Development multiple

Unidimensional multiple development enhances the separation of compounds with low ( 0.2), very similar R p values. The same solvent is used to repeatedly develop the paper in the same direction, with complete drying of the solvent between runs 

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Ascending, one-dimensional multiple development method (stepwise technique, drying between each run) in two mobile phase systems in a twin-trough chamber without chamber saturation (equilibration 30 min at 20-22°C) at a relative humidity of 60 — 70%. 

Figure 8.5 Schematic illustration of 2-D multiple development on a bilayer with the same mobile phase (Sj, Syi).

When multiple development is performed on the same monolayer stationary phase, the development distance and the total solvent strength and selectivity values (16) of the mobile phase (17) can easily be changed at any stage of the development sequence to optimize the separation. These techniques are typically fully off-line modes, because the plates must be dried between consecutive development steps only after this can the next development, with the same or different development distances and/or mobile phases, be started. This method involves the following stages ... 

The efficiency of the "D is partly a consequence of the zone refocusing mechanism, as depicted in Figure 8.7. Each time the solvent front traverses the stationary sample in multiple development it compresses the zone in the direction of development. The compression occurs because the mobile phase first contacts the bottom edge of the zone, where the sample molecules start to move forward before those... 

A detailed description of the versatility of multiple development techniques in one dimension has been given by Szabady and Nyiredy (18). These authors compared conventional TLC with unidimensional (UMD) and incremental (IMD) multiple development methods by chromatographing furocoumarin isomers on silica using chloroform as the monocomponent mobile phase. The development distance for all three methods was 70 mm, while the number of development steps for both of the "D techniques was five. Comparison of the effects of UMD and IMD on zone-centre separation and on chromatographic zone width reveals that UMD increases zone-centre separation more effectively in the lower Rf range, while IMD results in narrower spots (Figure 8.8). 

The basis of automated multiple development (AMD) is the use of different modes of multiple development in whieh the mobile phase eomposition (5j and Sy values) is ehanged after eaeh, or several, of the development steps. Figure 8.11 illustrates the prineiple of AMD employing a negative solvent-strength gradient (deereasing 5-p values). 

Unfortunately, the fact that, in addition to the 2-D separation, a further increase in separation performance might be obtained by the use of multiple development... 

D TLC combined with multiple development is therefore a promising route which should lead to real improvements in planar chromatography in the near future. 

Theoretically, 3-D OPLC in combination with multiple development is the most powerful technique of instrumental planar chromatography. Unfortunately, suitable instrumentation is at an early stage of development. 

Figure 8.19 illustrates another example of the versatility of multidimensional OPLC, namely the use of different stationary phases and multiple development ("D) modes in combination with circular and anticircular development and both off-line and on-line detection (37). Two different stationary phases are used in this configuration. The lower plate is square (e.g. 20 cm X 20 cm), while the upper plate (grey in Figure 8.19) is circular with a diameter of, e.g. 10 cm. The sample must be applied on-line to the middle of the upper plate. In the OPLC chamber the plates are covered with a Teflon sheet and pressed together under an overpressure of 5 MPa. As the mobile phase transporting a particular compound reaches the edge of the first plate it must-because of the forced-flow technique-flow over to the second (lower) stationary phase, which is of lower polarity. 

Figure 8.20 Combination of bilayer plates and multiple development teclmiques in which total solvent sti ength and mobile phase selectivity are changed simultaneously, in the first direction (a), S- and are varied in n re-chromatograpliic steps, while in the peipendicular, (second) direction (b), and are again varied in m re-clnomatographic steps, to give (c).

Figure 8.21 Schematic diagram of the combination of bidirectional, multiple development and coupled layers in decreasing polarity (A > B > C).

On the basis of theory and experimental observations it can be predicted that a zone capacity of ca. 1500 could be achieved by 2-D multiple development. Because the same result can be achieved by application of 2-D forced-flow development on HPTLC plates, it can be stated that the combination of stationary phases, FFPC and "D offers a fruitful future in modem, instmmental planar chromatography. 

B. Szabady and Sz. Nyiredy, The versatility of multiple development in planar cliro-matography , in Diinnschicht-Chromatographie in memoriam Prof. Dr. Hellmut Jork),... 

K. Burger, Online coupling HPLC-AMD (automated multiple development) , Awa/ytw 18 1113-1116(1990). 

E. Menziani, B. Tosi, A. Bonora, P. Reschiglian and G. Eodi, Automated multiple development high-performance thin-layer chromatographic analysis of natural phenolic compounds , 7. Chromatogr. 511 396-401 (1990). 

M. T. Belay and C. E. Poole, Determination of vanillin and related flavor compounds in natural vanilla exti acts and vanilla-flavored foods by thin layer chromatography and automated multiple development , Chromatographia 37 365-373(1993). 

Fig. 2 The steps in the process of thin-layer chromatography that have been instrumentalized and automated to a large degree in the recent past. PMD = Programmed Multiple Development, AMD = Automated Multiple Development, DC-Mat or ADC = Automatic Development Chamber.

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