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