Elimination of Typical Problems with Use of OPLC

It is of practical importance to summarize the most important distorting effects which arise in OPLC and to describe means of eliminating these problems.

Linear separations require specially prepared chromatographic plates with chamfered edges that are impregnated with a suitable polymer suspension, to prevent solvent leakage at overpressure. For proper preparation of the chromatographic plate, the surface from which the stationary phase has been scratched must be fully cleaned from particles. If this is not achieved, a narrow channel may be formed under the

Figure 7 Elimination of typical problems in OPLC. (A) 'Break-in effect' - a consequence of improper impregnation of the chromatographic plate; (B) 'meniscus effect' - a consequence of improper impregnation of the chromatographic plate; (C) lack of the appropriate inlet pressure for linear separation.

Figure 7 Elimination of typical problems in OPLC. (A) 'Break-in effect' - a consequence of improper impregnation of the chromatographic plate; (B) 'meniscus effect' - a consequence of improper impregnation of the chromatographic plate; (C) lack of the appropriate inlet pressure for linear separation.

consequence of this effect - which occurs either in the concave or convex form, depending on the physical properties of the solvents used - the eluent flows more slowly or more quickly on both edges of the chromatographic plate, again distorting quantitative results.

Before starting the separation with the optimized mobile phase, the mobile phase inlet valve is closed and the eluent pump is started to establish an appropriate solvent pressure. The separation is then started by opening the inlet valve; this ensures the rapid distribution of the mobile phase in the inlet channel necessary for linear migration of the mobile phase. If the inlet pressure is too low and the mobile phase does not fill the inlet channel totally, the start of the separation is similar to that for circular development; the distorted linear separation obtained is shown in Figure 7C. No preparation of the plate is needed for offline circular separations.

If multi-component mobile phases are used in un-saturated TLC, the fronts arising from the components can have a decisive influence on the separation. This effect can be substantial in OPLC; the secondary fronts appear as sharp lines because no vapour phase is present. Compounds of the mixture migrating with one of the fronts form sharp, compact zones whereas tailing or fronting can be observed for compounds migrating directly in front of or behind the a front. With multi-component mobile phases the 'multi-front effect' can appear in two forms. In the first (Figure 8A), one or more fronts can occur between compounds to be separated. In the second, all the compounds to be separated migrate behind the lowest front (Figure 8B), and the fronts do not influence the separation. As the position of the fronts is constant, if the chromatographic conditions are constant, possibly undesirable effects of the multi-front effect can be monitored and taken into account by applying the spots or bands stepwise. Thus for linear separations the sample is applied at different distances (s = 1, 2, ..., n) from the mobile phase inlet channel (Figure 8C). In circular OPLC the samples are applied at points on concentric circles (or rings) with their centres at the mobile phase inlet (Figure 8D). Quantitative evaluation is usually made more difficult, but not impossible, by the multi-front effect, because the phantom peaks formed at the fronts can be measured densitometrically in the substance-free zones at the sides of the chromatographic plates, and thus the values are taken into account. It must be mentioned that the multi-front effect also has a

Figure 8 'Multi-front effect' - a consequence of the use of multicomponent mobile phases. (A) The fronts occur between the compounds to be separated; substances migrating with one of the fronts form sharp, compact zones; (B) the compounds to be separated all migrate behind the lowest front, so the fronts do not influence the separation; (C) diagonal application of the samples (as bands) for linear separations to check the place of the different fronts; (D) eccentric application of the samples (as spots) for circular separations to check the place of fhe different fronts.

a front ji front -/front 8 front s front

Figure 8 'Multi-front effect' - a consequence of the use of multicomponent mobile phases. (A) The fronts occur between the compounds to be separated; substances migrating with one of the fronts form sharp, compact zones; (B) the compounds to be separated all migrate behind the lowest front, so the fronts do not influence the separation; (C) diagonal application of the samples (as bands) for linear separations to check the place of the different fronts; (D) eccentric application of the samples (as spots) for circular separations to check the place of fhe different fronts.

a front

'disturbing zone'

Figure 9 The 'disturbing zone' as a consequence of different air/gas volume ratios adsorbed by the surface of the stationary phase and dissolved in the eluent.

positive effect in preparative separations, because compounds that migrate with a front can be eluted in a very small amount of mobile phase.

If OPLC separation is started with a dry layer, distorted substance zones can sometimes be observed in different RF ranges, depending on the mobile phase used and the velocity of the mobile phase. This effect appears during the chromatographic process as a zigzag zone across the width of the plate, perpendicular to the direction of development as a result of the different refractive indices of the solvents in front of and behind this zone. This phenomenon, termed the 'disturbing zone', is depicted in Figure 9. The extent of this phenomenon depends on the interrelationship between gas physically bound to the surface of the sorbent and gas molecules dissolved in the mobile phase. Because modification of the location of the 'disturbing zone' is possible within a very narrow range, the only solution to this problem is to conduct a prerun. For separation of nonpolar compounds this can be performed with hexane; for separation of polar substances the prerun can also be performed with hexane or with any component of the mobile phase in which the components are unable to migrate. The selection of this solvent might be considered during optimization of the mobile phase.

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