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SiliaPlate - TLC Practical Guide

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Select a Stationary Phase

As almost 80% of all separations can be performed using silica gel plates, it is suggested to try using this coating. However, for acid sensitive compounds, alumina is probably a better choice (useful for amine purification). If you are working with highly polar compounds, reversed-phase mode is more suitable.

Select a Mobile Phase (Solvent Systems)

The selection of the mobile phase (also called solvent system or eluent) is perhaps the most important parameter to achieve efficient thin layer chromatography separation. It is based on the compound’s solubility with the solvent and the difference in the affinity for the mobile phase versus the stationary adsorbent (silica).

In normal phase chromatography, where non-polar solvents such as hexane or pentane are used, non-polar compounds will move up the plate while most polar compounds will stay on the baseline. Inversely, polar solvents will allow polar compounds to move off the origin. The most suitable solvent system is the one that moves all components off the baseline with Rf values between 0.15 and 0.85 (ideally, close to 0.2 - 0.4).

Remember that it is not always possible in TLC but should be possible in flash chromatography where solvent gradients can be used.

For most applications, a common solvent system to start with is 1:1 Ethylacetate (EtOAc) / Hexane. Varying the ratio can have a pronounced effect on the Rf. If it is not working, then try: Methanol (MeOH) / Dichloromethane (DCM) (1:99 – 10:90); or toluene with acetone, EtOAc, or DCM.

Remember: To increase the compound’s Rf, increase the polarity of the mobile phase; increase the ratio of the polar solvent or choose another solvent. Inversely, to decrease Rf, decrease the polarity of the eluent.

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%).

Reversed-phase mode

In reversed-phase chromatography, the typical solvent systems are:

  • Mixtures of water or aqueous buffers and water miscible organic solvents such as acetonitrile (ACN), methanol, and tetrahydrofuran (THF). Other solvents can be used such as ethanol (EtOH) & isopropanol (IPA).
  • If needed, to improve peak shape in flash chromatography, 0.1% of acetic, formic or trifluoroacetic acid (TFA) can be added to the solvent system.

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.

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.


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.

SiliaPlate Cospoting

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.



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