Chromatography is technique for separation, purification and testing of compounds.

This slide include details about chromatography which is a technique for separation and purification of compounds.

1. Chromatography Shiza A rshad

2. What is Chromatography? Chromatography is the technique for the separation, purification, and testing of compounds The term “chromatography” is derived from Greek, Chroma meaning, “color,” and graphein meaning “to write.” However, modern chromatography is used to separate a wide range of compounds, not just colored ones

3. Principle of Chromatography The principle behind chromatography is based on the differential separating of compounds between a mobile phase (which moves) and a stationary phase (which remains fixed) As the mobile phase carries the mixture through the stationary phase, different components of the mixture move at different rates depending on their interactions with the stationary phase Components that interact strongly with the stationary phase move more slowly, while those that interact weakly move faster, leading to the separation of the mixture

4. Example If we had two reactants ‘A’ and ‘ B’ We let them react with each other, under certain reaction conditions, to form a product ‘ C’ After the reaction was complete, we ended up with a reaction mixture that contained unreacted A, unreacted B and my desired product C Now the task was to separate out A, B and C to isolate and analyze pure product C

5. Term Definition Mobile phase or carrier solvent moving through the column Stationary phase or adsorbent substance that stays fixed inside the column Eluent fluid entering the column Eluate fluid exiting the column (that is collected in flasks) Elution the process of washing out a compound through a column using a suitable solvent Analyte mixture whose individual components have to be separated and analyzed

6. Working / Procedure: The basic process of chromatography involves the following steps : Preparation of the Stationary Phase: This phase can be a solid or a liquid. It is either packed into a column, coated on a plate, or used as a film Introduction of the Sample: The mixture to be separated (sample) is introduced into the system, typically in a liquid form Flow of the Mobile Phase: The mobile phase, which can be a liquid or a gas, is passed through the stationary phase. It carries the components of the mixture with it Differential Migration: As the mobile phase moves through the stationary phase, different components of the mixture migrate at different rates based on their attractions for the stationary phase Detection and Analysis: The separated components are detected and often quantified using various detectors. In some forms of chromatography, the separated components are collected for further analysis

7. Types: 1. Paper Chromatography: An early form of chromatography where the stationary phase is a piece of paper. The mobile phase is usually a liquid solvent that moves through the paper by capillary action, carrying the sample with it 2. Thin-Layer Chromatography (TLC): Similar to paper chromatography, but the stationary phase is a thin layer of a solid (like silica) coated on a glass or plastic plate.

8. Types: 3. Column Chromatography: A column is packed with a solid stationary phase, and the mobile phase (liquid or gas) passes through it, carrying the sample. The different components are collected as they elute from the column 4. Gas Chromatography (GC): The mobile phase is a gas, and the stationary phase is often a liquid film on a solid support. It’s commonly used for volatile compounds

9. Column Chromatography

10. Gas Chromatography

11. Types: 5. High-Performance Liquid Chromatography (HPLC): A highly refined form of liquid chromatography where the mobile phase is a liquid, and a high-pressure pump is used to push it through a tightly packed column. This method offers high resolution and fast separations 6. Ion Exchange Chromatography: Separates ions and polar molecules based on their affinity to the ion exchanger, which is a charged stationary phase 7. Affinity Chromatography: Uses specific interactions between a molecule (like an enzyme and its substrate) and a ligand attached to the stationary phase to purify specific compounds

12. Uses of Chromatography Purification of compounds: Isolating pure substances from mixtures Quantification : Measuring the concentration of components within a mixture Identification : Determining the identity of compounds within a mixture Analysis of complex mixtures: Decomposing complex mixtures into their individual components Quality control: Ensuring the purity of products in industries like pharmaceuticals, food and beverage, and chemicals

13. Chromatography in the Field of Nutrition Analyzing Food Components: It is used to identify and quantify nutrients, vitamins, and minerals in food products. For example, HPLC can be used to determine the concentration of vitamin C in fruits and vegetables Detecting Contaminants: Chromatography can detect harmful substances like pesticides, heavy metals, or food additives in food products. Gas chromatography (GC) is often used for this purpose. Studying Metabolites: In nutritional biochemistry, chromatography helps in studying metabolites (e.g., glucose, amino acids) in biological samples like blood or urine, providing insights into a person’s nutritional status and metabolic health Quality Control of Nutritional Supplements: Ensuring that supplements contain the correct amounts of active ingredients and are free from contaminants