TLC Tips & Tricks
1. Choice of Solvent System (Mobile Phase)
2. Choice of TLC Layer (Stationary Phase)
3. Pre-Conditioning TLC Plates
4. Correct sample application
5. Drying TLC Plates
6. How to Saturate TLC Chambers
7. Spraying TLC Plates for Derivatization
8. Quantitative Evaluation with TLC Scanners
The choice of solvent system is critical in thin-layer chromatography. Follow the guidelines and table below to find the most suitable mobile phase for your separation.
In the table below, the solvents are listed in increasing order of elution strength (according to Halpaap’s eluotropic series).
|Solvent||Velocity coefficient, k (mm²/s)|
|13||Isobutyl methyl ketone||9.1|
|17||Ethyl methyl ketone||13.9|
|Sorbent||TLC plate silica gel 60 F254 Merck|
|Type of chamber||N-chamber with chamber saturation|
|Room temperature||22 °C|
|Migration distance of solvent||100 mm|
|Source: Applied Thin-Layer Chromatography, Elke Hahn-Deinstrop, page 71|
To help you select the optimal stationary phase for your analysis, the table below shows the most popular pre-coated TLC layers available and their typical applications.
|Sorbent material||Chromatographic principle||Typical applications|
Adsorption chromatography due to polar interactions
|Alkaloids, steroids, terpenes, aliphatic, aromatic and basic compounds|
|Cellulose||Depending on acetyl content transition from normal phase to reversed phase chromatography||Anthraquinones, antioxidants, polycyclic aromatics, carboxylic acids, nitrophenols, sweeteners|
|Kieselguhr||Commonly impregnated for reversed phase separations||Aflatoxins, herbicides, tetracyclines|
|Standard silica gel, also with concentrating zone||Normal phase chromatography||Most frequent application of all TLC layers, Aflatoxins|
|Silica gel G, impregnated with ammonium sulfate||Surfactants, lipids (neonatal respiratory syndrome)|
|Silica gel 60, impregnated with caffeine for PAH determination||Charge transfer complexes||Polycyclic aromatic hydrocarbons (PAH) acc. to German drinking water specification|
|Cyano-modified layer CN||Normal phase and reversed phase chromatography||Pesticides, phenols, preservatives, steroids|
|DIOL-modified layer||Steroids, hormones|
|Amino-modified layer NH2||
Anion exchange, normal phase and reversed phase chromatography
|Nucleotides, pesticides, phenols, purine derivates, steroids, vitamins, sulfonic acids, carboxylic acids, xanthines|
|RP-2, RP-8, RP-18||Nonpolar substances (lipids, aromatics)|
|Silica gel 60 silanized||Polar substances (basic and acidic pharmaceutical active ingredients)|
|RP-18 W/UV254, wettable||Normal phase and reversed phase chromatography||Aminophenols, barbiturates, preservatives, nucleobases, PAH, steroids, tetracyclines, phthalates|
|Spherical silica gel|
|LiChrospher® Si 60||Normal phase chromatography||Pesticides, phytopharmaceuticals|
|Source: Applied Thin-Layer Chromatography, Elke Hahn-Deinstrop, pages 22-23|
Pre-conditioning TLC layers protects them from humidity, which could otherwise diminish their activity and affect chromatogram results.
The correct sample application on TLC plates is essential for accurate and reproducible separations. Below are a few ways you can avoid errors.
Important: Keep exposure of plates to blowers as short as possible to protect the layer from airborne dirt particles.
TLC development can be performed in saturated or unsaturated chambers. Chromatography in unsaturated chambers results in evaporation of the solvent from the layer, particularly near the front. This leads to higher solvent consumption, and higher Rf values.
Chamber saturation method