Incremental multiple development

Multiple developments can be classifled into nniform mnltiple developments (UMD, also called nnidimensional multiple developments) for methods in which the migration distance and mobile phase composition are constant and incremental multiple development (IMD) with mostly ascending migration distances. However,... 

FIGURE 11.32 Densitogram illustrating various techniques of development of Heracleum sphondylium fruit extract on silica with AcOEt + H (1 9) (a) by isocratic method, (b) incremental multiple development with 10-mm steps. For abbreviations, see Figure 11.1. (For details, see Wawrzynowicz, T., Czapinska, K., and Markowski, W., J. Planar Chromatogr., 11, 388-393, 1998.)... 

Two-dimensional separations in planar chromatography are rather trivial to perform. All unidimensional multiple development techniques employ successive repeated development of the layer in the same direction, with removal of the mobile phase between developments. The main variants are multiple chromatography and incremental multiple development. The basis for automated multiple development (AMD) is the automation of unidimensional, incremental, multiple development with a reverse solvent strength gradient [998]. 2D TLC finds limited use, and is mainly a qualitative technique. 

Another HPTLC method has been developed for the separation of kaempferol and quercetin in the extract of Ginkgo biloba leaves showing beneficial effect in brain diseases. Leaves were refluxed with methanol for 30 min then filtered. The filtrate was refluxed with 25 per cent HC1 for 60 min then neutralized with ammonia and the clear supernatant was applied for HPTLC. Silica plates were predeveloped in chloroform-methanol (1 1, v/v). Separation was performed with toluene-acetone-methanol-formic acid (46 8 5 1, v/v) as the mobile phase using incremental multiple development. A densitogram illustrating the good separation charactersitics of the system is shown in Fig. 2.42. The relative standard deviation (RSD) of the method was low (1.37 and 1.40 for kaempferol and quercetin,... 

In all techniques of multiple development, the plate is repeatedly developed in the same direction, with intermittent evaporation of the mobile phase between the consecutive developments (Fig. 2b). If the layer is developed many times to the same distance with the same eluent, the technique is called unidimensional multiple chromatography. A variation of this technique, called incremental multiple development, consists in the stepwise change of the development distance that is the shortest in the first step and is then increased, usually by a constant increment (equal dis-... 

For incremental multiple development, the first development is the shortest and each subsequent development is incremented by (usually) a fixed distance or variable distances arrived at by trial-and-error, to obtain the desired separation. The last development is the longest and, in most cases, corresponds to the maximum useful development length for the layer. 

Optimum conditions for isocratic multiple development are summarized in Table 6.10. The outcome of separations by multiple chromatography is the most predictable (see section 6.3.7) but is rarely the best approach for distributing sample zones throughout the whole chromatogram, and incremental multiple development is generally preferred. Incrementing the solvent entry position while simultaneously increasing... 

Figure 6.9. Separation of a mixture of estrogens by incremental multiple development with variable (A) and fixed (B) solvent entry position, and by multiple chromatography (C). A nine step sequence with the mobile phase cyclohexane-ethyl acetate (3 1, v/v) was used for (A) and (B) and seven 7 cm developments for (C), all on a silica gel HPTLC layer. The estrogens, in order of migration, are 17p-dihydroequilenin,...

Figure 6.10. Comparison of normal development and incremental multiple development with a decreasing solvent strength gradient (AMD) for the separation of poly(ethylene glycol) 400 as its 3,5-dinitrobenzoate ester. The AMD separation employed a 15- step gradient with methanol, acetonitrile and dichloromethane as...

Multiple development techniques [unidimensional multiple development (UMD) and incremental multiple development (IMD)] were also used to separate the components of a reference solution of amino acids in blood plasma on cellulose plates eluted with acetonitrile-water (8 2 y/y)P ... 

Other variations have been described by Szepesi and Nyiredy (1996) and Nyiredy (1996). Incremental multiple development (IMD) (Szabady et al., 1995 and 1997) involves rechromatography with the same composition mobile phase for distances that increase, usually by the same amount (linearly). If development occurs in the same direction with the same distance but different mobile phases having distinctive strength and selectivity, the method is termed gradient multiple development (GMD) this method most significantly increases the separation capacity of the system. In bivariate multiple development, the development distance and mobile phase composition are varied simultaneously for successive runs this method, which is effective for samples of differing polarity, has been used especially for preparative layer chromatography plates. 

Szabady, B., Ruszinko, M., and Nyiredy, Sz. (1997). Prediction of retention data when using incremental multiple development techniques. Chromatographia 45 369-372. 

Recently, Szabady et al. (40) reported that by using incremental multiple development (IMD), a variation of UMD in which rechromatography is performed over increasing development distances with the same mobile phase composition, the R/value can also be calculated. Using IMD with linearly increasing distances, the following formula for prediction of Rf values can be used ... 

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