Thin Layer Chromatography
- What type of TLC plate to choose?
- What type of TLC backing to choose?
- How to cut TLC plates on glass support?
- How to read your chromatogram?
- Stains for TLC Plates
- Streaking or elongated spot rather than a defined spot?
- Unable to see any spots on the TLC?
- How to monitor a reaction in presence of similar Rfs for both starting materials and product of interest?
- What is the co-spotting method?
- What to do if compounds stay too close to the baseline or solvent front?
Origin of TLC plates is a little obscure, but it was probably developed in the 40’s. Thus, it is a mature technology and is continually predicted to soon be in regression. Yet, because of major advantages such as speed of analysis, low cost, small quantity of compound needed and high sample throughput capability (up to 20 samples simultaneously), sales are constantly increasing, despite the threat of an emerging technological breakthrough that’s been expected for 30 years but which ultimately never came.
Like column chromatography, TLC is a solid-liquid partitioning technique, in which the sample is applied to the plate as a small spot near the base of the plate. The moving liquid phase is then allowed to ascend the plate, causing the sample to partition between moving and stationary phase.
HOW TO PREPARE YOUR PLATE
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.
HOW TO PREPARE YOUR SAMPLE
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.
HOW TO APPLY YOUR SAMPLE
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.
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.
TLC PLATE VISUALIZATION
If components of the reaction are colored, no visualization method is required (spots can be seen directly on the silica layer).
However, most of the time it is not the case, therefore one of the methods described below should be used to reveal the spots.
Non-destructive methods
As a general visualization procedure, before treating the TLC plate with any destructive methods, UV-active compounds can be viewed under an ultraviolet lamp (usually for polyconjugated compounds like benzophenones and anthracenes).
Furthermore, an iodine chamber can be useful for thiols, phosphines and alkenes but it works in about 50% of cases for alkanes. It is recommended to circle the spots with a pencil on the TLC plate prior to visualization by destructive methods.
Destructive methods
For compounds that are not UV-active, there are several varieties of stains that can be used depending on the nature of the compound of interest. To use a stain, simply dip the TLC plate into the staining solution as quickly as possible, and then immediately absorb the excess stain with paper and heat carefully with a heat gun or on a hot plate at 110°C until spots are revealed.
Read more:
> TLC plate visualization methods
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