# Unit 1: Chromatography-I (CH261) ## Classification and Principle of Chromatography - **Chromatography**: a separation technique in which components of a mixture distribute between a **stationary phase** and a **mobile phase**; components moving at different rates separate. - **Classification**: - By mobile phase: **gas** (GC) and **liquid** (LC) chromatography. - By mechanism: **adsorption**, **partition**, **ion exchange**, **size exclusion**, **affinity**. - By technique: **planar** (paper, TLC) and **column** (column, GC, HPLC). - **Principle of migration**: each solute has a distribution constant $K = C_s/C_m$. A solute with a high $K$ is held longer and moves slowly; with a low $K$ it moves faster. Differences in migration rates give separate bands. - **Retardation (retention) factor** $R_f$ = distance moved by solute / distance moved by solvent front. ## Adsorption Phenomenon - **Adsorption**: accumulation of molecules on the surface of a solid (adsorbent). Components of the mixture are adsorbed with different strengths by polarity and are then desorbed (eluted) by the solvent. - Adsorption affinity rises with polarity of the solute: alkanes < alkenes < ethers < esters < ketones < aldehydes < amines < alcohols < phenols < acids. - Physical adsorption is by weak van der Waals forces (reversible); chromatography uses this reversible nature. ## Nature of Adsorbents - **Properties**: high surface area, uniform particle size, chemically inert, insoluble in solvents, colourless, activated by heating, binds reversibly. - **Common adsorbents** (in increasing strength): sucrose, starch, talc, cellulose, **calcium carbonate**, **magnesium oxide**, **silica gel** (slightly acidic, most used), **alumina** (acidic, neutral or basic forms), **activated charcoal** (non-polar, adsorbs non-polar compounds), Fuller's earth. - **Activity**: depends on water content (Brockmann scale I to V); heating removes water and raises activity. ## Solvent Systems - Solvents arranged by increasing **eluting power (eluotropic series)**: petroleum ether/hexane < cyclohexane < carbon tetrachloride < toluene < benzene < chloroform < diethyl ether < ethyl acetate < acetone < propanol < ethanol < methanol < water < acetic acid. - Non-polar solutes need non-polar solvents; polar solutes need more polar solvents. **Mixed solvents** (e.g. hexane:ethyl acetate) tune the polarity. ## Thin Layer Chromatography (TLC) - **Advantages**: fast (15 to 60 min), simple, cheap, requires microgram quantity, high resolution, many detection methods, can use corrosive reagents, multiple samples on one plate. - **Preparation of plates**: a slurry of adsorbent (silica gel G with a binder such as **gypsum (calcium sulphate)**) in water is spread with a **spreader** or applicator to uniform thickness (0.25 mm) on glass plates; dried in air and **activated at 100 to 120 C** for 30 min. - **Spotting**: sample spotted by a capillary 1 to 2 cm from the bottom, on a pencil line. - **Development of chromatogram**: the plate placed vertically in a closed chamber saturated with solvent vapour, with the solvent level below the spots; solvent rises by **capillary action**; plate removed when the front nears the top; solvent front marked. **[Figure: TLC development]** A plate in a covered jar with solvent below the spots, showing separated spots and the solvent front. - **Detection of spots**: coloured compounds seen directly; **UV lamp** (254 and 366 nm) for fluorescent compounds or plates with fluorescent indicator; **iodine vapour** (brown spots); **spray reagents** such as **ninhydrin** (amino acids, purple), sulphuric acid charring, 2,4-DNP (carbonyls), ferric chloride (phenols), anisaldehyde. - **$R_f$ values** $= \frac{\text{distance moved by the spot}}{\text{distance moved by the solvent front}}$, between 0 and 1. - **Factors affecting $R_f$**: nature of adsorbent, particle size and layer thickness, solvent composition and purity, chamber saturation, temperature, amount of sample (overloading gives tailing), activation, development distance, pH. - **Applications**: purity check, monitoring reactions, identification of compounds by comparison of $R_f$ with standards, selection of a solvent for column chromatography, analysis of drugs, plant extracts, pesticides, amino acids, sugars and dyes, preparative separation. ## Paper Chromatography - A form of **partition chromatography**: the stationary phase is **water adsorbed on cellulose fibres** of the paper, and the mobile phase is an organic solvent. - **Choice of paper**: **Whatman No. 1** (general), No. 3 (thicker, preparative), uniform thickness, high purity, suitable flow rate and low ash. - **Solvent systems**: mixtures such as n-butanol : acetic acid : water (4:1:5, BAW) for amino acids, phenol : water, isopropanol : ammonia : water. - **Development techniques**: - **Ascending**: solvent rises in the paper by capillary action; simple and common. - **Descending**: solvent flows down from a trough by gravity and capillarity; faster, better for large $R_f$ differences. - **Radial (circular)**: spot at the centre of circular paper, solvent fed by a wick; concentric rings or sectors. - **Two-dimensional**: developed in one direction, dried, rotated 90 degrees and developed in a second solvent; resolves complex mixtures such as amino acids in protein hydrolysate. - **Applications**: separation of amino acids, sugars, organic acids, alkaloids, vitamins, inorganic cations, drug and dye analysis. ## Likely Exam Questions 1. Classify chromatographic methods. (2 marks) 2. What is $R_f$ value? Which factors affect it? 3. Describe the preparation and development of a TLC plate. 4. Name adsorbents and solvents used in TLC. (2 marks) 5. Explain ascending, descending and two-dimensional paper chromatography. 6. List the applications of TLC and paper chromatography.