Preparing a thin layer chromatography (TLC) plate primarily involves applying the sample to be analyzed onto the plate's surface.
The crucial step in preparing a TLC plate before chromatography begins is the precise application of the sample. This ensures the sample is in the correct starting position for separation by the mobile phase.
The Sample Spotting Process
Based on standard practices in Thin Layer Chromatography, including the method described:
Using a capillary tube, a small amount of a concentrated solution of the sample is deposited near the bottom edge of a TLC plate. The solvent is allowed to completely evaporate before the next step. A vacuum chamber may be necessary for non-volatile solvents.
Here's a breakdown of this process:
Key Steps for Sample Application
- Sample Preparation: The substance(s) to be analyzed are typically dissolved in a volatile solvent to create a concentrated solution. A higher concentration means less volume is needed, resulting in a smaller, tighter spot.
- Plate Marking: A light pencil line is often drawn near the bottom edge of the TLC plate (typically about 1-1.5 cm from the edge) as a baseline. This marks where the sample spots will be applied. Spots must be placed carefully on this line and spaced apart to prevent merging.
- Sample Deposition: Using a fine capillary tube or micropipette, a small amount of the sample solution is carefully applied as a spot on the baseline marked on the plate.
- Tip: Apply the solution in small increments, allowing the solvent to evaporate between applications, to keep the spot size small. A small spot leads to better separation (resolution).
- Solvent Evaporation: The solvent carrying the sample must be allowed to completely evaporate from the spot before the plate is placed in the development chamber. This is critical because residual solvent can interfere with the mobile phase's movement and lead to distorted spots or poor separation.
- Evaporation can occur naturally in air, or gentle heat (like from a heat gun or oven, if the compounds are stable) can be used.
- For solutions made with non-volatile solvents, a vacuum chamber may be necessary to effectively remove the solvent.
Why is Spotting Important?
Proper sample application is fundamental to successful TLC.
- Small Spot Size: A tight, small spot at the origin leads to discrete, well-separated spots or bands as the mobile phase moves up the plate. Large, smeared initial spots result in smeared final separation.
- Correct Placement: Applying the spot too low can cause it to be submerged in the mobile phase reservoir in the developing chamber, washing the sample off the plate instead of developing it.
- Complete Evaporation: Ensures that only the solid or liquid sample residue remains at the origin, ready to interact with the stationary and mobile phases effectively.
By following these steps, the TLC plate is prepared with the sample correctly positioned at the origin, ready for the chromatographic separation process.
