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is a family of analytical chemistry techniques for the separation of mixtures. It involves passing a sample in the "mobile phase", often in a stream of solvent, through the "stationary phase", some form of material that will provide resistance between the components of the sample and the material.
After the sample is flushed or displaced from the stationary phase, the different components will elute from the column at different times. The components with the least affinity for the stationary phase will elute first, while those with the greatest affinity for the stationary phase will elute last.
The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time ( tr ) . The time taken for the mobile phase to pass through the column is called ( to or tm )
RD to tr B tr A tr .
defined as the molar concentration of analyte in the stationary phase (Cx)s divided by the molar concentration of the analyte in the mobile phase (Cx)m . is termed the partition coefficient. K.X X X Distribution of analytes between phases [Cx]s K = ----------[Cx]m The equilibrium constant.
Vs + [Cx]m.Component Mol. in stationer phase [Cx]m .= ---------------------------------K (Vs/Vm) + 1 [Cx]s/[Cx]m . Vs/Vm + 1 . by (Cx)m. in mobile phase = -------------------------------∑ mol.Vm 1 1 = --------------------.Vm div. Vm = ----------------------------[Cx]s. Fraction in mobile phase: ∑ mol.
phase k’ is the measure of coloumn retention k’ = K Vs/Vm • K not depend to concentration • k’ depend to …… ? .[Cx]s/[Cx]m .= capacity factor = k’ ∑ mol. comp. phase -------------------------------------. comp. in stat. Vs/Vm = ∑ mol. in mob.
X µ flow X X L To = L/µ Tr = L/u [ 1+k’] .
.There are two theories of chromatography. •the plate theories and •rate theories.
the mobile and stationary phases. and is defined by the following equation: . The plate theory describes the chromotography system.Plate theory The plate theory of chromotography was developed by Archer John Porter Martin and Richard Laurence Millington Synge. as being in equilibrium. The partition coefficient K is based on this equilibrium.
species A elutes faster than species B. α = k‘B / k‘A When calculating the selectivity factor.We define a quantity called the selectivity factor. α . The selectivity factor is always greater than one. which describes the separation of two species (A and B) on the column. .
called theoretical plates. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 . Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves the column by transfer of equilibrated mobile phase from one plate to the next.The plate model supposes that the chromatographic column is contains a large number of separate layers.
. The effect is that one of the phases moves in steps across the other. It had a long series of 50 to 100 separators mounted on a motor-driven rack which shook all of them vigorously. with solutions of separated components eventually being poured out of the last separator in the series.If you need to separate two or more solutes with partition coefficients that are not very different. then tilted to pour one of the phases into the next separator (all 50 to 100 of them) for the next extraction step. That process was automated by Lyman Craig with the "Craig countercurrent distribution apparatus" (usually called a "Craig"). you will need many separatory funnels and many people to shake them. stopped to let the phases separate.
Craig Model Vs Vm .
you will need many plate numbers (N). If you have fix coloum. you can reduce H (heigt equivalent therotical plate) H = L/N .If you need to separate two or more solutes with partition coefficients that are not very different. and you need more N.
N=X ω N=2X N = 4X .
N = [4tr/ω]2 tr ω .
where w is the peak width. . then the HETP is HETP = L / N The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution.If the length of the column is L.
which assumes that equilibration is infinitely fast). .The Rate Theory of Chromatography The processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model.
A. B. . HETP = A + B / u + C u where u is the average velocity of the mobile phase.Van Deemter equation for plate height. and C are factors which contribute to band broadening.
because different paths are of different lengths. This will cause broadening of the solute band. The band of analyte is broadened. If the velocity of the mobile phase is high then the analyte spends less time on the column. the worse the broadening becomes. Solute molecules will take different paths through the stationary phase at random. B . and the analyte has a strong affinity for the stationary phase.Eddy diffusion The mobile phase moves through the column which is packed with stationary phase.Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase.A . The higher the velocity of mobile phase. . This causes band broadening. then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. which decreases the effects of longitudinal diffusion. Analyte diffuses out from the center to the edges. C . If the velocity of the mobile phase is high.Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center.
Van Deemter plots A plot of plate height vs. . average linear velocity of mobile phase. Such plots are of considerable use in determining the optimum mobile phase flow rate.
A and B.Resolution A measure of how well species have been separated is provided by measurement of the resolution. is defined as Baseline resolution is achieved when R = 1. The resolution of two species.5 .
Instead. An increase in N. the number of theoretical plates. . the selectivity factor and the retention factors of the two solutes. To obtain high resolution. the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles.which may not be desirable. the three terms must be maximised. to increase the number of plates.It is useful to relate the resolution to the number of plates in the column. by lengthening the column leads to an increase in retention time and increased band broadening .
optimising k' and increasing N is not sufficient to give good separation in a reasonable time. k' is optimised first. When α is close to unity. and then α is increased by one of the following procedures: • Changing mobile phase composition • Changing column temperature • Changing composition of stationary phase • Using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase) . The selectivity factor. In these cases. separations can be greatly improved.It is often found that by controlling the capacity factor. k'. α. can also be manipulated to improve separations. This can be achieved by changing the temperature (in Gas Chromatography) or the composition of the mobile phase (in Liquid Chromatography).
Elution chromatography can be carried out under •isocratic (constant mobile phase composition).• elution (isocratic and gradient). •gradient (continuous change in mobile phase composition) or step elution conditions. • frontal • displacement chromatography. .
. Components of a mixture may be interacting with the stationary phase based on charge. relative solubility or adsorption.Chromatography is a separation method that exploits the differences in partitioning behavior between a mobile phase and a stationary phase to separate the components in a mixture.
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