thin layer chromatography
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THIN LAYER CHROMATOGRAPHY (TLC)AND
COLUMN CHROMATOGRAPHY
M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar.
You will have to separate three components of paprika.
The three components can be easily identified because they are colored (absorb visible light).
They have different polarities.
They can be separated using column chromatography.
You can monitor the separation using thin layer chromatography.
What is chromatography….
Chromatography• Very useful technique in organic chemistry based on differential adsorption.
• Used to separate components in a mixture (solid or liquid).
• It depends on the polarity of the ingredients involved --- intermolecular forces!!
• Thin layer chromatography (TLC) is used to analyze components and purity of a mixture.
• Thin layer chromatography (TLC) is also used to monitor the progress of a reaction.
Chromatography
What do we need to perform a chromatographic separation?
• Adsorbent: Silica gel (silicon dioxide), also called “stationary phase”. • Eluent: solvent used to move your compound trough the silica gel, also called the mobile phase.
• Your compound mixture to be separated.
• Patience and chemical intuition.
Chromatography• More polar molecules “stick” to the adsorbent longer.• Less polar molecule separate more easily from the adsorbent. • When this happens, separation occurs.
SiOH
SiOH
SiOH
SiOH
SiOHSiOHSiOHSiOHSiOH
SiOH
SiOH
SiOH
Stationary phase To be separated
O
Eluent (mobile phase)
Chromatography• More polar solvent move the molecules more efficiently• Less polar move the molecules less efficiently• Separation occurs
Most polar
Least polarAlkanesTolueneDiethyl etherChloroformAcetoneEthyl acetateEthanolMethanol (CH3OH)
Column Chromatography
Load the silica gel plus eluent into the column…this is called “column packing”
Column Chromatography
Using a Pasteur pipette, load your compound that was dissolved in a minimum of solvent onto the silica.Your test solution will then add the eluent. Do not let your column run dry!!
Thin Layer ChromatographyTypical TLC chamber
We will use beaker with watch glass or aluminum foil
Thin Layer ChromatographySpotting TLC plate
•Use different capillary for each solution.
• make solution of approx. 1-2 mg of sample in 1 ml of solvent.
• Spot 2-3 times
•Try to make small spots
Insert filter paper to saturate atmosphere with solvent
Keep the lid on!!
Thin Layer Chromatographypreparation of chamber
Mark a line about 1 cmfrom the bottom with pencil
It is important to use pencil
Place TLC plate in chamber Let things develop!
Don’t let the solvent front run offThe top of the plate!!
Pull it out and mark the solvent front before it evaporates
Mark spots with pencil!
Good, bad and ugly
• First TLC shows”overloading" due to too much sample.
• Second shows good separation.
• Third shows almost not enough compound,but OK