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)

CHROMATOGRAPHY

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.

Chromatography History

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

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

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

TLC & HPTLC

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

HPLC (High Performance Liquid Chromatography)

Instrumentation

Isocratic System Binary Gradient system System of HPLC Isocratic System Binary Gradient system

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.

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

Separation Mechanisms

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

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