Chromatography & Solvent Extraction (NTCH13/02) Why separations matter: from "what" to "how much" In the lab and industry, we often face mixtures. First we ask: what is present? (qualitative). Then: how much? (quantitative). Chromatography and solvent extraction are workhorse methods to separate components cleanly so we can identify and/or measure them. Chromatography can be qualitative (spot identity by R f ) or quantitative (peak areas in HPLC/GC). Solvent extraction shifts a solute between two immiscible liquids to isolate or clean up a component. Qualitative vs quantitative: chromatography can tell you both — spot identity (what) and peak area (how much). In diagnostics, a quick urine dipstick (qualitative) flags presence of glucose/protein. If positive, a blood test quantifies the exact glucose or creatinine level to guide treatment. clinical If a qualitative test is strongly positive, it means a large quantity is present. Qualitative tells presence/absence only. Intensity of a color or spot is not a reliable measure of amount without calibration. Every measurement has error. Precision and accuracy depend on method, calibration, and handling. Quantitative analysis always gives perfect, absolute values. Core idea of chromatography Chromatography separates components because they distribute differently between a stationary phase and a moving mobile phase. A component that sticks (adsorbs/partitions) more to the stationary phase moves slower; one that prefers the mobile phase moves faster. Over distance or time, they separate into zones/bands. Two big families: - Adsorption chromatography: stationary phase is a solid (silica gel, alumina). Separation by differential adsorption. - Partition chromatography: stationary phase is a liquid bound on a solid support (e.g., water on cellulose paper). Separation by differential partition. A separation technique where components distribute between a stationary phase and a mobile phase, leading to different migration speeds and separation. Chromatography The fixed phase (solid or liquid on a support) that a component can adsorb to or dissolve in; higher affinity means slower movement. Stationary phase The phase that flows (liquid or gas), carrying components along; higher affinity means faster movement. Mobile phase Why components separate: the one that loves the stationary phase lags; the one that prefers the mobile phase runs ahead. Concept diagram of chromatography principle on white background. Left: stationary phase (silica strip) labeled; bottom: mobile phase arrow upward. Two solute spots (blue, yellow) at origin; blue strongly interacts with stationary phase and lags; yellow weakly interacts and moves higher. Include labels: 'strong adsorption → slower', 'weak adsorption → faster'. Clean 2D vector style; red curved arrows for interactions; no internal text beyond labels. 2026-05-26T17:05:13.630Z gpt-image-2 Adsorption chromatography: Column chromatography In column chromatography, the stationary phase is a solid adsorbent (commonly silica gel, SiO2, or alumina, Al2O3) packed in a vertical glass column. The mobile phase (eluent; a solvent or solvent mixture) flows by gravity. A mixture loaded at the top separates into colored or invisible bands as different components travel at different speeds. Fractions are collected as the bands elute. Polarity thumb-rule on silica (polar): less polar compounds elute first with nonpolar solvents; more polar compounds elute later (they adsorb more to silica). A polarity gradient (start hexane; then add ethyl acetate, etc.) can speed and resolve separations. 2026-05-26T17:05:14.210Z Diagram of a gravity column: glass column with cotton plug, sand layer, silica gel bed, sand, sample layer. Mobile phase reservoir and collection vials. Three colored bands (yellow at top, green middle, blue lower) moving down. Arrows for flow direction. Labels for 'stationary phase (silica)', 'mobile phase (eluent)', 'fractions'. Clean vector, neutral palette. Column chromatography setup: bands separating as eluent passes through silica. gpt-image-2 Basic steps in a gravity column Pack column uniformly with silica/alumina; wet with initial eluent (no air gaps). Load sample as a narrow band (pre-adsorbed on silica or dissolved in minimal solvent). Elute with chosen solvent/gradient; maintain steady flow. Collect fractions; monitor each by TLC; pool pure fractions; remove solvent. Start nonpolar (e.g., hexane) and slowly increase polarity (add ethyl acetate). Too polar too early makes all compounds zip out together with poor separation. tip Thin-layer chromatography (TLC) TLC uses a thin layer of adsorbent (silica gel or alumina) coated on a plate (glass/plastic). A tiny spot of mixture is applied near the bottom (origin). The plate stands in a closed jar with shallow solvent. The mobile phase rises by capillarity; components separate as spots. We measure R f (retention factor) to compare compounds under identical conditions. It is a unitless ratio between 0 and 1 and is characteristic for a given stationary phase, solvent, and temperature. TLC retention factor Measure distances from the origin line along the travel direction. gpt-image-2 TLC plate: origin line, separated spots, solvent front, and R f measurement arrows. Clean TLC schematic: plate with origin line at bottom, three spots (A,B,C), solvent front near top. Rulers/arrows showing d compound and d solvent front from origin. Include a development chamber at side (with lid). Labels: 'origin', 'solvent front', 'spot A', ' R f =A distance/solvent distance'. Vector style, no clutter. 2026-05-26T17:05:14.212Z UV indicator plates: quench under 254 nm shows dark spots. Iodine vapour chamber: many organics form brown spots. Ninhydrin spray: amino acids give purple/blue spots on heating. Visualising TLC spots R f never exceeds 1; more polar analytes usually show lower R f on polar silica with the same solvent. Increasing solvent polarity generally increases all R f values. neet-alert Partition chromatography: Paper chromatography In paper chromatography, cellulose paper holds a thin film of water, acting as the stationary liquid phase. A nonpolar or moderately polar mobile phase (e.g., butanol–acetic acid–water mix) rises by capillary action. Solutes partition between the water (on paper) and the moving solvent; those preferring the moving phase travel farther. R f is computed the same way as in TLC. gpt-image-2 2026-05-26T17:05:14.527Z Paper chromatography: sample spot at origin, solvent rising by capillarity, separated colored spots. Vertical strip of paper partially dipped in solvent within a closed jar. Origin line with sample spot; after development, multiple colored spots at different heights; solvent front marked. Labels for 'stationary: water on cellulose', 'mobile: solvent', 'capillary rise'. Vector schematic. remember TLC uses a solid adsorbent (silica/alumina) — adsorption chromatography. Paper uses bound water on cellulose — partition chromatography. Advanced (NEET context): GC, HPLC, ion-exchange Gas chromatography (GC): mobile phase is an inert carrier gas (e.g., He, N2). Stationary phase is a high-boiling liquid coated on an inert solid inside a capillary column. Separation depends on volatility and interaction with the stationary phase. Used for volatile organics; coupled to mass spectrometry (GC–MS) for identification (e.g., fragrance profiling, pesticide residues). High-performance liquid chromatography (HPLC): high-pressure pump pushes liquid mobile phase through a packed column (often silica modified with C18 in reversed-phase). Highly quantitative; used in pharmaceutical quality control and sports doping tests (HPLC–MS). Ion-exchange chromatography: resins with fixed charges exchange counter-ions with solutes; separates ions by charge/affinity. Used for water softening and separating closely related ions, including lanthanides (cross-link to NTCH11). gpt-image-2 2026-05-26T17:05:14.277Z Block diagram of HPLC: two solvent bottles (A/B) → mixer → high-pressure pump → injector/loop → column (C18) in oven → UV detector → computer. Arrows for flow; labels for each module. Clean vector, white background, red arrows for flow. HPLC schematic: solvent reservoirs, high-pressure pump, injector, column, detector, and data system. Column (adsorption) Silica gel/alumina (solid) Liquid solvent (e.g., hexane→ethyl acetate) Differential adsorption/polarity Purifying organic reaction mixtures; natural product isolation TLC (adsorption) Silica/alumina on plate Liquid solvent by capillarity Differential adsorption; R f Quick monitoring of reactions; identity check Paper (partition) Water on cellulose paper (liquid film) Liquid solvent by capillarity Differential partition between water and solvent Amino acid/dye separations; teaching labs GC High-boiling liquid on inert support (inside capillary) Inert carrier gas (He/N2) Volatility and interaction with stationary phase Essential-oil profiling; pesticide residue analysis HPLC Packed particles (e.g., C18-bonded silica) Pressurised liquid solvent Partition/adsorption under pressure Pharma QC; forensic-toxicology; doping tests Ion-exchange Charged resin (cation/anion exchanger) Aqueous mobile phase with counter-ions Charge-based ion exchange affinity Lanthanide separation; water softening; protein purification Type Stationary phase Mobile phase Separation basis Typical application Common chromatography methods at a glance Method Solvent extraction (liquid–liquid extraction) Solvent extraction separates a solute between two immiscible liquids (often water and an organic solvent like diethyl ether). The solute prefers one layer based on solubility and, for acids/bases, ionisation. Shake, allow layers to separate, and drain the desired layer. Example: elemental iodine (I2; SMILES: I[I]) distributes between water and carbon tetrachloride (CCl4; SMILES: ClC(Cl)(Cl)Cl). Iodine is far more soluble in CCl4 (violet solution) than in water (brownish), so extraction into CCl4 is efficient. gpt-image-2 Separating funnel operation: add immiscible solvents, shake–vent–settle, drain layers safely. Schematic of a separating funnel showing two immiscible layers (top organic, bottom aqueous), with labels 'organic layer (less dense)' and 'aqueous layer (more dense)'. Steps: shake, invert, open stopcock to vent, then allow to separate and drain bottom layer. Include arrows and safety note icon. Vector style. 2026-05-26T17:05:15.481Z 2026-05-26T17:05:15.348Z Two test tubes side by side: left labeled 'CCl4 layer' with deep violet color; right labeled 'Water layer' with brownish color. Caption label 'I2 distribution'. Clean realistic-vector hybrid, no clutter. Iodine partitions: violet in CCl4 (organic), brown in water (aqueous). gpt-image-2 Define clearly which phase is 1 and 2 (e.g., 1 = organic, 2 = aqueous). For neutral solutes, K D is constant at a given temperature. Partition coefficient (distribution constant) Fraction of solute remaining in the aqueous phase after n extractions with fresh organic solvent (define K D = C organic / C aqueous ; V a = volume of aqueous phase; V o = volume of organic per extraction). Overall fraction extracted = 1 - q n . Multiple extractions beat one big extraction Fix K D orientation (e.g., K D = C org / C aq ) and volumes ( V a , V o ). Compute fraction remaining after one extraction: V a /( V a + K D V o ). For n identical extractions, raise to power n. Overall fraction extracted = 1 − fraction remaining. How to tackle extraction calculations Multiple small extractions are more efficient than a single large one with the same total solvent volume. This follows directly from partition equilibrium. neet-alert Iodine (diiodine; I2; SMILES: I[I]) CCl4 / Water ≫ 1 (prefers CCl4) Extract iodine from aqueous mixtures; demo of partitioning Benzoic acid (benzenecarboxylic acid; SMILES: O=C(O)c1ccccc1) Diethyl ether / Water ≫ 1 (neutral acid prefers ether); ≪ 1 when deprotonated Back-extraction with NaHCO3 to move into water as benzoate Aniline (benzenamine; SMILES: Nc1ccccc1) Diethyl ether / Water ≫ 1 (neutral base prefers ether); ≪ 1 when protonated Extract into aqueous HCl as anilinium chloride; then basify to recover System Solute (IUPAC; common) Solvent pair (organic/aqueous) Qualitative K D trend Use Typical solvent-extraction systems ( K D defined as C org / C aq ) R f = d compound / d solvent front is between 0 and 1. A spot cannot overtake the solvent front. R f can be greater than 1 if the compound races ahead on TLC. TLC is adsorption on a solid (silica/alumina). Paper chromatography is partition with water on cellulose as the stationary liquid. TLC and paper chromatography are the same technique. For the same total solvent volume, multiple smaller extractions remove more solute, as shown by q n = ( V a /( V a + K D V o )) n. One big extraction is as good as multiple small extractions. Context box: classical quantitative formulas (preservation) These classical elemental analysis formulas (Liebig, Dumas, Kjeldahl, Carius) belong to quantitative analysis. They are shown here for continuity if you cross-read NTCH13/03. They are not core to chromatography/solvent extraction but useful in the overall theme of qualitative vs quantitative testing. Qualitative lab tests often show characteristic color changes or flames to indicate presence of elements/ions. Quantitative setups (titration, Kjeldahl, analytical balance) prioritise precision for exact amounts. Key terms Chromatography Separation based on different distribution of components between stationary and mobile phases. Stationary phase Fixed phase (solid or liquid on a support) that interacts with analytes. Phase that moves through/over the stationary phase (liquid or gas). Mobile phase Stationary phase is a solid; analytes adsorb with varying strengths. Adsorption chromatography Partition chromatography Stationary phase is a liquid bound to a support; analytes partition between two liquids. Adsorption chromatography in a packed column; fractions collected as bands elute. Column chromatography Adsorption chromatography on a thin layer (silica/alumina) on a plate. TLC (thin-layer chromatography) Partition chromatography using water on cellulose as the stationary liquid. Paper chromatography R f (retention factor) Ratio of distance travelled by compound to distance of solvent front (0 to 1). GC (gas chromatography) Gas mobile phase; separation based on volatility and interaction with stationary film. HPLC High-performance liquid chromatography; pressurised liquid mobile phase through packed columns for high-resolution separations. Ion-exchange chromatography Charged resin exchanges ions with analytes to separate by charge/affinity. Transfer of a solute between two immiscible liquids based on solubility/ionisation. Solvent extraction Separating funnel Apparatus to shake, settle, and separate immiscible liquid layers safely. Ratio of equilibrium concentrations in two phases (must define orientation explicitly). Partition coefficient ( K D ) Real-world uses: HPLC in pharma QC, GC for fragrance/essential oils, pesticide-residue monitoring in food and agriculture, forensic-toxicology and sports doping control, and protein purification via columns in biotech. remember