TLC Plates - User Guide
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Thin Layer Chromatography (TLC) is a widely used technique for analyzing and optimizing chemical separations. This guide provides a step-by-step approach to running a TLC plate, selecting an appropriate solvent system, and optimizing mobile phase composition. By following these guidelines, users can achieve more effective separations and enhance their purification processes.
- • Running a TLC Plate: A Step-by-Step Guide - Learn the essential steps to prepare, run, and analyze a TLC plate for efficient compound separation.
- • Guidelines for Solvent System Selection - Understand the key factors in choosing the right solvent system to improve resolution and separation efficiency.
- • Optimizing Mobile Phase Selection - Discover how to fine-tune the mobile phase composition for better chromatographic performance.
Running a TLC Plate: A Step-by-Step Guide
Running a TLC plate is a simple process that, when followed correctly, ensures precise and reliable results. Follow these detailed steps for optimal outcomes:
1. Prepare Your Materials
Before starting, ensure you have:
- • A SepaFlash™ TLC Plate
- • Capillary tubes for spotting samples
- • A developing chamber
- • Your mobile phase (solvent system)
- • A UV lamp or stains for visualization
2. Prepare the TLC Plate
- • Cut the TLC plate to the appropriate size or use a precut TLC plate.
- • Mark the baseline: draw a light baseline 0.5 - 1 cm above the plate’s bottom edge with a pencil. Ensure sample spots are above the solvent level in the elution chamber.
- • Define sample spots: mark evenly spaced dots on the baseline, keeping 5 mm from the edges and between spots.
3. Prepare the Sample
- • Dissolve a small amount of the compound (1 - 2 mg) in a suitable solvent.
- • Dip the capillary into the sample and touch it to the baseline mark. Let it dry, repeating if needed for a more concentrated spot.
4. Prepare the Developing Chamber
- • Add your chosen mobile phase (see optimizing solvent section) to the chamber. The solvent should be deep enough to cover the bottom of the chamber but below the baseline of the TLC plate.
- • Place a piece of filter paper inside the chamber to saturate the atmosphere with solvent vapor.
5. Develop the Plate
- • Carefully place the TLC plate vertically into the chamber with the baseline above the solvent level.
- • Cover the chamber and allow the solvent to ascend the plate until it is about 1 cm from the top edge. This is the solvent front.
- • Remove the plate and immediately mark the solvent front with a pencil before it evaporates.
6. Visualize the Results
- • UV visualization: If your plate has a UV indicator, observe under UV light to locate the separated compounds. Mark the visible spots with a pencil.
- • Chemical stains: For compounds not visible under UV, apply a stain (see TLC stains section) and develop the plate according to the stain’s instructions.
7. Analyze and Interpret the Results
- • Measure the distance traveled by each spot from the baseline (compound distance) and the distance traveled by the solvent front.
- • Calculate the Rf value for each spot as explained in the image at right.
- • Compare the Rf values to known standards for identification.
Guidelines for Solvent System Selection
The ideal solvent system moves all components off the baseline, achieving Rf values between 0.15 and 0.85, with optimal separation typically observed at Rf values of 0.2 to 0.6.
Starting point for most applications is ethyl acetate / hexane (1:1). Adjust the ratio to optimize Rf values. Remember that to increase Rf values, you need to increase the polarity of the mobile phase by raising the ratio of the polar solvent or selecting a more polar solvent.
To decrease Rf values, lower the polarity of the mobile phase.Alternative systems that can be tried are methanol / dichloromethane (ratios between 1:4 and 1:9) or toluene with acetone, dichloromethane, or ethyl acetate.
In normal phase chromatography, a typical mobile phase for:
- • Standard compounds: 10 - 50 % ethyl acetate / hexane
- • Polar compounds: Best separated with 100 % ethyl acetate or 5 - 10 % methanol / dichloromethane
- • Non-polar compounds: Perform well with 5 % ethyl acetate (or ether) / hexane or 100 % hexane
- • Basic compounds: Adding up to 2 % triethylamine or 10 % ammonia will help
- • Acidic compounds: Adding up to 2 % acetic or formic acid improves separation
In reversed-phase chromatography, common solvent systems consist of water or aqueous buffers combined with organic solvents such as acetonitrile, methanol, or tetrahydrofuran. Additionally, solvents like ethanol and isopropanol are versatile alternatives for various applications.
Optimizing Mobile Phase Selection for Thin-Layer Chromatography
The choice of the mobile phase, also known as the solvent system or eluent, is critical for achieving efficient TLC separation. This selection depends on the compound’s solubility in the solvent and the difference in its affinity for the mobile phase versus the stationary adsorbent (typically silica or alumina).
In normal-phase chromatography, non-polar solvents such as hexane or pentane are typically used. Under these conditions:
- • Non-polar compounds readily migrate up the plate
- • Polar compounds tend to remain closer to the baseline
Conversely, when polar solvents are used, polar compounds are more likely to migrate further from the origin. Compounds with highly polar groups are strongly adsorbed and elute less readily than less polar or polarizable compounds.
Adsorption strength generally follows the order of polar functional groups listed at right, but variations may occur based on each compound’s overall structure. In reversed-phase chromatography, the behavior is reversed, with polar solvents facilitating the migration of non-polar compounds.
| Common developing solvents | Polarity | Organic compounds polarity by functional group |
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