a b Chroma Graphein Chromatography

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2 a b Chroma Graphein Chromatography

3 (Stationary Phase) (Mobile Phase)

4

5 CHROMATOGRAPHY GAS SFC LIQUID GSC GLC Column Planar NP RP IEC SEC TLC Paper Normal Phase Reverse Phase GPC GFC Thin Layer Chromato.

6

7 Normal phase LS Reverse phase LS δ δ+ 30 µ Si - O - H Silica Gel

8 -COOH -OH -NH 2 C=O -OCH 3 -Cl

9 Polar solvent Non-Polar solvent

10 GRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY OH A + B + C OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO Solvent(mobile or moving phase) Sample (A+B+C) Column Solid Particles (packing materialstationary phase) OOOOO A OOOO OOOOOOOOOO OOOOOOOOOO OOOOO B OOOO OOOOOOOOOO OOOOOOOOOO OOOOOOOOOO OOOOO C OOOO OOOOOOOOOO OOOOOOOOOO HO HO HO OH O OH Eluant (eluate)

11

12 (Normal Phase) (Reversed Phase)

13 O O O O Si O Si O Si O H O O O O Si O Si O Si O H O O O O O O O Si O Si O Si O H O O O O Si O Si O Si O H O O O (CH 2 ) 17 CH 3 (CH 2 ) 17 CH 3 (CH 2 ) 17 CH 3 : (ODS )

14 Types of Chromatography MOBILE PHASE LIQUID FORM AT L iquid-l iquid Chromatography (Partition) L iquid-solid Chromatography (Adsorption) STATIONARY PHASE Liquid Solid N ormal Phase Reverse Phase N ormal Phase Reverse Phase M obile Phase - N onpolar Stationary phase - P olar M obile Phase - P olar Stationary phase - N onpolar

15 FOUR BASIC LIQUID CHROMATOGRAPHY The 4 basic liquid chromatography modes are named according to the mechanism involved: 1. Liquid/Solid Chromatography (adsorption chromatography) A. Normal Phase LSC B. Reverse Phase LSC 2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC B. Reverse Phase LLC 3. Ion Exchange Chromatography 4. Gel Permeation Chromatography (exclusion chromatography)

16 LIQUID SOLID CHROMATOGRAPHY OH HEXANE Si - OH CH 3 - C CH 3 CH 3 OH CH 3 C-CH 3 CH 3 CH 3

17 Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation. LIQUID-LIQUID CHROMATOGRAPHY ODPN(oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC NCCH 3 CH 2 OCH 2 CH 2 CN(Normal) CH 3 (CH 2 ) 16 CH 3 (Reverse) The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase

18 SELECTING AN OPERATING MODE Sample Type Positional isomers Moderate Polarity Molecules Compounds with Similar Functionality Ionizable Species Compounds with Differing Solubility Mixture of Varying Sized Molecules LC Mode LSC or LLC LSC or LLC LSC or LLC IEC LLC GCC

19 Schematic Diagram of Liquid Chromatography

20 Detectors 1. Ultraviolet Detector nm 254 nm 2. Reflective Index Detector Universal Detector

21 High Performance Liquid Chromatography

22 High Performance Liquid Chromatography

23 Chromatogram of Organic Compounds from Fermented Cabbage

24 Chromatogram of Orange Juice Compounds

25 Retention Time Time required for the sample to travel from the injection port through the column to the detector. Response D B A C Retention Time

26 SELECTIVITY (α) Ratio of Net Retention Time of 2 components. (Equilibrium Distribution Coefficient) α = - X 2 X 0 X - 1 X 0

27 Selectivity Selectivity Response X 2 X 1 X Retention Time

28 RESOLUTION EQUATION R = V - V 2 1 1/2(W + W ) 1 2 Response V 2 V 1 W W Volumes W W 2

29 HEIGHT EQUIVALENT TO A THEORETICAL PLATE Length of a column necessary for the attainment of compound distribution equilibrium (measure the efficiency of the column). X Theoretical plates (N) = 16 ( ) Y 2 X Y

30 RESOLUTION

31

32 EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT TO A THEORETICAL PLATE 2 V 2 4 V 0 V W 1 W 2 W W 3 4 V 3 V 4 V0 = 1.02(Minutes) V1 = 4.92 V2 = 6.59 V3 = 8.17 V4 = 9.14 W1 = 1.0 (Minutes) W2 =1.0W3 = 1.0 W4 =1.0

33 GENERAL FACTORS INCREASING RESOLUTION 1. Increase column length 2. Decrease column diameter 3. Decrease flow-rate 4. Pack column uniformly 5. Use uniform stationary phase (packing material) 6. Decrease sample size 7. Select proper stationary phase 8. Select proper mobile phase 9. Use proper pressure 10. Use gradient elution

34 Distribution Coefficient (Equilibrium Distribution Definition: Concentration of component A in stationary phase Concentration of component A in mobile phase Different affinity of these 2 components to stationary phase causes the separation.

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