1. Thin Layer Chromatography
This technique is called Thin-layer chromatography because a thin layer
of solid material that is coated onto a small glass plate.
2. History :
• Michael Tswett is credited as being the father of chromatography.
Tswett developed his ideas in the early 1900’s.
• 1958 : Ergon stahl introduced a standard equipment for preparing
uniform thin layers of unknown thickness.
3. Introduction :
• It is one of the simplest, fastest, easiest and least expensive of several
• TLC is a form of Liquid chromatography.
• TLC is used in quantitative and qualitative analysis of organic
compounds and to test their purity.
5. Consisting of :
1. A mobile phase
2. A stationary phase ( a plate or a strip coated with a form of silica
• But nowadays,
• There is a more sophisticated stationary phases and more complexed
• Ready made plates are available.
6. TLC is very much like HPLC :
• The stationary and mobile phases are similar.
• In TLC it is not possible to change the characteristics of the mobile
phase on the fly, but by proper mixing, a large variety of mobile
phase can be achieved.
• Stationary phases of various polarities are also available
commercially. (Readymade plates)
7. Stationary Phase :
• Thin layer of solid material combined with binders coated on glass,
plastic or aluminum plates.
• Size of plate : from microscope slide to 6”
• Coatings :
range from few microns up to 1-2mm.
It may contain an internal fluorophore; it’s a substance that will
fluoresce when exposed to UV light.
8. How fluorophore works?
• When a non-fluorescent sample is loaded – it will quench or blot out
the fluorescence under the sample and it will be seen as a dark spot.
• how heavily a spot has been made
• Its position
• As stated earlier, TLC plates (also known as chromatoplates) can be
prepared in the lab, but are most commonly purchased
• Silica gel and alumina are among the most common stationary phases, but
others are available as well.
• Many plates incorporate a compound which fluoresces under short-wave
UV (254 nm).
• The backing of TLC plates is often composed of glass, aluminum, or plastic.
• Glass plates are chemically inert and best withstand reactive stains and
heat, but are brittle and can be difficult to cut.
11. Conti …
• Aluminum and plastic plates can be cut with scissors, but aluminum
may not withstand strongly acidic or oxidizing stains, and plastic does
not withstand the high heat required to develop many stains.
• Aluminum and plastic plates are also flexible, which may result in
flaking of the stationary phase.
12. Conti …
• Polarity of S phase : range from moderately polar to very non-
polar (so both normal and reverse phase chromatography are
13. Mobile phase :
• As in HPLC, it is an organic liquid (or water) or a solution of two or
• Polarity : range from very non-polar to very polar.
• pH : buffered to maintain pH or ionic strength.
14. Conti …
• Usually mobile phase cannot be
altered during TLC run
• In 2-D TLC : plate is developed using
one mobile phase and then turned
900 and developed in a different
15. The process :
• Carried out in a chamber
• Dimensions depend upon size of the coated plate (glass/plastic)
• Plate must fit so it doesn’t touch the sides of chamber
• Arrangement may use 5 * 10 cm plate in a 400 ml chamber
• Piece of filter paper is put inside chamber up against side (this will
absorb some of M phase and chamber is saturated with M phase)
• Top of chamber is tightly covered (paraffin is used as it works well as a
16. Conti …
• Analyte is dissolved in a small amount of solvent (volatile),
chloroform/methanol often used.
• Spot :
• Spot of dissolved analyte is mark on plate
• Kept as tiny as possible
• Narrow capillary can be used for this
• spot can be over spotted, but 1st spot should be dried first.
• After the plate is loaded with sample(s), small amount of M phase is
put in the chamber (must be enough to travel up the plate)
• When m phase nearly reached top of the plate, process is stoped by
removing and drying the plate.
• Some common techniques for visualizing the results of a TLC
plate include :
2.Iodine Staining: is very useful in detecting carbohydrates since it
turns black on contact with Iodine
3.KMnO4 stain (organic molecules)
4.Ninhydrin Reagent: often used to detect amino acids and proteins
18. Conti …
• For example, tetrahydrocannabinol, the active ingredient in
marihuana, shows up as a bright, orange-red spot when sprayed with
a reagent known as Fast Blue BB.
• Most nitrite (NO2) containing compounds, such as most explosives,
will turn red when a two-step reagent sequence, known collectively
as Griess reagent is sprayed on them.
20. Retention Factor (Rf ) Value :
• The behavior of a compound on a TLC is usually described in
terms of its relative mobility or Rf value.
• Rf or Retention factor is a unique value for each compound
under the same conditions.
• The Rf for a compound is a constant from one experiment to the
next only if the chromatography conditions below are also
thickness of the adsorbent
amount of material spotted
21. Conti …
• Since these factors are difficult to keep constant from
experiment to experiment, relative Rf values are generally
• Relative Rf” means that the values are reported relative to a
• The Rf value is calculated using the following equation:
Rf = distance traveled by the component
distance traveled by the solvent
22. Applications of TLC :
1.In monitoring the progress of reactions
2.Identify compounds present in a given mixture
3.Determine the purity of a substance.
• Analyzing ceramides and fatty acids
• Detection of pesticides or insecticides in food and water
• Analyzing the dye composition of fibers in forensics
• Assaying the radiochemical
purity of radiopharmaceuticals
• Identification of medicinal plants and their constituents
23. Advantages of TLC :
• It is a simple process with a short development time.
• It helps with the visualization of separated compound spots
• It helps in isolating of most of the compounds.
• The separation process is faster and the selectivity for
compounds is higher (even small differences in chemistry is
enough for clear separation).
• The purity standards of the given sample can be assessed
• It is a cheaper chromatographic technique.
• More than one sample can be run at the same time.
24. Limitations of TLC :
• It cannot tell the difference between enantiomers and some
• In order to identify specific compounds, the Rf values for the
compounds of interest must be known beforehand.
• TLC plates do not have long stationary phases. Therefore, the
length of separation is limited compared to other
Notas del editor
Never under any circumstances touch the face of a TLC plate with your fingers as contamination from skin oils or residues on gloves can obscure results. Instead, always handle them by the edges, or with forceps.