Chromatography

Chromatography is a family of separation techniques that resolve mixtures by exploiting differential affinity of each component for a fixed stationary phase and a moving mobile phase. Substances with strong attraction to the stationary phase lag behind; those that favour the mobile phase move ahead. The same physical principle underlies every variant you will meet at A-Level: thin-layer chromatography (TLC) on a silica plate, column chromatography for preparative purification, paper chromatography for amino acids, gas chromatography (GC) for volatile organics, and high-performance liquid chromatography (HPLC) for non-volatile or thermally fragile compounds. The retention factor Rf quantifies TLC behaviour; retention time t_R and peak area quantify GC and HPLC. Chromatography is the anchor for Required Practical 12 (purity assessment of a transition-metal complex) and is the workhorse separation technique in every modern analytical, pharmaceutical, environmental, and forensic laboratory.

Spec mapping (AQA 7405): This lesson maps directly to §3.3.16 (chromatography — TLC, column, GC, HPLC; Rf values; retention times; quantitative analysis). It is the anchor lesson for Required Practical 12 (preparation of a transition-metal complex and its analysis by TLC for purity), which couples back to §3.2.5 (transition metals) and §3.3.14 (organic synthesis route purity checking). Paper chromatography is referenced in §3.3.13 (amino acids and proteins). Cross-references to mass spectrometry (§3.3.15) underpin GC-MS and LC-MS coupling. Refer to the official AQA 7405 specification for exact wording.

Assessment objectives: AO1 recall covers the definitions of stationary and mobile phase, the Rf formula, and the basic instrumentation of GC and HPLC. AO2 application is tested by calculating Rf values from given measurements, predicting retention orders from polarity arguments, and computing percentage composition from GC peak areas. AO3 evaluation — the hardest mark in this topic — requires students to choose the right technique for a given separation problem (TLC vs HPLC vs GC), to justify visualisation methods, and to interpret chromatographic evidence in the context of synthesis purity or forensic identification.

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The General Principle: Partition Between Two Phases

Every chromatographic method, regardless of scale or hardware, relies on the same molecular-level idea. A sample component continuously moves between two phases:

• The stationary phase, fixed in place — silica gel (SiO₂), alumina (Al₂O₃), C₁₈-bonded silica, or a high-boiling liquid coated on a solid support.
• The mobile phase (also called the eluent), which flows through or over the stationary phase — a liquid solvent (TLC, column, HPLC) or an inert carrier gas (GC).

Each molecule of the sample spends some fraction of its time stuck to (or dissolved in) the stationary phase and the rest of its time travelling with the mobile phase. The molecule only moves while it is in the mobile phase. Therefore the net rate at which a component traverses the system depends on the ratio of time spent in each phase — quantified by the partition coefficient K:

K = (concentration in stationary phase) / (concentration in mobile phase)

A component with a small K (low affinity for the stationary phase) moves quickly. A component with a large K is retained for longer and moves slowly. If two components have different K values, they separate. If their K values are identical — as for enantiomers on a non-chiral phase — they co-elute and the method fails.

Key Definition: Adsorption is the surface phenomenon in which molecules stick to a solid (the dominant mechanism on silica and alumina). Absorption is the bulk dissolution of molecules into a liquid (the dominant mechanism for a liquid-coated GC column). Both fall under the umbrella of partition between two phases.

The strength of interaction with silica is controlled by polarity. Silica is highly polar (free hydroxyl groups on its surface), so polar compounds (alcohols, carboxylic acids, amines) are held more strongly than non-polar compounds (alkanes, aromatic hydrocarbons). A non-polar eluent (e.g. petroleum ether) elutes only the most non-polar components; gradually increasing eluent polarity (e.g. adding ethyl acetate or methanol) pushes more polar components off the stationary phase.

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Thin-Layer Chromatography (TLC)

TLC is the workhorse of organic chemistry: fast, cheap, and informative. A small glass, plastic, or aluminium plate is precoated with a thin (~0.25 mm) layer of silica gel or alumina, often containing a UV-fluorescent indicator (typically a manganese-activated zinc silicate, denoted F₂₅₄ for fluorescence at 254 nm).

Running a TLC plate — the practical method

1. Mark the baseline. Using a soft pencil (not pen — ink contains dyes that run on the plate), draw a faint line ~1 cm from the bottom of the plate.
2. Spot the samples. Dissolve the sample in a small volume of volatile solvent (CH₂Cl₂, ethyl acetate). Apply a tiny spot to the baseline using a fine capillary tube. Allow the solvent to evaporate, then re-spot to concentrate. Keep spots small (<2 mm) — large spots smear and reduce resolution.
3. Prepare the developing tank. Pour ~5 mm depth of eluent into a sealed glass jar lined with filter paper (to saturate the atmosphere with solvent vapour). The solvent level must be below the baseline.
4. Develop the plate. Stand the plate upright in the tank with the baseline above the solvent. Replace the lid. Solvent rises by capillary action, dragging sample components up the plate at rates dictated by their partition coefficients.
5. Mark the solvent front. When the solvent front is ~1 cm from the top, remove the plate and immediately mark the front with a pencil. The front retreats as the plate dries, so this mark is essential.
6. Visualise. Coloured spots are seen directly. Colourless spots are revealed by:
   • UV lamp (254 nm): the F₂₅₄ indicator fluoresces green; UV-absorbing compounds appear as dark spots quenching the fluorescence.
   • Iodine vapour: place the dry plate in a sealed jar containing a few I₂ crystals; many organic compounds adsorb iodine and appear as brown spots.
   • Ninhydrin spray (0.2% in ethanol): heat to ~100 °C; primary amines and amino acids develop a purple (Ruhemann's purple) colour. Proline gives a distinctive yellow colour.
   • Phosphomolybdic acid or vanillin/H₂SO₄ stains: char broadly any organic compound on heating.

The retention factor Rf

The position of each spot on the developed plate is summarised by its retention factor:

Rf = (distance from baseline to centre of spot) / (distance from baseline to solvent front)

Properties of Rf:

• Always 0 ≤ Rf ≤ 1 — a spot cannot travel further than the solvent front.
• A spot stuck at the baseline has Rf = 0 (very strong affinity for the stationary phase).
• A spot at the solvent front has Rf = 1 (no interaction with the stationary phase).
• Rf is characteristic of a compound under fixed conditions: stationary phase, eluent composition, temperature, and even plate thickness all affect Rf.
• Identification is by co-elution with an authentic standard run on the same plate, NOT by quoting an Rf number from the literature.

Exam Tip: A common 2-mark question asks why Rf values from different plates are not directly comparable. Answer: Rf depends on solvent composition, plate batch, temperature, and chamber saturation. Always run a known standard on the same plate.

Worked Rf calculation

A TLC plate is developed with 4:1 hexane:ethyl acetate. After development, the solvent front is 8.4 cm above the baseline. Three spots are visible at:

Spot	Distance from baseline
A	1.8 cm
B	4.2 cm
C	6.7 cm

Calculate the Rf of each spot and predict the relative polarities.

Rf(A) = 1.8 / 8.4 = 0.21 Rf(B) = 4.2 / 8.4 = 0.50 Rf(C) = 6.7 / 8.4 = 0.80

Interpretation: A has the strongest affinity for the polar silica stationary phase — the most polar component (likely an alcohol or carboxylic acid). C has the weakest — the most non-polar component (likely a hydrocarbon or ester). B is intermediate. If a literature value for a suspected product (e.g. an ester) is Rf = 0.48 under the same conditions, then spot B is consistent with that compound, but co-spotting an authentic sample is still required for confirmation.

Applications of TLC

• Reaction monitoring. Spot the reaction mixture alongside the starting material; the appearance of a new spot (and disappearance of the SM spot) tracks progress.
• Purity assessment. A pure compound gives one spot under multiple eluent systems. Impurities appear as additional spots and lower the apparent yield.
• Identification. Co-elution with an authentic standard — the standard and unknown spots should co-spot and have identical Rf in at least two different solvent systems.
• Required Practical 12 (RP12). After preparing a transition-metal complex (e.g. [Cu(NH₃)₄(H₂O)₂]²⁺ or a cobalt amine complex), TLC against the starting material confirms that the complex is formed and free of unreacted Cu²⁺ or by-product.

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Column Chromatography

Column chromatography is preparative TLC — same principle, but at a scale that allows the user to collect the separated components as physical fractions. A vertical glass column is plugged at the bottom with cotton wool or a sintered glass frit, then packed with silica gel slurry. The dissolved mixture is loaded on top, and eluent is added continuously to maintain solvent flow.

The method

1. Pack the column. Mix silica gel with the starting eluent to a slurry; pour into the column; tap to settle and avoid air bubbles.
2. Apply the sample. Dissolve the sample in a minimum volume of the starting eluent and pipette onto the silica bed. Alternatively, dry-load by absorbing the sample onto a small amount of silica and tipping the impregnated silica onto the column.
3. Elute. Add solvent at a steady rate. Maintain a constant head of solvent above the silica — the column must never run dry, or channels form and resolution collapses.
4. Collect fractions. As the eluent drips out the bottom, collect into numbered test tubes (5–20 cm³ each).
5. Analyse fractions by TLC. Spot each fraction on a TLC plate. Combine fractions containing the same single spot; evaporate solvent under reduced pressure (rotary evaporator) to recover the pure compound.

Eluent gradients

For mixtures spanning a wide polarity range, the eluent polarity is gradually increased during the run. A typical gradient on silica might run from 5% ethyl acetate in hexane (elutes non-polar components first), through 25%, 50%, to 100% ethyl acetate, and finally to 5% methanol in ethyl acetate (elutes polar components last). This is the bedrock workflow of synthetic organic chemistry: every published natural-product synthesis includes dozens of column chromatographies.

Practical aside: Modern synthesis labs increasingly use flash chromatography — pressurised column chromatography with finer silica — and automated flash systems (Biotage, CombiFlash) which run pre-programmed gradients and collect fractions automatically based on UV absorbance. The underlying principle is unchanged.

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Paper Chromatography

Historically the first chromatographic separation of amino acids (Martin and Synge, Nobel Prize 1952), paper chromatography uses cellulose fibres as the stationary phase. Water trapped in the cellulose network acts as the true stationary phase — mechanistically this is a partition (liquid–liquid) chromatography rather than the adsorption mechanism of silica TLC. A water-soluble mobile phase (often a butanol/acetic acid/water mixture) rises by capillary action.

Paper chromatography has largely been superseded by TLC for routine use (TLC plates are more reproducible and easier to visualise), but it survives in school laboratories for cheap separation of dyes and amino acids. It also remains relevant historically: Sanger's determination of the primary sequence of insulin (1953) used paper chromatography after partial hydrolysis to identify amino acid fragments.

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Gas Chromatography (GC)

GC separates volatile organic compounds by partitioning them between a flowing inert gas and a high-boiling liquid film coated on the inside of a long (10–100 m), narrow (0.1–0.5 mm) capillary column.

Instrumentation

1. Injector. A heated port (~250 °C) where a microlitre of sample is flash-vaporised.
2. Carrier gas. Helium (most common, but increasingly hydrogen as helium prices rise) or nitrogen flows at a constant pressure or constant flow rate.
3. Column. Coiled inside a temperature-controlled oven. The inner wall is coated with a thin liquid film (the stationary phase) — typically a polysiloxane such as polydimethylsiloxane (DB-1, non-polar) or polyethylene glycol (DB-WAX, polar). The column may be operated isothermally (constant T) or with a temperature programme that ramps T to elute high-boilers in reasonable time.
4. Detector. Most commonly the flame ionisation detector (FID): the eluent is burnt in a hydrogen flame; ions formed from organic carbon are collected at an electrode and measured as a current. FID is essentially universal for organic compounds and responds linearly over five orders of magnitude. Alternatives include the thermal conductivity detector (TCD) for non-FID-active species (water, permanent gases) and the electron capture detector (ECD) for halogenated compounds (used in pesticide analysis).

The gas chromatogram

A GC trace plots detector response (y-axis) against retention time t_R (x-axis). Each component appears as a peak. Two pieces of information are extracted:

• Retention time (t_R, in minutes): the time from injection to peak maximum. Used for qualitative identification by comparison with authentic standards run on the same column under the same conditions.
• Peak area (the integrated area under the peak): proportional to the amount of that component. Used for quantitative analysis. Note: peak height alone is NOT a reliable measure of quantity, because peak width also depends on column conditions.

Key Definition: Retention time t_R is the time between sample injection and peak maximum. It depends on column temperature, carrier-gas flow rate, column length and stationary-phase polarity. Comparison between laboratories requires the same column type and method; comparison within one run requires authentic standards spiked into the sample.

Worked example: percentage composition by GC

A reaction mixture is analysed by GC. The chromatogram shows three peaks:

Peak	t_R (min)	Peak area (arb. units)
A	2.3	1500
B	4.7	3500
C	8.1	5000

Total area = 1500 + 3500 + 5000 = 10 000.

• %A = (1500/10 000) × 100 = 15.0%
• %B = (3500/10 000) × 100 = 35.0%
• %C = (5000/10 000) × 100 = 50.0%

If authentic standards of starting material (t_R = 8.1 min) and product (t_R = 4.7 min) run under the same conditions match peaks C and B respectively, then 35% of the starting material has converted to product, 50% remains unreacted, and 15% is an unidentified by-product. A more rigorous quantitative analysis would correct for the response factor of each compound (FID response is not exactly equal per mole for all compounds; oxygen-containing molecules give slightly lower signals than pure hydrocarbons per carbon atom).

GC-MS — the gold standard

In GC-MS, the column effluent feeds directly into a mass spectrometer (typically an electron-impact quadrupole or time-of-flight instrument). Each peak in the chromatogram now has an associated mass spectrum, which is searched against a library (e.g. the NIST mass spectral database, ~300 000 reference spectra) for identification. GC-MS is the standard tool for forensic toxicology (drugs of abuse, poisons), environmental analysis (pesticide residues), doping control in sport (anabolic steroids), and arson investigation (accelerant residues in fire debris).

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High-Performance Liquid Chromatography (HPLC)

HPLC extends column chromatography to non-volatile, thermally labile, and ionic compounds that cannot be analysed by GC. The mobile phase is a liquid, but driven at high pressure (typically 100–400 bar) through a short (5–25 cm), narrow (2–4 mm) stainless-steel column packed with very fine particles (1.7–5 µm diameter). Fine particles produce sharp peaks (high theoretical plate count) but offer huge resistance to flow, hence the high pressure.

Instrumentation

• High-pressure pump delivers solvent at a constant flow rate (0.5–2 cm³ min⁻¹).
• Autosampler injector introduces a microlitre of sample without depressurising the system (six-port valve).
• Column (often a C₁₈-bonded silica in reversed-phase mode — see below).
• Detector: UV-visible diode array (most common; tracks absorbance at multiple wavelengths simultaneously), fluorescence (more sensitive for fluorophores), refractive index (universal but less sensitive), or mass spectrometer (LC-MS).

Reversed-phase HPLC

In normal-phase chromatography (silica), the stationary phase is polar and the eluent is non-polar; polar compounds are retained more strongly. In reversed-phase chromatography (the dominant mode in modern HPLC), the stationary phase is non-polar (C₁₈ alkyl chains chemically bonded to silica) and the eluent is polar (water/acetonitrile or water/methanol mixtures). Non-polar compounds are retained more strongly; polar compounds elute first. Reversed-phase HPLC is used for analysing pharmaceuticals, peptides, plant metabolites, vitamins, food additives, and environmental contaminants.

LC-MS — modern bioanalytical workhorse

When the HPLC effluent feeds into a mass spectrometer (via electrospray ionisation, ESI, or atmospheric pressure chemical ionisation, APCI), the combined technique — LC-MS — is the dominant tool in pharmaceutical drug discovery (pharmacokinetic studies, metabolite identification), proteomics (peptide mapping), and clinical chemistry (therapeutic drug monitoring, neonatal screening for inborn errors of metabolism). LC-MS combines the separating power of HPLC with the mass-resolution and structural information of MS.

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Comparison of Chromatographic Methods

Feature	TLC	Column	GC	HPLC
Mobile phase	Liquid	Liquid (gravity)	Inert gas	Liquid (high pressure)
Stationary phase	Solid (silica/alumina on plate)	Solid in column	Liquid film on column wall	Solid (fine particles) or bonded silica
Scale	Analytical (µg)	Preparative (mg–g)	Analytical (ng)	Analytical or semi-prep
Speed	<30 min	Hours	Minutes	Minutes
Quantitative?	Semi (spot intensity)	Yes (fraction mass)	Yes (peak area)	Yes (peak area)
Sample requirement	Soluble	Soluble	Volatile, thermally stable	Soluble (in eluent)
Identification	Co-spotting with standard	Spectroscopy of fractions	Retention time + MS	Retention time + UV/MS
Typical use	Reaction monitoring (RP12)	Synthesis purification	Volatile organics, forensics	Pharmaceuticals, biomolecules

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Required Practical 12: TLC for Purity in Inorganic Synthesis

RP12 is the chromatography anchor in AQA 7405. Students prepare a transition-metal complex (commonly a cobalt(III) ammine or a copper(II) ammine), then use TLC — or paper chromatography — to assess purity by comparing against starting materials and known by-products.

A typical RP12 workflow:

1. Synthesise [Co(NH₃)₆]Cl₃ (or another assigned complex) by an established literature route.
2. Spot the product, the starting Co²⁺ or Co³⁺ salt, and a known by-product side-by-side on a TLC plate.
3. Develop the plate using an aqueous/organic eluent (e.g. 50:50 propan-2-ol/aqueous NH₃). Note that for charged inorganic species the mobile phase typically requires an electrolyte modifier.
4. Visualise: coloured cobalt complexes are seen directly (yellow [Co(NH₃)₆]³⁺, purple [Co(NH₃)₅Cl]²⁺, etc.).
5. Calculate Rf for each spot; compare unknown product to authentic samples.