1. HPLC (High Performance Liquid Chromatography)

2. LIQUID CHROMATOGRAPHY
A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

3. Schematic Diagram of Liquid Chromatography

4. High Performance Liquid Chromatography

5. 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 (uncommon)
 2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
 3. Ion Exchange Chromatography
 4. Gel Permeation Chromatography (exclusion chromatography)

6. HPLC columns (Stationary phase)
Stationary phase: silica (SiO2ㆍxH2O)
Active adsorption site: silanol (Si-O-H)
Bare silica: Adsorption chromatography
Boned stationary phase

7. Silica gel (SiO2) and alumina (Al2O3)

8. LIQUID SOLID CHROMATOGRAPHY
Normal phase LS
Reverse phase LS
d- d+
Si - O - H
30 m
Silica Gel
The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.

9. The elution process In adsorption chromatography
Elution can be described as a displacement of solute from the stationary phase by solvent.
Elution strength

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

11. Partition chromatography
Columns for boned phase chromatography.
Reverse and normal phase chromatography
Reverse and normal chromatography are distinguishable based on the relative polarities of the mobile and stationary phase.

12. Elution strength

13. SOLVENTS Polar Solvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane > Cyclohexane

15. Effect of particle size on the HPLC analysis

16. Types of Chromatography
LIQUID
MOBILE PHASE
Liquid-Solid
Liquid-Liquid
FORMAT
Chromatography
(Adsorption)
Chromatography
(Partition)
Solid
Liquid
STATIONARY
PHASE
Normal Phase
Reverse Phase
Normal Phase
Reverse Phase
Mobile Phase -
Nonpolar
Mobile Phase -
Polar
Stationary phase -
Polar
Stationary phase -
Nonpolar

17. HPLC Injector

18. Isocratic and gradient elution
Isocratic elution
Gradient elution.

21. ION-EXCHANGE CHROMATOGRAPHYSO 3 - Na +
Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).

22. Ion exchange chromatography
Packing materials
Mobile phase
Solute retention
Application
A pH is very important in Ion exchange chromatography.

23. MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS+ + SO 3 Na H N 3
COOH
Ion-exchange Resin - + SO H N 3 3 -
COO
pH4.5 + Na

24. Chromatography of Amino Acids

25. GEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.

26. Detectors
1. Ultraviolet Detector nm nm 2. Reflective Index Detector Universal Detector