TLC Introduction - Grace Davison Discovery Sciences - #1

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188 www.discoverysciences.com prep | flash | tlc tlc Thin Layer Chromatography to Preparative Chromatography One of the first steps in scale-up of preparative liquid chromatography separations is selection of an appropriate mobile phase. Two methods are commonly used to determine the proper mobile phase composition: Thin Layer Chromatography (TLC) or High Performance Liquid Chromatography (HPLC). The use of TLC will be discussed here to deal with the successful correlation between the TLC separation to the preparative silica column. TLC is a liquid-solid adsorption technique where the mobile phase ascends the thin layer of stationary phase coated onto a backing support such as glass by capillary action. There is a similar relationship to column chromatography where the solvent travels down through the column’s adsorbent. The similar relationship allows TLC to be a rapid method for determining solvent composition for preparative separations. Steps for Method Development Choose Stationary Phase Choose a scalable TLC plate, preferably that has an identical media as the preparative column. Choose between normal and reverse phase based on sample polarity and solubility. Choose a Mobile Phase Criteria for Choosing a Preparative Solvent • Solubility • Affinity • Resolution 1. Solubility Many solvent systems provide the minimal solubility for the sample, but to elute a sample from a column the mobile phase must have a greater solubility for the sample, as the sample concentration is usually very high. When possible, it is best to dissolve the sample in the mobile phase. The first step in solvent selection is determination of the solubility of the sample. The desired mobile phase would provide the greatest solubility, while providing affinity for the sample on the stationary phase. Solvent Solubility Screening Table Water Methanol Ethanol Acetone Diethyl Ether Ethyl Acetate Dichloromethane Toluene Chloroform Cyclohexane Petroleum Ether Hexane 2. Affinity To achieve a separation, the sample must have a relatively equal affinity for the solvent and the packing material. If the sample has a higher affinity for the stationary phase than the solvent, the sample will remain at the origin (Rf value will be too low). 3. Resolution Resolution is improved by optimizing the affinity between sample, solvent, and support. The optimum solvent for separating two or more compounds will maximize the difference in the compounds. Most TLC and preparative mobile phase systems contain a polar solvent and a chromatographically dissimilar less-polar solvent. As a guide for method development, a substitution in the polar solvent often results in a change in resolution, while a change in the less-polar solvent results primarily in a change in Rf of the sample components. The table below shows some common tendencies of various functional groups to adsorb onto the silica. Affinity of Functional Groups for Silica Gel -NH2 Amine -COOH Carboxylic Acid -COH Alcohol -CONH2 Amide -C=O Carbonyl -C=-CO2R Ester -C-O-C Ether -C1 Halocarbons -CC- Hydrocarbons Select Visualization Technique Once a mobile phase is selected, visualization techniques will need to be determined. Common techniques include SWUV, I2 /SWUV, I2 /KI for Nitrogen containing compounds, H2SO4/LWUV, H2SO4/PMA for non-nitrogen containing compounds. Perform TLC Analysis Look up the affinity for the type of compound as well as the solvent strengths to find a starting point for method development or look up a reference from a similar structure, then adjust the mobile phase composition to adjust the Rf. It is common to try 3–6 solvent systems for the first round of method development. Review the results after visualization and adjust the Rf if necessary, increase the separation and evaluate visualization techniques to make sure you are seeing all necessary compounds. Optimizing TLC Separations for Preparative Separations The optimum separation of compounds by TLC is usually achieved when Rf values are between 0.3–0.5. Rf = Distance from origin to center of spot Distance from origin to solvent front Generally, adjusting the compound’s Rf between 0.3–0.5 is done first for a TLC separation. For scale-up to preparative separations, the TLC solvent system’s polarity must be decreased to lower the Rf between 0.15–0.35. This Rf range is optimal for a preparative separation, in terms of sample load, resolution, residence time, and solvent usage. Determination of Column Volumes (C.V.) The equation C.V. = 1/ Rf relates the TLC values and the preparative LC column volumes to elute each component. This equation is only a guideline and the relationship between the Rf values and the column volume will vary in use. Generally, the LC column volumes will be equal to or less than the calculated values. The elution volume will also be dependent upon the sample load and solvent used to solubilize the sample. Increasing Polarity Increasing Affinity TLC Introduction