Instrumentation of Thin Layer Chromatography

Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed.
Thin-layer chromatography is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose.

1. Instrumentation
of
Thin layer chromatography
Presented By:
Tanmoy Sarkar
ID: 181130
3
rd
Year 1
st
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 commerciallyproduced thin adsorbents are:
Fig: TLC plate

4. ➢A solvent or solvent mixture which moves on the stationary phase.
➢The solvent travels through the stationary phaseby 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. ➢Slurryispreparedbymixingtheadsorbent.
➢Asmallamountofinertbinderlikecalciumsulfate(gypsum)andfluorescentcompounds
canbeaddedforbetterdetection.
➢Thismixtureisspreadedonanunreactivecarriersheet,usuallyglass,thickaluminumfoil,
orplastic.
➢Theresultantplateisdriedandactivatedbyheatinginanovenfor30minutesat110°C.
Preparation of TLC Plates

6. ➢Alineismarkedabout1cmabovethebottomoftheplatewiththehelpofapencil.
➢Solutionofsampleorstandardisspottedusingacapillarytubeormicropipette.
➢Thespotmustbeasnarrowaspossibleforbetterseparationandminimumtailing.
Marking & spotting on TLC Plates

7. ➢To equilibrate the atmosphere of empty space in chamber,
it is kept closed with lidwith 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 compoundsand 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
DC