1. Satish Pradhan Dnyanasadhana College, Thane (W)
T.Y.BSc SEMESTER V BOTANY PAPER IV UNIT III CHROMATOGRAPHY-COLUMN – ION EXCHANGE, HPLC
By Mr. Swapnil Kadam
Department of Botany
Satish Pradhan Dnyanasadhana College, Thane (W)

2. CHROMATOGRAPHY

3. Concept of Chromatography
Chromatography is an analytical method in which compounds are physically separated prior to measurement.
The main purpose of chromatography is to separate and quantify the target sample in the matrix.

5. Chromatography History

6. Types of Chromatography
Adsorption Chromatography :-
Mobile Phase :- Liquid
Stationary Phase :- Solid (Adsorbing Surface)
Partition Chromatography :-
Stationary Phase :- Liquid (Film as support)

7. Important Basic Terms
Polarity :- 1. Strong Dipole 2. More Hydrogen Bonds
Solubility:- Tendency to Dissolve
Affinity :- Natural Attraction toward something (Column)
Volatility :- Tendency of Molecules to entre the vapour state or evaporate

8. Sophisticated Analytical Instruments For Chromatography
High Performance Thin Layer Chromatography(HPTLC)
High Performance Liquid Chromatography (HPLC)
Chromatography
Gas Chromatography (GC)
Super Critical Fluid Chromatography

9. TLC & HPTLC

12. What is Column Chromatography ?
Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds.

17. HPLC (High Performance Liquid Chromatography)

19. Instrumentation

20. Isocratic System Binary Gradient system
System of HPLC
Isocratic System
Binary Gradient system

22. Some Important Terms
Chromatogram: A plot of detector signal output versus time or elution volume.
Peak: The visual representation on the chromatogram based on the detector's electrical response due to the presence of a sample component inside the flow cell.
Retention time: The time taken by the analyte peak to reach the detector after sample injection.
Qualitation: An analysis process which is designed to identify the components of a substance or mixture.
Quantitation: An analysis process which is designed to determine the amounts or proportion of the components of a substance.

23. Why Use HPLC ? High Resolution High Sensitivity (ppm-ppb)
Good repeatability
Small sample size
Moderate analysis condition
No need to vaporize the sample like GC
Easy to fractionate the sample and purify
Non destructive

24. Separation Mechanisms

27. Stationary Phase :- Polar Mobile Phase :- Non-Polar
Normal Phase Mode
Stationary Phase :- Polar
Mobile Phase :- Non-Polar
Reversed Phase Mode
Stationary Phase :- Non-Polar
Mobile Phase :- Polar

37. Thank You!