Development Mode

The ascending mode, in which the mobile phase moves up the plate, is most frequently used with a maximum separation distance of 18 cm. The angle at which the plate is supported during development affects the rate of development and the shape of the bands. As the angle of the plate decreases towards the horizontal development mode, the flow of the mobile phase increases, but so also does spot distortion. An angle of 75° is recommended as optimum for development. Because descending development has no significant advantages in terms of resolution, it is rarely used.

The advantages of circular development of compounds in the lower RF range are well known, but it has not been accepted for preparative separations, because the mobile phase velocity would be too slow. However, it is possible to start development not directly from the centre, but from a circle of 2 cm radius, i.e. the mobile phase inlet is not a point, but a circle. Because the size of the mobile phase inlet and the velocity of the mobile phase are related linearly, a relatively high mobile phase velocity can be achieved over a separation distance of 8 cm. Recently a new device has been described which enables a suitable mobile phase velocity to be used in the circular development mode (Figure 7). A solvent reservoir

Figure 7 Schematic diagram of a circular preparative chromatography chamber. 1, glass plate; 2, chromatoplate; 3, support block; 4, magnet; 5, Teflon ring; 6, solvent reservoir; 7, quartz glass cover plate; 8, Teflon ring; 9, solvent.

made of steel and a silicone sealing ring are placed on the layer and fixed by a magnet located below the chromatographic plate. To start the separation, adsorbent is scratched from the centre of the plate and the recess produced filled with mobile phase. The device can be used for different types of chamber (N, UM). The entry of sample and mobile phase is regular over the whole cross-section of the preparative layer, irrespective of whether the sample is applied as a liquid or a solid. The device ensures rapid, efficient separation with all the advantages of circular development. The resolution is significantly higher than that obtained from linear development. With the UM chamber the glass cover plate is placed directly on the surface of the chromatographic plate. In the N chamber, the cover plate is placed on a 19 cm diameter metal ring, the height of which can be varied between 0.5 and 2 cm, depending on the type of chamber applied. To start development the solvent reservoir is filled with the appropriate mobile phase and the level of this is kept constant by applying a constant hydrostatic pressure by means of a second reservoir. To stop development the inlet from the second reservoir is turned off.

Anticircular development is accepted in analytical TLC for increasing resolution in the higher RF range. Because a special device is necessary for such separations, this development mode is rarely used.

Although the different types of multiple development (MD) are rarely used for preparative purposes, the advantage of the method should be understood. In MD the first development length is the shortest and subsequent developments are performed over longer development distances. The last migration distance is the longest and corresponds to the useful development length of the chromatographic plate; it also depends on the nature of the mobile phase. The removal of the mobile phase between development steps is performed by careful drying of the plate. The dried layer is returned to the development chamber for repeated development under the same chromato-graphic conditions as for earlier development steps.

The most important aspect of MD techniques is the spot-reconcentration mechanism. In each development step the solvent front first contacts the lower part of the chromatographic zone formed in the previous chromatographic step. The molecules at this part of the zone start moving with the mobile phase toward the molecules in the upper part of the chromatographic zone - those still ahead of the solvent front. As the mobile phase front reaches the upper part of the zone, the narrow band developed as a result of the zone reconcentration mechanism migrates and broadens by diffusion in the mobile phase, as in conventional planar chromatography.

In terms of development distance and mobile phase composition, MD techniques can be classified into four basic categories - UMD, IMD, GMD, and BMD (Figure 8). Unidimensional multiple development (UMD) is the repeated development of the chromatographic layer over the same development distance (D) with the same mobile phase (same values of ST1 and SV1). In the modification of UMD known as incremental multiple development (IMD) re-chromatography is performed over increasing development distances (D1 p D5) with the same mobile phase (same values of ST1 and SV1). In gradient multiple development (GMD), successive chromatographic development steps are performed with a change in solvent strength and selectivity (ST1, SV1; ST4, SV4) over the same development distance (D is constant). GMD is required for analysis of multicomponent mixtures spanning a wide polarity range. The most complex multiple development technique is bivariate multiple development (BMD), in which development distance and mobile phase composition are varying simultaneously (D1, ST1, SV1; D4, ST4, SV4) during successive chromatographic developments. Needless to say, the solvent strength and selectivity of the mobile phase can be changed independently of each other. For the analysis of less complex mixtures of wide polarity range, the preferred technique is BMD with a mobile phase gradient of decreasing solvent strength, when the final chromatographic separation can be detected as a single chromatogram. In BMD the shortest development is performed first with the mobile phase of strongest solvent strength; the chromatographic distance is increased and the solvent strength reduced during successive steps of the chromatography, until finally the last development step is performed over the longest development distance with the weakest mobile phase.

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