TLC

TLC Plate Preparation

Using a pencil, lightly draw a straight line parallel to the width of the plate at about 1 cm from the base end of the plate. Sample application will be done on this line called baseline or origin.

Note: Never use a pen because ink can move with some solvents used as eluent.

Sample preparation

Thorough sample preparation is a prerequisite for an optimal and efficient TLC separation. Typical sample preparation processes could consist in a sample crushing, filtration, extraction or concentration of the product of interest.  Samples should not be spotted neat\.  Neat means "pure" compound.  They should be diluted by 50-200 times.  So add a drop of compound or few crystals to about 2-3 mL of solvent.

Sample Application

Sample preparation will differ depending on the nature of the plate (analytical or preparative). For analytical plates, because thin layer chromatography is extremely sensitive, it is really important to apply a small quantity using a glass capillary (or a micro pipette) to get optimal resolution. For preparative plates, apply a series of small adjacent spots to form a band or a streak using a glass capillary (or a microliter syringe). In both cases, a spotting guide can be used to facilitate sample application.

Co-spotting

For analytical chromatography, co-spotting is frequently used for similar polarity products. This consists to apply on the same spot, the starting material and reaction mixture as shown by the image below.

TLC Plate Development

The most commonly used method to perform thin layer chromatography separation is to place vertically the TLC plate inside a sealed developing chamber to ensure solvent saturation. Place approximately 0.5 cm of the suitable solvent system inside the chamber. Slowly place the TLC inside the chamber and allow the eluent to travel up the plate until it gets to 1 cm from the top of the plate. Immediately remove the plate and draw a line along the solvent front.

Note: for optimal solvent saturation, a filter paper can be added inside the TLC chamber. This also prevents eluent evaporation. The solvent level needs to be below the baseline; otherwise the spots will be dissolved.

Rules of Thumb

• Standard compounds (most popular solvent system): 10 - 50% EtOAc/Hexane.
• Polar compounds: 100% EtOAc or 5 - 10% MeOH/DCM.
• Non-polar compounds: 5% EtOAc (or ether) / Hexane or 100% Hexane.
• For basic compounds: (amine or nitrogen containing), it could be useful or required to add a small quantity of triethylamine (Et₃N) to the solvent mixture (0.1 – 2.0% but typical quantity is 0.1%) or 1 - 10% ammonia (NH₃) in MeOH/DCM.
• For acidic compounds: it could be useful to add acetic (AcOH) or formic acid (FA) to the solvent mixture (0.1 - 2.0%).

Problem 1: Your sample is streaking or elongated.

Solution:

• • Your sample may be overloaded. Run the separation again with a more diluted sample solution.
  • For base-sensitive compounds, try adding acetic or formic acid to the mobile phase (0.1–2.0%).
  • For acid-sensitive compounds, try adding triethylamine to the mobile phase (0.1–2.0%) — or 1–10% ammonia in methanol (MeOH)/dichloromethane (DCM).
  • For highly polar compounds, try using a specialized silica TLC plate such as C18 for reversed phase.

Problem 2: The spots on your TLC plate are not visible.

Solution:

• • Even if your plate contains a fluorescent material your compound may not be UV-sensitive, so try another method – such as the staining methods described further below.
  • Your sample may be too diluted. Concentrate your sample spot by spotting several times in the same location,being sure to let dry between applications.
  • Make sure that the solvent level in the tank is lower than the spotting line to avoid sample dissolution.
  • Another reason may be that the compounds are volatile and may have evaporated from the plate. In this case, visualizing the product by TLC will be difficult.

Problem 3: You have spots with the same R_f values in your samples.

Solution:

• • Try “co-spotting”. Apply the starting material (standard) and the sample in the same spot and compare the R_f values of the resulting separation.
  • If necessary, change your solvent system. Three classes of solvent mixtures provide significantly different results:
    • Polar/hydrocarbon (e.g., ethyl acetate (EtOAc)/hexane or heptane, ethyl ether/petroleum ether).
    • Polar/dichloromethane (e.g., polar solvents: ether, EtOAc, MeOH)
    • Polar/benzene (or toluene) (e.g., polar solvents: ether, EtOAc, MeOH)