6. • Paper chromatography is an analytical method used to separate
colored chemicals or substances.
• A paper chromatography variant, two-dimensional
chromatography involves using two solvents and rotating the
paper 90° in between.
• This is useful for separating complex mixtures of compounds
having similar polarity, for example, amino acids.
INTRODUCTION
7. • The principle of separation is mainly partition rather than
adsorption.
• Cellulose layers in filter paper contains moisture which acts
as stationary phase & organic solvents/buffers are used as
mobile phase.
principle
8. 1. Solvent trough –Isopropyle alcohol
+
Distilled water
2. Airtight chamber
3. Whattmann filter paper number
instrumentation
9. Whattmann filter paper & make point
Put drop of sample mixture at point using capillary tube
Dry it in open air
Suspend the filter paper in chromatographic chamber
Take out from chamber
Calculate Rf value
PROCEDURE
12. The separation depends on the relative affinity of
compounds towards stationary and the mobile phase.
principle
13. 1.TLC plates
2. TLC chamber
3. Mobile phase
4. A filter paper
instrumentation
14. Make Thin mark at the bottom of the TLC plate
Applied samples solutions are on the spots marked
Pour Mobile phase into the TLC chamber
Place TLC plate in TLC chamber
Allow to dry the Plates
Visualization of spot under UV rays.
(Calculate the Rf value)
PROCEDURE
15. 1. To check the purity of given samples.
2. Identification of compounds like acids, alcohols, proteins,
alkaloids, amines, antibiotics, and more.
3. To evaluate the reaction process by assessment of
intermediates, reaction course, and so forth.
APPLICATION
19. Column is filled with ion exchanger, sample, buffer.
Particles will come down the column along with Buffer.
Particles are analyse by spectroscopically.
PROCEDURE
20. ◘ Softening of water
◘ Demineralisation of water
◘ Purification of solutions free from ionic impurities
◘ Separation of inorganic ions
◘ Separation of sugars, amino acids
APPLICATION
22. • The porous beads act as “traps” or “sieves” and function to
filter small molecules which become temporarily with in the
pores.
• Large molecules pass around or are excluded from the beads.
principle
23. Stationary phase - porous beads.
The mobile phase – Tris buffer.
The columns
The pump-syringe pumps, reciprocating pumps.
Detectors.
COMPONENTS & PROCEDURE
32. PROCEDURE
Unplug the stopper on the bottom
Take the lid off the top, that will the buffer
to drain out
Plug the column
Load sample evenly around the surface of beads
Add little amount buffer
Collect the elute & analyse the particles
spectrophotometrically
42. ULTRA CENTRIFUGATION
Theodor Svedberg invented the analytical
ultracentrifuge in 1925,and won the nobel
prize in chemistry in 1926 for his research on
colloids and proteins using the
ultracentrifuge.
43.
44.
45. • A particle , whether it is precipitate ,macro molecule or cell
organelle when rotated at high speed is subjected to a
centrifugal force
principle
