Development two dimensional

The usual two-dimensional chromatography is a multiple development method in which the sample is spotted in the corner of a square of paper and ascending or descending development is made successively with two different solvents in two directions perpendicular to each other. Between developments, the paper is thoroughly dried to remove all traces of the first solvent. The best separations of complex mixtures are obtained if different types of solvents are employed. 

Two-dimensional paper chromatography of standard mixtures produces chromatography maps that may be kept as permanent records. Normal biological samples, e.g., urine, are run, and the maps are retained so that patterns of certain important compounds, such as amino acids, are available for reference. In this way, abnormal spots can be identified easily when they occur. 

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Repeated chromatography in a third dimension after completion of two-dimensional development. Here, development in the first, second, and third dimensions can be envisaged as occurring on three plates arranged in the form of a cube the plate is again dried between developments. 

Secondly it is possible to carry out functional chromatography within the framework of a two-dimensional development [6-8]. The first separation is followed by an in situ reaction of the sample substance on the layer the chromatogram is then developed perpendicular to the direction of the first chromatogram (SRS technique). The decision concerning the type of alcohol, is then made on the basis of the positions of the chromatogram zones esters migrate appreciable further than their parent alcohols and acids. 

FIGURE 5.23 Example of a two-dimensional development (plate turned by 180°), first development for removal of lipophilic substances and second development for separation. 

Two-dimensional development is commonly used in analytical TLC for the separation, identification, and sometimes, quantification of complex lipid species especially when one-dimensional development does not provide satisfactory separation where some individual hpids coelute in a number of common solvent systems. 

Silica gel plates also have been used for the separation of 16 different eye pigments of Drosophila melanogaster using two-dimensional development in nonpolar solvent systems [55]. Although not very common, two-dimensional development may be nsed in preparative scale on thick-layered plates for further analysis. 

One-dimensional multiple development and two-dimensional development Multiple developments through one or two dimensions can be applied to separate certain components in sequence, with detection at each step. This gives a theoretical increase in the capacity of the spots, so it is ideal for the separation of mixtures with a large number of components. In addition, it is a useful tool to confirm the purity of a given component. Though hyphenated HPLC could serve as a multiple separation technique, TLC takes the lead in this area by its faster separation and choice of different mobile phases and detection methods through each run. 

BuOH = butyl alcohol MeOH = methyl alcohol THF = tetrahydrofuran. c Compositional heterogeneity (CH) was determined. d Descending solvent strength with development path. e Two-dimensional development. f Ascending solvent strength with development path. 

Figure 1. Determination of molecular weight distribution for narrow polystyrene standard. Curve I, calibration of Rf vs. M curve II, densitogram of two dimensionally developed sample curve III, molecular weight distribution Mw/Mn = 1.02 (12).

Two-dimensional development, in which a second mobile phase is run perpendicular to the first mobile phase flow on a square plate that is dried after the first development. The field has recently been reviewed by Guiochon and co-workers.24 A variation of this technique using a bed with two stationary phases was shown in Figure 10.4. 

Consden et al. 14 published the two-dimensional development of a planar (paper) chromatogram. Later on. the method was widely used to improve a wide range of planar separation methods. Using the same stationary and mobile phase, the spot capacity has been multiplied with a factor of 1.44 (the square root of 2) (18-2(). Two-dimensional chromatography can be performed by using the same or different stationary and mobile phases. By changing the mobile phase composition the mode of development... 

Two-dimensional developments can be easily carried out using the elution-type development in the first direction, drying the plate, and then carrying out displacement-type development in the. second direction. The elution-type development in the first directional run results in the e.s.sential pre-separation of the. sample components. while the second dimensional run separates the components with similar chromatographic characteristics. 

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