Distribution coefficients column chromatography

The capacity ratio of a solute (k ) was introduced early in the development of chromatography theory and was defined as the ratio of the distribution coefficient of the solute to the phase ratio (a) of the column. In turn the phase ratio of the column was defined as the ratio of the volume of mobile phase in the column to the volume of stationary phase in the column. 

The upper curve shows the adsorption isotherm that normally occurs in liquid chromatography separations where the concentration of solute in the system is very low. The isotherm is linear and thus the distribution coefficient is constant at all concentrations of solute in either phase. It follows that as the peak velocity is inversely related to the distribution coefficient, all solute concentrations travel at the same velocity through the column and the peak is symmetrical. 

In chromatography the components of a mixture are separated as they pass through a column. The column contains a stationary phase which may be a packed bed of solid particles or a liquid with which the packing is impregnated. The mixture is carried through the column dissolved in a gas or liquid stream known as the mobile phase, eluent or carrier. Separation occurs because the differing distribution coefficients of the components of the mixture between the stationary and mobile phases result in differing velocities of travel. 

Gel Permeation Chromatography. Samples were filtered on columns of Bio-Gel P-6DG (Bio-Rad Laboratories), and columns of Sephadex G-10, G-25, and G-50 (Pharmacia Corp.) using deionized water as eluant. Gel filtration properties are expressed in terms of the distribution coefficient calculated from the relationship = (V - V )/ (V - V ) where the the volume at which a component elutes, is the void volume, and is the total volume of the system. Blue dextran 2,000 and xylose were used to determine and Vj respectively. 

In gas or liquid column chromatography, the adsorbent particles are packed into a column, after which a small amount of fluid containing several solutes to be separated is applied to the top ofthe column. Each solute in the applied fluid moves down the column at a rate determined by the distribution coefficient between the adsorbent and the fluid and emerges at the outlet of the column as a separated band. Liquid column chromatography is the most common method used in the separation of proteins and other bioproducts. 

A chromatography column of 10 mm i.d. and 100 mm height was packed with particles for gel chromatography. The interparticle void fraction e was 0.20. A small amount of a protein solution was applied to the column and elution performed in an isocratic manner with a mobile phase at a flow rate of 0.5 cm min. The distribution coefficient A of a protein was 0.7. An elution curve of the Gaussian type was obtained, and the peak width W was 1.30 cm . Calculate the Hs value of this column for this protein sample. 

Any chromatographic process is associated with the distribution of the analyzed substance between the mobile and the stationary phase. In liquid chromatography the solvent, with the volume V0 in the interparticle space, moving along the column at a certain speed, is the mobile phase, and a porous adsorbent, with an overall pore volume of V, is the stationary phase. The distribution coefficient, Kd, equal to the ratio between the concentration of the substance in the stationary and the mobile phase, determines the retention volume, VR, of a given substance in the column according to the basic chromatographic equation... 

The concept of plate theory was originally proposed for the performance of distillation columns (12). However, Martin and Synge (13) first applied the plate theory to partition chromatography. The theory assumes that the column is divided into a number of zones called theoretical plates. One determines the zone thickness or height equivalent to a theoretical plate (HETP) by assuming that there is perfect equilibrium between the gas and liquid phases within each plate. The resulting behavior of the plate column is calculated on the assumption that the distribution coefficient remains unaffected by the presence of other... 

Zone chromatography is a variant of the zone melting method, in which the mixture being separated is introduced into a column with a solid solvent and a molten zone is passed repeatedly along the length of the column to separate mixtures into separate bands of their components. Zone chromatography has been used for the separation of mixtures of lanthanides for preparative and analytical purposes The chelates used were mixtures of hydrated / -diketonates and their adducts with 2,2 -bipyridyl (bipy) and acety-lacetonimines. The distribution coefficients of different chelates and binary mixtures have been determined . 

Following elution of the isotachic train and the displacer solution from the column, the column must be regenerated and reequilibrated with the carrier before any subsequent displacement separation. This reequilibration step can be lengthy and is frequently considered a major Umitation to efficient displacement operation. Displacement chromatography requires the competitive isotherms of the solutes and the displacer to be convex upward and to not intersect each other. (See the entry Distribution Coefficient for related information.)... 

The second analytical procedure is somewhat similar, but a continuous stream of gas is passed over the sample and through the trap. This produces a much larger sample of the volatile substances of interest and, thus, can often detect trace materials. The adsorbed components are desorbed by heat in the same manner and passed directly onto a gas chromatography (GC) column. The results are stiU determined by the distribution coefficient of each solute between the sample matrix and the air and, thus, the quantitative results remain comparative or relative, but not absolute. 

A distribution coefficient is used widely in various areas involving two-phase systems [43,44] to describe behaviour of immobilized enzymes, electrode systems, different kinds of chromatographic separation and, in particular, makes it possible to correlate analytically parameters describing equilibria on a surface with parameters of column and thin-layer chromatography, whose success is determined mostly by extensive use of pristine and modified silicas as adsorbents and supports. 

The effect of N, or plate number, becomes important when considering both the V/, volume and the elution volume in SPE. Figure 4.12 shows how the breakthrough curve varies with plate number. The values are taken from a technical brochure by Waters Chromatography that compares several of their SPE cartridges with different particle sizes. The for both columns was the same value of 200 mL. However, the varied from 25 mL for the column with 10 plates to 125 mL with the column with 50 plates. Thus, although the distribution coefficient, k, was the same for both columns, the efficiency of the column was better for the column with 50 plates. This result means that the column with more plates will recover more analyte than the less efficient column. 

As mentioned earher, the plate theory has played a role in the development of chromatography. The concept of "plate" was originally proposed as a measmement of the performance of distillation processes. It is based upon the assumption that the column is divided into a number of zones called theoretical plates, that are treated as if there exists a perfect equilibrium between the gas and the Hquid phases within each plate. This assumption imphes that the distribution coefficient remains the same fi-om one plate to another plate, and is not affected by other sample components, and that the distribution isotherm is hnear. However, experimental evidences show that this is not true. Plate theory disregards that chromatography is a dynamic process of mass transfer, and it reveals httle about the factors affecting the values of the theoretical plate number. In principle, once a sample has been introduced, it enters the GC column as a narrow-width "band" or "zone" of its composite molecules. On the column, the band is further broadened by interaction of components with the stationary phase which retains some components more than others. Increasing... 

In order to achieve separation in chromatography, the analytes must be retained in the column. The fundamental relationship between the retention volume (Fr), the quantity of stationary phase (Fj) and the distribution coefficient (A ) is expressed in the following equation ... 

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