1. Thin layer chromatography uses a stationary phase, such as silica gel or alumina, coated onto a plate. A mobile phase, such as a solvent or solvent mixture, is run up the plate to separate the components of a sample spotted onto the plate. 2. Key instruments used in TLC include TLC plates, forceps, pencil, ruler, filter paper, micropipette, and a developing chamber. Samples are spotted onto the plate using a micropipette and developed in the chamber containing the mobile phase and filter paper. 3. Detection of separated components is done using visualizing agents like iodine or UV light, or specific reagents that react with certain functional groups to

1. Instrumentation
of
Thin layer chromatography
Presented By:
Tanmoy Sarkar
ID: 181130
3rd Year 1st Term
Pharmacy Discipline
Khulna University
Khulna

2. Instruments used in TLC
Stationary Phase
Mobile Phase
TLC plate
Forceps, pencil and ruler
Filter paper
Micropipette
Developing container’
- chamber/ jar/ glass beaker
Analyte
Detecting & visualizing agent

3. Stationary Phase
Adsorbents Acidic or Basic
Components to be
separated
Silica gel Acidic
Acidic and neutral
substances
Alumina Basic Basic and neutral
Cellulose Powder Neutral Soluble compounds
➢ The phase which the mobile phase passes over
➢ Made of coated with adsorbents.
➢ Used to provide facility for the adsorption of analyte on TLC film.
➢ Some commercially produced thin adsorbents are:
Fig: TLC plate

4. ➢ A solvent or solvent mixture which moves on the stationary phase.
➢ The solvent travels through the stationary phase by capillary action.
➢ The ability of mobile phase to move up is depend on the polarity itself.
➢ Commonly used: Ethyl acetate, Hexane
mobile Phase
Solvent Polarity
Hexane 0.1
Carbon tetrachloride 1.56
Isopropyl ether 1.83
Toluene 2.4
Chloroform 2.7
Diethyl ether 2.8
Isopropanol 3.92
Ethyl Acetate 4.4
Acetone 5.1
Acetonitrile 5.8
Water 10.2

5. ➢ Slurry is prepared by mixing the adsorbent.
➢ A small amount of inert binder like calcium sulfate (gypsum) and fluorescent compounds
can be added for better detection.
➢ This mixture is spreaded on an unreactive carrier sheet, usually glass, thick aluminum foil,
or plastic.
➢ The resultant plate is dried and activated by heating in an oven for 30 minutes at 110 °C.
Preparation of TLC Plates

6. ➢ A line is marked about 1 cm above the bottom of the plate with the help of a pencil.
➢ Solution of sample or standard is spotted using a capillary tube or micropipette.
➢ The spot must be as narrow as possible for better separation and minimum tailing.
Marking & spotting on TLC Plates

7. ➢ To equilibrate the atmosphere of empty space in chamber,
it is kept closed with lid with the mobile solvent.
➢ Filter Paper has to be placed inside the chamber.
➢ After placing the plate in the chamber, close it with lid.
➢ Use of forceps is a must for handling the plate.
➢ The spotting area should not be immersed in mobile phase.
DEVELOPING CHAMBER

8. ➢ For colorless solutes, visualization or detection techniques are required, such as:
❑Non-specific:
✓ UV Chamber for fluorescent compounds
✓ Iodine Chamber Method
DETECTING AGENTS
❑Specific:
✓ Ninhydrin: method for amino acids.
✓ Bromocresol Green: method for carboxylic acids.
✓ Iodine vapor: method for hydrocarbons
✓Dragendorff’s reagent: method for alkaloids, nitrogen-
containing compounds and lipids.
✓Antimony trichloride: method for cardiac glycosides
✓Aniline phthalate: method for Sugar
Fig: TLC UV Lamp

9. Some precaution
A. Handle with forceps
B. Do not immerse the
spotting line in solvent
C. Spotting should be as
narrow as possible
D. Carefully choose the
solvent
A B
D
C