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12 FLASH CHROMATOGRAPHY AND LOW PRESSURE CHROMATOGRAPHIC TECHNIQUES FOR SEPARATION OF PHYTOMOLECULES 2. Optimize the TLC solvent mixture so that the compound of interest has an R f ≈ 0.15-0.35 and ΔR f >0.20. These conditions will provide the most reliable starting point for a successful flash chromatography separation. a) Adjust the solvent selectivity to provide an ΔRf>0.20. Solvent selectivity is defined as the ability to affect the retention of one compound in the mixture relative to the others, therefore affecting ΔR f and number of CV. Experimenting with different solvent combinations to obtain the desired TLC separation usually reveals appropriate conditions for effective flash chromatography separation. Different solvent mixtures such as hexane:ethyl acetate (1:1) and hexane:dichloromethane (1:2) may provide different solvent selectivities while providing similar solvent strengths. Different solvent mixtures can even reverse the elution order of some of the components in the sample. It is interesting to note that ΔR f and ΔCV may vary greatly relative to one another for a given separation. ΔCV predicts column capacity, i.e. the amount of material that can be effectively separated in a single column loading (Table 2). Greater the ΔCV, the better the effective capacity of the column. b) Adjust the solvent strength to obtain an Rf between 0.15 and 0.35 (CV, 3-6). Solvent strength refers to the solvent’s simultaneous effect on the retention of all compounds in the mixture; therefore, solvent strength affects R f and CV. Once the optimum separation has been established by modifying solvent selectivity, it may be useful to move some or all of the compounds off the flash column as quickly as possible by increasing solvent strength. Often, slight changes in solvent strength can make large differences in retention. In some cases, a lower-strength mobile phase provides improved separations. It is important to remember that the sample loading solvent should have equal or lower elution strength than the starting strength of the mobile phase. Additional adjustments to the selectivity and strength of the flash solvent system may be necessary to optimize the separation and to achieve a CV≈3-6 and a ΔCV>1. This can often be achieved by using a less polar solvent system or by decreasing the proportion of polar modifier. Table 2: Approximate capacity of a 20 g/70 ml ISOLUTE Flash Si column (Biotage) ΔCV Sample load, g 6 1.0 2 0.5 1 0.25 198
EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS 12.3 Isocratic versus Gradient Chromatography Two types of solvent systems are used in fl ash chromatography: isocratic and gradient. The most common is an isocratic (meaning “same solvent strength”) system where a single-strength mobile phase mixture brings about the desired separation. If the mixture is complex and contains compounds that differ greatly in column retention times, chemists may use a gradient solvent system that changes solvent composition during the course of elution. For example, in a normal phase system equipped with a silica column, a non-polar solvent such as hexane is applied to elute non-polar compounds. Then, a more polar solvent such as ethyl acetate is added to the hexane to elute the more polar compounds. The percentage of the polar solvent in the mixture is increased until all components of the mixture have eluted. In a step-gradient system, the various solvent concentrations are typically changed in large increments (or steps). Alternatively, a linear gradient can be employed whereby a continuous linear change in the concentrations of the solvent (and thus mobile phase strength) is achieved. Chemists can often achieve effective separations more rapidly by using gradient solvent systems. Chemists must select miscible solvents for use in gradient solvent systems. A common solvent system for fl ash separations using polar sorbents such as silica is hexane and ethyl acetate, where ethyl acetate is the more polar solvent. Increasing elution strength in normal phase mode Relative Solvent Strength Hexane Toluene Diethyl ether Dichloromethane Acetone Tetrahydrofuran Ethyl acetate Acetonitrile Isopropanol Ethanol Methanol Water Increasing elution strength in reversed phase mode Figure 1: Step-gradient system 199
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CONTRIBUTORS Chapter 6 Aqueous Alco
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EXTRACTION TECHNOLOGIES FOR MEDICIN
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7 DISTILLATION TECHNOLOGY FOR ESSEN
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9 SOLID PHASE MICRO-EXTRACTION AND
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