2. PLANAR CHROMATOGRAPHY Ferosekhan. S FNB-41

3. Chromatography Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate Analyze Identify Purify Quantify ComponentsMixture

4. Mechanisms Of Separation  Partitioning equilibrium ( SP: liquid, MP: liquid/gas )  Adsorption equilibrium ( SP: solid, MP: liquid )  Exclusion equilibrium ( SP&MP: liquid )  Ion Exchange equilibrium ( SP: solid ion-exchanger, MP: liquid electrolyte )  Affinity equilibrium ( SP: immobilised ligand, MP: liquid)

5. Classification Of Chromatography chromatography Liquid chromatography Gas chromatography Flat chromatography Column chromatography Paper chromatography Thin layer chromatography TLC Ascending Two dimensionlal Circular descending Open column e.g Partition chromotography Adsorption chromotography Ion exchange chromotography Gel filtrtion chromotography Affinity chromotography High performance liquid chromotography

6. Classification  Column Chromatography the stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or by gravity.  Planar Chromatography the stationary phase is supported on a flat plate or the interstices of a paper and the mobile phase moves through the stationary phase by capillary action or by gravity.

7. Planar Chromatography - Types  Thin layer chromatography (TLC) separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)  Paper Chromatography (PC) separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)

8. THIN LAYER CHROMATOGRAPHY (TLC)

9. Thin layer chromatography (TLC)  In TLC, any substance that can be finely divided and formed into a uniform layer can be used.  Both organic and inorganic substances can be used to form a uniform layer for TLC.  Organic substances include: cellulose, polyamide, polyethylene  Inorganic: silica gel, aluminum oxide and magnesium silicate

11. Cont…  TLC to separate lipids  Surface of the plate - very thin layer silica – SP  Silica – polar (stationary phase)  Spot the material at the bottom of the TLC plate cellulose Al 2 O 3

12. Cont…  Place the plate into a glass jar - small amount of a solvent  This solvent - moving phase.  Remove the plate from the bottle when the solvent is close to the top of the plate.

13. Thin-Layer Chromatography: A Two- Component Mixture More polar! Less polar! solvent front origin mixture solvent front component B component A origin solvent front component B component A origin Increasing Development Time

14. Thin Layer Chromatography TLC plate silica gel - silicon dioxide (SiO 2 ) x (a common, inexpensive stationary phase) bulk (SiO 2 ) x These exposed OH units give silica gel a relatively polar surface. surface

15. Four Stages in TLC 1. Sample Application - Capillary used to spot solution of each sample. 2. Development - This is when the separation actually occurs. 3. Visualization - viewed under UV light. 4. Interpretation of Result - Comparison of retention factors.

16. TLC plate “finishing line”  1 cm. A. Draw “guide lines” lightly with pencil “starting line”  1 cm. B. Dissolve solid sample in MeOH C. Use TLC capillary to transfer and spot dissolved sample Sample A B C Ref. Ref. Ref. 1. Sample Application (spotting)

17. 2. Development of TLC Plate TLC plate TLC Developing Chamber (just a glass jar with solvent in it!) A. Place spotted TLC plate in developing chamber B. Developing solution is drawn up the plate by capillary action C. Remove TLC plate when solvent reaches top line Developing solution (mobile phase) } {keep capped} NOTE: During this ~20 min. developing stage, compounds in the original spots are being pulled through the silica gel.

18. 3. Visualization of TLC Results A. Allow solvent to evaporate from surface of TLC plate. C. Mark spots with a pencil while viewing under UV. UV B. View results under UV light. look for grayish spots on the fluorescent green background

19. 4. Interpretation of TLC Results A. Determine retention factors (R f ) for each spot detected. B. Use R f ’s of reference spots to identify the other components. distance spot has moved distance solvent has moved _______________________ R f = = XYXY Y - - - - - - - - - - - - - - - - - - - - - - - X3X3 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - X1X1 X2X2 How do you interpret any other spots? 4 1 3 2 ? ?

20. Applications 1. Separation of carbohydrates: Mobile phase: acetonitrile : water (85:15) Detection: sulfuric acid : methanol (1:3) heat for 10 min at 110 C to see brown spots

21. Separation of Total Lipid into different Classes Mobile Phase: hexane: diethyl ether: formic acid (80:20:2) Cholesteryl esters TAG Free fatty acids Cholesterol 1,3-DAG 1,2-DAG Monoacyl glycerols Phospholipids

22. Separation of Triacylglycerols Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1) Tristearin 2-oleodistearin 1-stereodiolein Triolein Trolinolein With HUFA

24. Paper Chromatography  Purpose Use the technique of paper chromatography to separate a homogeneous mixture into its individual components  Uses Separation Identification  Chromatography paper Stationary phase  Solvent Mobile phase

25. Paper Chromatography

26. Cont…  Paper chromatography is a variant of partition chromatography procedure in which the cellulose support is in the form of a sheet or paper  Cellulose contain a large amount of bound water even when extensively dried  Partitioning occurs between the bound water and the developing solvent

27. Cont…  In paper chromatography the mixture to be separated is spotted onto the paper and dried  Then the solvent flows along the sheet either by gravity ( descending chromatography ) or capillary attraction (ascending chromatography )

28.  Place 25 mL of solvent in a 600 mL beaker. Cover the beaker and set it aside. 25 mL 1 cm 2 mm  Obtain a piece of chromatography paper and draw a line 1 cm from the bottom with a pencil.  Place a small spot of each indicator on the line. Procedure

29.  Spot and label each of the four indicators and one of the unknowns.  The spots should be about 2 cm apart. 2 cm  When the spots have dried, re-spot each one. Cont…

30.  When the spots have dried, form the paper into a cylinder with the spots facing out. Staple the edges together being careful to keep them straight and not allowing them to touch.  Place the cylinder into the 600 mL beaker and replace the cover. Be sure the cylinder is not touching the sides of the beaker. Cont…

31.  Let the chromatogram develop until the solvent is 2 cm from the top of the paper.  Remove the chromatogram from the beaker and immediately mark the solvent front with a pencil.  Allow the chromatogram to dry before going to the next step. Cont…

32.  Take the chromatogram to the hood and lightly mist it with water. Place it in the ammonia chamber.  Remove the cylinder from the ammonia chamber and unroll it. Immediately circle the colored regions with a pencil. Cont…

33.  Determine the R F values for each colored spot in the knowns and the unknown. a b c d R F(a) = a d  Use your computed R F values to identify the components of your unknown.

34. Paper Chromatography-Applications  Separation of amino acids Mobile phase: butanol : acetic acid: water(4:1:1) Detection: spray with ninhydrin reagent  Separation of carbohydrates: Mobile phase: ethylacetate : pyridine water(10:4:3) Detection: 1. silver nitrate (1 ml in 200 ml of acetone) 2. 40% NaOH in methanol gives brown spots