Introduction to Thin Layer Chromatography
Thin layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. The main components of a TLC system include a glass, plastic, or aluminum backed silica gel plates coated with a thin layer of adsorbent material usually silica gel, aluminum oxide, or cellulose as the stationary phase. The mixture is applied as a small spot or band near one edge of the plate and the plate is then placed into a developing chamber containing a small amount of adsorbing solvent (mobile phase). As the mobile phase travels up the plate by capillary action, different analytes travel at different rates depending on how strongly they adhere to the stationary phase. Substances that differ in their distribution coefficients will therefore migrate different distances. After the mobile phase travels a set distance the components will be separated into spots at different positions on the plate. The separated spots can then be visualized under UV light at specific wavelengths or by dipping the plate in color reagents to indicate the composition of the original mixture.
TLC works on the principle that the different components of a mixture move up the TLC plate at different rates depending on how they partition between the mobile phase and stationary phase. The partition coefficient, K, is the ratio of concentrations of a compound in the mobile and stationary phases when the two phases are in equilibrium. Compounds with higher K values (less polar) exhibit higher Rf values. Rf is defined as the ratio of the distance travelled by the compound to the distance travelled by the solvent front. Compounds with lower K values (more polar) typically remain closer to the origin. TLC is used for confirming the identity and purity of samples and the resolution is usually sufficient for analytical purposes.
Advantages of Thin Layer Chromatography
There are several key advantages that make TLC a useful technique:
Simple and inexpensive – No expensive equipment is needed. Plates, mobile phases and reagents can be prepared easily in any lab. Costs are low compared to other methods like HPLC or GC.
Rapid – Separation and analysis of substances can be done within 30 minutes to 1 hour depending on the mobile phase used. This allows for high sample throughput.
Versatile – A wide variety of stationary and mobile phases can be employed to suit different sample properties. Plates can also be developed in one direction or by two dimensional development.
Sensitive – Detection limits in the low nanogram to low microgram range are possible depending on detection technique used. Spots as small as 50-100 ng can often be detected.
Semi-quantitative – Density of spots can give an indication of relative quantities/purity which is useful for analytical applications.
Portable – Plates can be developed anywhere without need for elaborate infrastructure. Field testing is possible.
Multisample – Several samples can be run simultaneously on a single plate allowing for easy comparison.
Low toxicity – Mobile phases used like organic solvents, water are not highly toxic in amounts used compared to eluents used in other methods.
Thin Layer Chromatography Sample Applications
Some common applications of TLC in research include:
Separation and confirmation of components in herbal extracts and plant secondary metabolites – Used extensively in herbal and natural product research. Can fingerprint extracts and indicate purity.
Analysis of pharmaceutical samples – Used for identification and purity testing of crude drug samples, detection of impurities, stability studies etc.
Food analysis – Detection of adulterants, identification of flavors and aroma compounds, separation of food dyes are some uses in the food industry.
Clinical and Forensic analysis – Screening of body fluids like urine, blood, etc. for drugs of abuse, toxins. Also used in forensic toxicology in confiscated drug samples.
Characterization of reaction products – Separation and confirmation of components obtained after chemical reactions helps in optimization and structure elucidation.
Separation of amino acids, sugars, lipids etc. – Common analytical applications include analysis of simple biomolecules and their derivatives.
Thin layer radiochromatography – Incorporates radioactive tracing to locate compounds of interest that cannot be visually detected on plates.
Microscale TLC – Used for analysis when only small amounts of samples are available. Plates have smaller sizes and coatings.
Two dimensional TLC – Improves resolution by developing first in one direction, followed by 90 degree rotation and second development.
Immobilized enzymes TLC – Used to study enzyme kinetics, inhibition, inhibitor screening by incorporating enzymes onto plates.
Imaging TLC scanning – New detection methods employ imaging scanners for digitized quantification and processing of TLC plate results.
Thin Layer Chromatography Detection Methods
Several detection methods can be used to visualize separated components on TLC plates depending on the nature of analytes. Some common techniques used include:
UV visualization – Many organic compounds fluoresce or change color when viewed under short (254 nm) or long (366 nm) wavelength UV light.
Iodine vapor – Used for detection of unsaturated compounds which form colored complexes with iodine vapor. Spots appear brown.
Dragendorff’s reagent – Alkaline picrate solution used to detect alkaloids which appear orange. Other reagents based on diazonium salts or mercury compounds also widely applied.
Ninhydrin – Used for detecting amino groups in amino acids which are visualized as purple spots.
Anisaldehyde – General purpose reagent used to detect a wide range of compounds including sugars, steroids, terpenoids which develop various colors on heating.
Dinitrophenylhydrazine – Develops violet/pink spots on reaction with carbonyl groups to detect aldehydes, ketones in samples.
Bromocresol green – Used to detect acidic and neutral compounds on TLC plates. Blue or yellow spots produced.
Fluorescent indicators – Spray reagents containing fluorescing tags are used to detect specific compound classes like flavonoids under UV light.
ELISA detection – New immunodetection methods interface TLC plates with ELISA to sensitively visualize target analytes.
Mass spectrometry – TLC plates can be directly coupled to MS detectors for identification after separation without need for chemical derivatization.
Documentation and Thin Layer Chromatography Evaluation
Precise documentation of experimental details is essential when performing and reporting TLC separations. Key parameters to record include:
Stationary phase material used (RP-18 silica, cellulose, alumina etc.) and plate batch or lot numbers.
Mobile phase composition and volume used for development.
Method of sample application (spotting, banding and volumes).
Direction and distance of mobile phase travel or solvent front (Rf).
Detection method employed (wavelengths if UV or specific chemical spray used).
Rf values of all resolved spots.
Photographs of plates under normal and UV light (if applicable).
Analysis of results includes determining Rf, number of spots, resolution between spots, reproducibility of the method. Control standards should be run for comparison. Effect of parameters like mobile phase composition, proportion on separations must be characterized. Method can be validated by spiking samples, running recovery tests for quantitative purposes. TLC is a powerful yet simple technique useful as a primary screening step or confirmatory identification step in various fields of research. Proper method development and documentation is key to fully realizing its analytical capabilities.
Andrew Stroud