Thin Layer Chromatography (TLC)
Separation and retention on TLC plate
Each compound is defined by its retardation factor Rf (unitless) that corresponds to its relative migration compared to the solvent. Rf values lie between 0 and 1. Rf values often depend on the solvent use in the TLC experiment and temperature
The efficiency N of the plate for a compound whose migration distance is x and spot diameter w is given by
Retention factor is defined as the distance travelled by the individual component divided by the total distance travelled by the solvent
Component is a mixture
the same plate can be used to determine the component of mixture by rotating it when one set of component seperation is done
Knowing the polarity
Rule of thumb: The component that travels the least distance on the TLC plate is the most polar, since it binds to the silica most tightly
The component that travels the maximum distance is the least polar; it binds to the silica least tightly and is most soluble in the non-polar solvent (mobile phase), and hence moves up the plate with the solvent
Mobile Phase of TLC
The organic solvent mixture of low polarity is used. Highly polar solvents are avoided to minimize adsorption of any components of the solvent mixture. Use of water as a solvent is avoided as it may loosen the adhesion of a layer on a glass plate
Solvents with an increasing degree of polarity are used in liquid-solid or adsorption chromatography
Property of stationary phase depends on size of the particles, their specific surface area, the volume of the pores and the distribution of particle.
TLC plates can also be prepared to contain chemical groups with net positive or negative charges on the surface. This type of plate is used for ion-exchange TLC
Characteristics of TLC
TLC corresponds to a three-phase system between which equilibriums are established: solid (stationary), liquid (mobile) and vapour phases
The adsorption phenomenon of the stationary phase is substantially reduced once a large part of the adsorption sites are occupied. This creates an effect of elongation of the spots.
As a result, the Rf (retardation factor) of a compound in the pure state is slightly different from the Rf of the same compound present in a mixture migration velocity
This is done by placing the plate under the lens of a densitometer (or scanner) that can measure either absorption or fluorescence at one or several wavelengths.
This instrument produces a pseudo-chromatogram that contains peaks whose areas can be measured. In TLC a spot is usually detectable if it corresponds at least to a few ng of a compound UV absorbent
High-performance thin layer chromatography (HPTLC)
High-performance thin layer chromatography HPTLC is an improvement of the technique where the sorbent material (e.g. silica gel 60) has a finer particle size and a narrower particle size distribution than conventional TLC. HPTLC plates have an improved surface homogeneity and are thinner
The resolution is improved, analysis times are shorter and it is sufficient to apply nanolitres or nanograms of sample (Nano-TLC)
TLC versus HPLC
Compared with HPLC, TLC is able to treat more samples in the same time period by the setting
up of analyses in parallel on the same plate.
The plate, only used once and disposable, allows for rapid sample preparation with less risk of loss or contamination. It is very useful for biological samples
Making of TLC Plates
TLC plate can be made by various methods of coating. Automated methods are now used for accuracy and precision
Pouring: The adsorbent of finely divided and homogeneous particle size is made into slurry and is poured on a plate and allowed to flow over it so that it is evenly covered
Dipping : This technique is used for small plates by dipping the two plates at a time, back to back in a slurry of adsorbent in chloroform or other volatile solvents. Exact thickness of layer is not known and evenness of layer may not be good
Spraying : Slurry is diluted further for the operation of sprayer. But this technique is not used now a days as it is difficult to get uniform layer
Spreading : All the above methods fail to give thin and uniform layers. Modern methods utilize the spreading devices for preparation of uniform thin layers on glass plates. Commercial spreaders are of two types (a) Moving spreader, (b) Moving plate type. It gives layer thickness from 0.2 to 2.0 mm
Activation of plates
After spreading plates are allowed to dry in air and further dried and activated by heating in oven at about 110 degree C for 30 minutes. By removing the liquids associated with layer completely, the adsorbent layer is activatethus its a must thing to do in TLC plate
Steps of Component Separation
Deposition of the sample
A small volume of sample (between a few nanolitres to a few microlitres), dissolved in a volatile solvent, is deposited close to the bottom of the plate
Developing the plate
The mobile phase rises up the stationary phase by capillarity, moving the components of the sample at various rates because of their different degrees of interaction with the matrix and solubility in the solvent
Identifying the spots
Manufacturers sell plates that contain a fluorescent salt of zinc which emits a bright green fluorescence when the plate is irradiated with a UV mercury vapour lamp = 254nm and observed in a viewing cabinet.
Another method to make compounds visible, almost universally used, consists of heating the plate after spraying it with sulfuric acid which leaves charred blots behind. e.g. Ninhydrin (2,2-dihydroxyindane-1,3-dione) in alcoholic solution for amino acids
Identification is usually carried out by scraping off the eluted spots on the chromatogram and eluting them in analytical columns. The identified compounds then quantified using several methods. These can be quantified by densitometer, spectrophotometer, auto-radiography and fluorescence
Thin Layer Chromatography (TLC)
Thin layer chromatography is another type of planar chromatography which is performed on a piece of glass plate that is coated with a thin layer of silica
TLC originated to overcome the problems faced in paper chromatography and is widely used in pharma and environmental applications.
Here, silica acts as the stationary phase and the solvent in which the plate is dipped and that runs up the plate by capillary action is the mobile phase.
TLC can also be done in normal as well as reverse phase;
Normal Phase TLC - The stationary phase i.e. silica is very polar in nature, while the solvent is less polar compared to silica
Reverse Phase TLC - Stationary phase in non or less polar and solvent is polar in nature. When using a plate of reversed polarity (‘RP-TLC’), the mobile phase will generally contain water. In this case it can be useful to add a salt such as lithium chloride which limits diffusion phenomena and thereby increases the resolution
In normal phase; the polar components of the analyte/sample will adhere to the silica tightly and thus travel slowly up the plate, while the less polar or non-polar components will not adhere that strongly to the silica and travel up the plate relatively fast with the solvent
The technique has been improved and fully automated with time. The development of automatic applicators and densitometers have led to nano-TLC, also called HPTLC, a highly sensitive technique which can be hyphenated with mass spectrometry
Densitometers are device used to measure optical density. In TLC it is used for quantitative measurement of analyte.
Principle of TLC
Basic principle of seperation is ADSORPTION
Separation is conducted on a thin layer 100–200µm of stationary phase, usually based upon silica gel and deposited on a rectangular plate made out of glass, plastic or aluminium of a few centimetres in dimensions.
To maintain the stationary phase on the support and to assure the cohesion of the particles, an inert binder like gypsum (or organic linker) is mixed into the stationary phase during the manufacture of the plate