General Elution Problem

Constant separation conditions, for example isothermal operation in GC and isocratic elution in LC, are unsuitable for separating samples containing components with a wide retention range. Employing average separation conditions will result in a poor separation of early-eluting peaks, poor detectability of late-eluting peaks, and excessively long separation times. In GC there is an approximately exponential relationship between retention time and solute boiling point under isothermal conditions. For mixtures with a boiling point range > c. 100°C it is impossible to identify a compromise temperature that will provide an acceptable separation. The solution in this case is to use temperature programming, flow programming, or both. Temperature programming is the most common and usually involves a continuous linear increase in temperature with time, although other programme profiles are possible, including segmented programmes incorporating isothermal periods. The reduction in separation time, increase in peak capacity, and nearly constant peak widths obtained are illustrated by the separation in Figure 18. The general elution problem in LC is solved using solvent-strength gradients. Here, the composition of the mobile phase is changed as a function of time. Binary or ternary solvent mixtures are commonly used as the mobile phase in which the relative composition of the strong solvent (that solvent with the capability of reducing retention the most) is increased over time. In SFC it is usual to programme the density, mobile-phase composition or temperature as a single factor, but it is also possible for some combination of parameters to be changed simultaneously. The goal remains the same, as indicated by the

Figure 18 Temperature programmed separation of fragrance compounds by GC on a 30 m x0.25 mm i.d. fused silica open-tubular column coated with DB-1, film thickness 0.25 ^m, helium carrier gas 25 cm s"1 and temperature program 40°C (1 min isothermal) then 40-290°C at 5°C min"1. (Reproduced with permission from J&W, copyright © J&W Scientific Inc.)

Figure 18 Temperature programmed separation of fragrance compounds by GC on a 30 m x0.25 mm i.d. fused silica open-tubular column coated with DB-1, film thickness 0.25 ^m, helium carrier gas 25 cm s"1 and temperature program 40°C (1 min isothermal) then 40-290°C at 5°C min"1. (Reproduced with permission from J&W, copyright © J&W Scientific Inc.)

Figure 19 Separation of Triton X-114 by SFC using programmed elution on a 10 cm x 2 mm i.d. column packed with 3 |im octadecylsiloxane-bondedsilica gel at 170°C with UV detection. (A) Carbon dioxide/methanol (2 # 0.125) mL min~1 at 210 bar; (B) as for (A) with pressure programmed form 130 to 375 bar over 8 min; and (C) using a mobile-phase composition gradient from 0.025 to 0.4 mL min~1 methanol over 8 min at 210 bar. (Reproduced with permission from Giorgetti A, Pericles N, Widmer HM, Anton Kand Datwyler P (1989) Journal of Chromatographic Science 27: 318, copyright © Preston Publications, Inc.)

density- and composition-programmed separation of oligomers in Figure 19.

Solvent-strength gradients in TLC are usually discontinuous and achieved through the use of unidimensional multiple development. This is accompanied by zone refocusing resulting in a larger zone capacity and easier-to-detect separated zones. All unidimensional multiple development techniques employ successive repeated development of the layer in the same direction with removal of the mobile phase between developments. Each time the solvent front traverses the sample zone it compresses the zone in the direction of development because the mobile phase contacts the bottom edge of the sample zone first where the sample molecules then start to move forward before those molecules ahead of the solvent front. Once the solvent front has reached beyond the zone, the refocused zone migrates and is broadened by diffusion in the usual way. When optimized, it is possible to migrate a zone a considerable distance without significant zone broadening beyond that observed for the first development. If the solvent composition is varied for all, or some, of the development steps during multiple development, then solvent strength gradients of different shapes can be produced. With increasing solvent-strength gradients it is usually necessary to scan the separation at a number of intermediate development steps corresponding to the development at which different components of interest are separated, since in later developments these zones may be merged again because of the limited zone capacity in TLC. Alternatively,

Figure 19 Separation of Triton X-114 by SFC using programmed elution on a 10 cm x 2 mm i.d. column packed with 3 |im octadecylsiloxane-bondedsilica gel at 170°C with UV detection. (A) Carbon dioxide/methanol (2 # 0.125) mL min~1 at 210 bar; (B) as for (A) with pressure programmed form 130 to 375 bar over 8 min; and (C) using a mobile-phase composition gradient from 0.025 to 0.4 mL min~1 methanol over 8 min at 210 bar. (Reproduced with permission from Giorgetti A, Pericles N, Widmer HM, Anton Kand Datwyler P (1989) Journal of Chromatographic Science 27: 318, copyright © Preston Publications, Inc.)

Figure 20 Separation of the 3,5-dinitrobenzoyl esters of poly(ethylene glycol) 400 by (A) a single conventional development and (B) by incremental multiple development with a step-wise gradient of methanol, acetonitrile and dichloromethane over 15 developments (Reproduced with permission from Poole CF, Poole SK and Belay MT (1993) Journal ofPlanar Chromatography 6: 438, copyright © Research Institute for Medicinal Plants.)

Figure 20 Separation of the 3,5-dinitrobenzoyl esters of poly(ethylene glycol) 400 by (A) a single conventional development and (B) by incremental multiple development with a step-wise gradient of methanol, acetonitrile and dichloromethane over 15 developments (Reproduced with permission from Poole CF, Poole SK and Belay MT (1993) Journal ofPlanar Chromatography 6: 438, copyright © Research Institute for Medicinal Plants.)

incremental multiple development can be used with a decreasing solvent-strength gradient. In this case, the first development distance is the shortest and employs the strongest solvent composition, while subsequent developments are longer and employ mobile-phase compositions of decreasing solvent strength. The final development step is the longest and usually corresponds to the maximum useful development length for the layer and employs the weakest mobile phase. In this way sample components migrate in each development until the strength of the mobile phase declines to a level at which some of the sample zones are immobile, while less retained zones continue to be separated in subsequent development steps, affording the separation of the mixture as a single chromatogram (Figure 20). Incremental multiple development with a decreasing solvent-strength gradient is easily automated.

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