots are uniformly distributed in <0,1> range (for example 0.25,0.5 and 0.75 for three solutes) should be characterized as the best situation possible.
The response factor can be expressed on a molar, volume or mass [1] basis. Where the true amount of sample and standard are equal: = where A is the signal (e.g. peak area) and the subscript i indicates the sample and the subscript st indicates the standard. [2]
In contrast to the similar concept called Retention uniformity, R d is sensitive to R f values close to 0 or 1, or close to themselves. If two values are not separated, it is equal to 0. For example, the R f values (0,0.2,0.2,0.3) (two compounds not separated at 0.2 and one at the start ) result in R D equal to 0, but R U equal to 0.3609.
The relative mobility (called Rf value or Rm value) is defined as the distance migrated by the protein band divided by the distance migrated by the buffer front. The distances are each measured from the beginning of the separation gel. The migration of the buffer front roughly corresponds to the migration of the dye contained in the sample buffer.
In most drug and pharmacokinetic studies, animals like rats, are subjected to a new drug in order to probe how the substance metabolizes in the body. By analyzing the rat’s urine or plasma with a triple quadrupole coupled to liquid chromatography, the concentration and fragmentation pattern of the new drug can be determined. [8]
The spot capacity (analogous to peak capacity in HPLC) can be increased by developing the plate with two different solvents, using two-dimensional chromatography. [8] The procedure begins with development of a sample loaded plate with first solvent. After removing it, the plate is rotated 90° and developed with a second solvent.
Chromatographic peak resolution is given by = + where t R is the retention time and w b is the peak width at baseline. The bigger the time-difference and/or the smaller the bandwidths, the better the resolution of the compounds.