Chemistry · Analytical and physical chemistry · Year 2

Extraction and chromatography

How is oil extracted from cottonseed, and how does a mixture separate into spots?

The situation

In oil and fat plants, oil is first pressed from cottonseed, and the rest is extracted with hexane: the oil passes into the organic solvent, and the solvent is distilled off and reused. In the quality-control laboratory of such a plant in Tashkent, the same phenomenon — the distribution of a substance between two phases — works on a small scale: in a separating funnel iodine passes from water into CCl₄, on a thin-layer plate amino acids and dyes separate into spots, in a column pigments split into coloured rings, and a gas chromatograph tells the composition of petrol in minutes. How do we separate a mixture that looks uniform into pure parts, and how do we measure the amount of each?

Topic

Separation methods: extraction and chromatography. Tashkent oil and fat plant: extracting oil from cottonseed with hexane; in the laboratory, spots of amino acids and dyes on a thin-layer plate,…

Key ideas

Partition coefficient
Rf and adsorbent activity
Resolution

Formulas

K = \frac{c_{org}}{c_{water}} = const

Nernst's distribution law: at equilibrium a substance distributes itself between two immiscible liquids in a constant ratio (if it is in the same molecular state in both).

q_{n} = {(\frac{V_{water}}{V_{water} + K V_{org}})}^{n}

The fraction left in the water after $n$ extractions, each with $V_{org}$ mL of solvent.

R_{f} = \frac{l_{spot}}{l_{front}} = \frac{1}{1 + k}

Thin-layer and paper chromatography: the ratio of the distance travelled by a spot to the distance travelled by the solvent front; $k$ is the retention factor.

k = \frac{t_{R} - t_{M}}{t_{M}}, N = 16 {(\frac{t_{R}}{w})}^{2}, H = \frac{L}{N}

Retention factor, number of theoretical plates (w is the width of the peak at its base) and plate height.

R_{s} = \frac{2 (t_{R 2} - t_{R 1})}{w_{1} + w_{2}} = \frac{\sqrt{N}}{4} \cdot \frac{α - 1}{α} \cdot \frac{k}{1 + k}

Resolution (Purnell equation): $R_{s}$ ≥ 1.5 — the peaks are fully separated; $α = k_{2} / k_{1}$ — selectivity.

Theory

Extraction: the distribution law

A substance distributes itself between two immiscible liquids, and at equilibrium the ratio of concentrations is constant: $K = c_{org} / c_{water}$ (Nernst's law). For iodine in the CCl₄–water system at 25 °C, $K$ ≈ 85: the pale yellow-brown iodine in the water passes into the lower layer and colours it violet. For the same total volume of solvent, extracting in several portions is more effective: each time the fraction $V_{water} / (V_{water} + K V_{org})$ is left in the water, and this fraction is raised to the power $n$ . For example, extracting from 100 mL of water once with 20 mL of CCl₄ removes 94 %, while two portions of 10 mL remove 98.9 %. If the substance associates in one phase (benzoic acid forms a dimer in benzene) or dissociates, the simple ratio stops being constant: for dimerisation $c_{org} / c_{water}^{2}$ ≈ const — this is how the molecular state of a substance is determined. Extraction from a solid: in a factory, the oil remaining in pressed cottonseed cake is washed out with hexane; in the laboratory the oil content is determined in a Soxhlet apparatus — the solvent evaporates, condenses in the condenser, washes the seed in the thimble and returns to the flask through the siphon; the cycle repeats. Then the solvent is distilled off and the oil is weighed. Distillation, Raoult's law, cryoscopy, ebullioscopy, osmosis, the Gibbs–Konovalov and Vrevsky laws and azeotropes are in the “Raoult's law, distillation and osmosis” experiment.

Chromatography: phases and classification

In chromatography a mixture moves along a stationary phase (an adsorbent, a liquid film, an ion exchanger) in a stream of a mobile phase (gas or liquid). A substance that is held more strongly by the stationary phase moves more slowly: its speed is $u / (1 + k)$ , where $k$ is the ratio of the amounts of the substance in the stationary and mobile phases. By the state of the phases: gas (gas–liquid, gas–solid) and liquid chromatography. By the separation mechanism: adsorption (absorption on a surface), partition (between two liquids), ion exchange, size-exclusion (gel filtration — by molecular size) and affinity (specific binding). By technique: column, thin-layer and paper chromatography. Optimisation rests on the Purnell equation: separation is improved by efficiency ( $N$ ), selectivity ( $α$ — stationary phase, eluent, temperature) and retention ( $k$ ≈ 2–10).

Thin-layer and paper chromatography (laboratory)

A start line is drawn in pencil 1.5 cm from the bottom of the plate, and the samples are applied as small spots with a capillary. The plate is lowered into a chamber saturated with eluent vapour (the eluent level must be below the start line!). The solvent rises by capillary forces ( $l_{front} \propto \sqrt{t}$ ); the front is marked and $R_{f}$ is calculated. As the eluent strength rises (the eluotropic series: hexane < toluene < chloroform < ethyl acetate < acetone < ethanol < water), all $R_{f}$ values on silica gel increase. Adsorbent activity is on the Brockmann scale: from I (most active, anhydrous) to V — 0, 3, 6, 10 and 15 % of water are added to Al₂O₃; the activity is found from which of a standard mixture of azo dyes move from the start. Nitroanilines and nitrophenols: in the o-isomers the polar groups are “shielded” by an intramolecular hydrogen bond — they are held little by the adsorbent and rise highest. Amino acids are separated on paper (the water in the cellulose is the stationary phase — partition) with n-butanol–acetic acid–water (4 : 1 : 5), and when ninhydrin is sprayed on and the paper heated they give violet spots (proline — yellow). The sugars of dried fruits (raisins, dried apricots, dried fruit slices) are extracted with hot water; Fehling's reagent detects glucose and fructose (sucrose is a non-reducing sugar); if we spray the plate with alcoholic H₂SO₄ and heat it, all carbohydrates char and give brown spots (on sugars — in the “Carbohydrates: glucose and starch” experiment).

Column and ion-exchange chromatography

A column is filled with adsorbent (silica gel, Al₂O₃) or an ion exchanger, the sample is applied at the top and washed down with eluent; the solution coming out is collected in fractions. By strengthening the eluent step by step (hexane → hexane–acetone → acetone), first the weakly held and then the strongly held substances are brought out: from leaf pigments, first orange β-carotene, then the green chlorophylls and yellow xanthophylls. Ion exchangers are polymers that contain ionic groups: a cation exchanger $R - SO_{3} H$ replaces cations in solution with H⁺, and an anion exchanger $R - N (CH_{3})_{3}^{+} Cl^{-}$ replaces anions with Cl⁻; the exchange capacity is the number of mmol of ions per 1 g of dry ion exchanger. In the Kraus–Moore method, chloride complexes are separated on an anion exchanger: Ni²⁺ does not form an anionic complex even in strong HCl and comes out at once; $[CoCl_{4}]^{2 -}$ (blue) is washed out with 4 M HCl and $[FeCl_{4}]^{-}$ with 0.5 M HCl. The adsorption of ions on a surface and ion exchange are also a topic of colloid chemistry (the Gibbs adsorption equation belongs to that course). Proteins are isolated by homogenisation, extraction and salting out (ammonium sulfate), then purified by ion exchange (the charge of a protein depends on pH and the isoelectric point; it is eluted with a salt gradient), gel filtration, affinity chromatography (enzyme–inhibitor, antibody–antigen) and HPLC; purity is checked by electrophoresis and mass spectrometry (the “Spectra: determining structure” experiment).

Gas and high-performance liquid chromatography

In a gas chromatograph a carrier gas (N₂, He) flows through the column; in the thermostatted column the substances separate and reach the detector one after another. The retention time is $t_{R}$ (for qualitative analysis, compared with a standard; the Kováts index $I$ is relative to n-alkanes), and $t_{M}$ is the time of an unretained substance. The width of a peak comes from diffusion and mass transfer: the Van Deemter equation $H = A + B / u + C u$ — there is an optimal flow rate. If the temperature is lowered, $k$ and usually $α$ increase, but the analysis takes longer. Detectors: the katharometer (thermal conductivity, TCD) detects all gases; the flame-ionisation detector (FID) only organic substances, very sensitive, and does not “see” N₂, CO₂ or H₂O; the electron-capture detector — halogen compounds; the mass spectrometer — structure. In HPLC a liquid eluent is pumped (10–40 MPa) through a column of fine (3–5 µm) particles; detectors: UV, refractive index (RI), fluorescence. Quantitative analysis: the peak area is proportional to the amount of substance; normalisation (with correction factors), external or internal standard methods.

Safety and the model

Safety: CCl₄ and benzene are toxic (benzene is a carcinogen): use them only in a fume cupboard, wearing gloves; today dichloromethane, hexane and toluene are often used instead. When shaking a separating funnel, release the pressure inside through the tap from time to time and do not point the end at people. Hexane and ethers are easily ignited — the Soxhlet is heated with an electric heater, with no naked flame. Ninhydrin stains the skin violet; spray or pour H₂SO₄ and HCl in a fume cupboard, wearing goggles; do not look at the UV lamp; for the FID, hydrogen is a flammable gas, and cylinders must be secured. Model: $K$ = 85 for I₂ (25 °C, literature value); the partition and dimerisation constants of benzoic acid, the fraction extracted in the Soxhlet cycle, the $\lg k$ and $S$ parameters in TLC, and the Brockmann activity coefficients (a scale drawn up for Al₂O₃ applied nominally to a silica-gel plate), the $k$ values in the column and the Van Deemter coefficients are illustrative; the $R_{f}$ of amino acids are approximate literature values; the Kováts indices are approximate for a non-polar phase; the compositions of the petrol, natural gas and dried-fruit samples are illustrative; detector sensitivities are simplified; time is shown sped up (× before the clock).

Did you know?

Archer Martin and Richard Synge received the Nobel Prize in Chemistry in 1952 for the discovery of partition chromatography; in that same year Martin, together with A. James, announced gas–liquid chromatography.
Ninhydrin was discovered in 1910 by the chemist Siegfried Ruhemann, who worked in Cambridge: it forms a violet dye with amino acids. Forensic scientists use this reaction to reveal invisible fingerprints on paper — sweat contains amino acids.

Inside the experiment

The experiment starts from a real-life situation: you predict the result, change the controls, run the experiment and draw a conclusion. Inside are missions, theory, a quiz and a guided tour in which the camera moves and explains the key points.

More experiments in this section

Thermochemistry and Gibbs energy — Year 1
Electrochemical analysis — Year 1
Qualitative analysis and gravimetry — Year 2
Titrimetric analysis — Year 2
Optical methods of analysis — Year 2
Spectra: determining structure — Year 2
Alloys: phase diagrams — Year 2
Chemical kinetics and Arrhenius — Year 2
Catalysis and enzymes — Year 2
Colloids and adsorption — Year 2