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I am grateful for all hints on other pitfalls and sources of error or on how to avoid them. XIV Introduction. John W. Dolan and Lloyd R.see url
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Paul C. Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. In chromatography, a physical separation method, the components of a mixture are partitioned between two phases. One of the phases stays in its place and is called the stationary phase, whereas the other moves in a denite direction and is called the mobile phase. According to the type of mobile phase we distinguish between gas chromatogra- phy, supercritical uid chromatography, and liquid chromatography. The separation is based upon the different partition coefcients of the sample components between the two phases.
It is helpful to divide the chromatographic column into small hypothetical units, the socalled theoretical plates. Within each plate a new partition equilibrium is established.
The narrower a theoretical plate, the more equilibrium processes can take place within a column of given length and the more demanding the separation problems which can be solved. The gure shows the separation of two compounds.
One of these prefers the mobile phase but also enters the stationary phase. For the other compound the preference is the other way round. Thanks to this large difference in their properties the two types of molecule can easily be separated. They are transported through the column by the ow of the mobile phase and thereby reach zones where new equilibria are formed again and again. This height depends on the packing quality of the column, on the mass transfer properties of the phases, and on the analytes involved.
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Plate height is a function of the particle diameter of the stationary phase. For good columns, plate heights are equal to ca. A ne packing, e. The column with the ne packing can therefore be used for more difcult separation problems. To judge a chromatogram it is necessary to calculate some data which can be easily obtained. In addition the breakthrough time or dead time, t0 , must be known although it can be a problem to measure it unambiguously.
In principle, the rst baseline deviation after injection marks t0. It depends only on the phase system the types of mobile and stationary phase and on the temperature. For HPLC separations should be 1. The plate number is a measure of the separation performance of a column. The equations given here are in principle only valid for symmetrical peaks.
From the plate number it is possible to calculate the height, H, of a theoretical plate e. The smaller a peak compared with its large neighbor the greater is the resolution necessary to separate them.
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It is important to realize that the resolution is inuenced by the three parameters. The separation factor has the largest effect. If a separation needs to be improved it is well worth the effort of increasing , although it is impossible to give a general proposal concerning how to do this. If the plate number is increased, the effect is only by the factor N; if the column length is, e.
Increasing the retention factor only has a notable inuence on resolution if k was small to start with. The upper gure presents several pairs of peaks separated with varying resolution. The graph below demonstrates how the resolution increases with increasing plate number for three different separation factors. The judgement and comparison of HPLC columns is best done with reduced, dimensionless parameters.
A test chromatogram is acquired which enables the theoretical plate number, N, of the column to be determined from a suitable peak with low tailing. It is also necessary to measure the breakthrough time, t0 , with a refraction index peak or with an otherwise suitable compound for reversed-phase separations, e. Lc is column length. It is, however, necessary to know the viscosity, , of the mobile phase.
Mixtures of water and organic solvents pass through a maximum of viscosity! It's quite readable and very thorough. I've used it to good effect for years. There are quite a number of smart people here Uwe, Tom, Bryan, Bruce, Merlin, Danko, and others come to mind who are willing to share. I haven't read Snyder's book yet just received it some weeks ago but for a really newbie maybe the following are a bit easier, giving an general overview and basic knowledge. I have to say that I just know the german version of the books, but I think the english doesn't differ a lot.