Chromatography

Spec Mapping — OCR H432 Module 6.3.1 — Chromatography, covering the principle of separation by partition between a stationary phase and a mobile phase (the underlying physics behind all chromatographic techniques); thin-layer chromatography (TLC) practical method — silica or alumina stationary phase on a plastic/glass plate, solvent mobile phase, capillary action; calculation and interpretation of retention factor Rf = (distance moved by spot) / (distance moved by solvent front) under specified solvent and temperature; visualisation methods for colourless spots (UV lamp, iodine vapour, ninhydrin for amino acids); gas chromatography (GC) practical method — inert carrier-gas mobile phase, high-boiling-point oil stationary phase coated on the inside of a heated capillary column, microlitre injection volumes, detector at column exit; retention time (tR) as the characteristic diagnostic time from injection to peak maximum, with quantitative analysis from peak area against a calibration curve; combined GC-MS for unambiguous identification of organic compounds by retention time PLUS mass spectrum fingerprint, including library search; forensic and anti-doping applications (refer to the official OCR H432 specification document for exact wording).

Synthesising a compound is only half the job — you also need to separate it from the by-products and identify what you have made. Chromatography is the analytical technique that answers both questions, and is the analytical workhorse of every modern chemistry, biochemistry, pharma, forensic and food-safety laboratory in the world. It is the technique that catches dopers at the Olympics, identifies trace pollutants in drinking water, monitors the purity of synthetic drugs, separates the components of crude oil, and resolves the dozens of amino acids in a biological sample. At A-Level you meet three variants: thin-layer chromatography (TLC) — a quick paper-and-solvent test used to monitor reactions and check purity, gas chromatography (GC) — a high-resolution technique for separating volatile compounds in a heated coiled column, and the combined GC-MS — gas chromatography directly hyphenated to a mass spectrometer, the gold-standard tool for identifying unknowns from complex mixtures. This lesson develops the underlying physical principle (partition between a stationary phase and a mobile phase) that all three share, the practical method and Rf interpretation of TLC, the retention-time and peak-area analysis of GC, and the combined GC-MS workflow including its forensic and anti-doping applications.

Key Definition — Chromatography: an analytical technique for separating the components of a mixture by repeated partitioning between a stationary phase (which does not move) and a mobile phase (which carries the sample through or over the stationary phase). Components that interact more strongly with the stationary phase travel slower than components that prefer the mobile phase, so the components separate along the direction of flow and emerge or migrate at different times/distances.

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1. The Big Idea — Partition Between Two Phases

All chromatographic techniques work on the same underlying principle:

Key Principle: A sample is split between a stationary phase (which stays put) and a mobile phase (which moves). Different components of the sample spend different amounts of time in each phase, so they travel at different speeds, and become separated along the direction of mobile-phase flow.

The strength of interaction between each component and each phase depends on the components' polarity, size and chemical affinities. So any two compounds with different structures will travel at different speeds, and can (in principle) be separated.

Technique	Stationary phase	Mobile phase
TLC	Silica or alumina on a plate	Liquid solvent (e.g. ethanol, cyclohexane)
GC	Viscous oil on an inert solid	Gas (N₂, He)
Column chromatography	Silica in a column	Liquid solvent
HPLC (university)	Silica or bonded phase	Liquid solvent under high pressure

The strength of interaction between a component and each phase depends on the component's polarity, size, and chemical functional groups. Polar molecules (alcohols, acids, amines, sugars) bind tightly to polar stationary phases (silica) and travel slowly; non-polar molecules (alkanes, halogenoalkanes, esters) prefer the non-polar mobile phase and travel quickly. Because every compound has its own unique pattern of functional groups and shape, every compound interacts slightly differently with the two phases — and so every compound has its own characteristic speed through the system. This is the molecular basis of the separation power of chromatography.

Mihail Tsvet (1872-1919) is generally credited with founding chromatography in 1903 when he used a column of calcium carbonate and petroleum ether to separate plant pigments into coloured bands — hence the name "chromatography" (Greek chroma = colour, graphein = to write) (paraphrase). The technique was largely ignored for 30 years until Archer Martin and Richard Synge rediscovered partition chromatography in the 1940s and shared the 1952 Nobel Prize in Chemistry for it (paraphrase). Their insight — that the strength of partitioning between two phases is the molecular origin of the technique — underpins every modern variant.

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2. Thin-Layer Chromatography (TLC)

2.1 Setup

• A small rectangular plastic or glass plate is coated with a thin layer of silica gel (SiO₂) or alumina (Al₂O₃) — this is the stationary phase.
• A pencil line is drawn near the bottom (not ink — ink would dissolve and move with the solvent!).
• A spot of the sample (dissolved in a volatile solvent) is applied to the line.
• The plate is stood in a beaker containing a small volume of solvent (the mobile phase) — the solvent level must be below the pencil line or the sample washes away.
• The beaker is covered with a lid or watch glass to prevent evaporation.

2.2 What happens

The solvent rises up the plate by capillary action, carrying the sample components with it. Each component partitions between:

• Adsorption on the silica gel (stationary phase) — more polar compounds stick harder.
• Dissolution in the solvent (mobile phase) — less polar compounds dissolve more.

Less polar components travel further up the plate because they spend more time in the moving solvent. More polar components travel less far because they are held more strongly on the silica.

When the solvent nears the top of the plate, the plate is removed and the solvent front is marked with pencil. The plate is then dried, and the spots are visualised (see Section 2.4).

graph LR
  A[Apply sample on pencil line] --> B[Stand in solvent, solvent level below line]
  B --> C[Solvent rises by capillary action]
  C --> D[Components separate by polarity]
  D --> E[Mark solvent front, dry, visualise]
  E --> F[Calculate Rf values]

2.3 Rf value — the diagnostic measurement

The retention factor (Rf) is the ratio of the distance travelled by the spot to the distance travelled by the solvent front:

$R_f = \frac{\text{distance moved by spot from pencil line}}{\text{distance moved by solvent front from pencil line}}$Rf​=distance moved by solvent front from pencil linedistance moved by spot from pencil line​

• Rf is always between 0 and 1 (the spot cannot move further than the solvent front).
• Rf is characteristic of a compound under specified conditions — solvent, stationary phase, temperature. Two different solvents will give two different Rf values for the same compound.
• Rf has no units — it is a ratio of two lengths.
• Polar compounds → low Rf (held strongly on silica); non-polar compounds → high Rf (travel with solvent).

Worked example 1: A spot of compound X moves 3.6 cm up the plate; the solvent front moves 7.2 cm. The Rf is 3.6 / 7.2 = 0.50.

Worked example 2 — separation of amino acids: A student spots a mixture of glycine, alanine, and phenylalanine on a TLC plate and runs in a butan-1-ol / acetic acid / water solvent. After visualising with ninhydrin (sprayed and gently heated), three purple spots appear at heights 1.5 cm, 2.4 cm and 5.4 cm respectively. The solvent front moved 6.0 cm.

• Glycine Rf = 1.5 / 6.0 = 0.25 (most polar — has only H side chain).
• Alanine Rf = 2.4 / 6.0 = 0.40 (medium polarity — methyl side chain).
• Phenylalanine Rf = 5.4 / 6.0 = 0.90 (least polar — bulky aromatic phenyl side chain).

The order matches our expectation that the bulkier and more non-polar side chain partitions into the mobile phase. Comparison of these Rf values against published values for pure amino acids confirms the identity of each spot.

Exam Tip: Measure the distance to the centre of the spot, and always measure from the pencil line (where the sample was applied), not the bottom of the plate. OCR markschemes are strict on this.

2.4 Visualising colourless spots

Many organic compounds are colourless and invisible on a white silica plate. Common ways to visualise them:

• UV lamp (254 nm or 365 nm): Most silica plates contain a fluorescent indicator; compounds that absorb UV show up as dark spots on a glowing background.
• Iodine vapour: Many compounds adsorb iodine, giving brown spots.
• Ninhydrin spray: Turns amino acids purple.
• Chemical stains: e.g. KMnO₄ spray turns alkenes brown.

2.5 Uses of TLC in practice

• Reaction monitoring: Spot the starting material, the reaction mixture, and the pure product side by side on a plate. Watch the starting material spot disappear as the reaction proceeds, and the product spot grow. This is the routine way to decide when to stop a reflux.
• Identification: Spot an unknown and a suspected pure compound; if they give the same Rf in two different solvents, they are almost certainly the same compound. Using two solvent systems guards against the coincidence that two different compounds might happen to share an Rf in one solvent.
• Purity check: A pure compound should give a single spot. Multiple spots indicate impurity (or that the compound has decomposed).
• Forensic and medical: Rapid screening of drugs, dyes, amino acids, plant alkaloids. A skilled chromatographer can identify a sample in minutes from an Rf comparison against a library of known standards.
• Teaching: TLC is cheap, fast and visually striking, which makes it the canonical introductory chromatographic technique in school laboratories.

2.6 Why two-dimensional TLC?

For mixtures of similar compounds (e.g. 20 amino acids in a protein hydrolysate), one-dimensional TLC is not enough — too many spots overlap. The trick is to run the plate once in one solvent, rotate the plate by 90° and run it again in a different solvent. Each spot has now been characterised by two independent Rf values, and the second run separates overlapping pairs in the orthogonal direction. The result is a 2D pattern of spots, each at a unique (Rf₁, Rf₂) coordinate. This is the basis of much of biochemistry's amino-acid and peptide analysis from the 1950s onward, though modern labs now use HPLC or capillary electrophoresis instead.

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3. Gas Chromatography (GC)

3.1 Setup

• Stationary phase: A long, thin, coiled capillary column (up to 100 m, wound into a coil) coated on the inside with a high-boiling-point oil (the stationary liquid). The column sits in a temperature-controlled oven.
• Mobile phase: An inert carrier gas — usually nitrogen or helium — flowing through the column at a controlled rate.
• A tiny volume of the liquid sample (microlitres) is injected into the hot injection port, where it vaporises.
• The vapours are swept along the column by the carrier gas.

3.2 What happens

Each component of the sample partitions between:

• Dissolving in the liquid stationary phase (more soluble → stays longer).
• Moving with the gas (less soluble → moves faster).

The solubility depends on the component's boiling point and its affinity for the stationary phase oil. Typically, lower boiling-point components travel faster through the column and emerge first; higher-boiling-point components lag behind.

As each component emerges from the end of the column, it passes a detector (e.g. a flame ionisation detector, FID, or a mass spectrometer in GC-MS) which produces an electrical signal. The result is a chromatogram: a plot of detector signal against time.

3.3 Retention time

The retention time (tR) is the time between injection and the peak maximum for that component. Under fixed conditions (column, temperature, gas flow) it is characteristic of each compound — like a fingerprint.

• Shorter retention time = component emerged quickly = low affinity for stationary phase (or low bp).
• Longer retention time = component held up in column = high affinity (or high bp).

Worked example — police breath-test confirmation: Roadside police breath-testers give an instantaneous estimate of blood-alcohol concentration but are not legally definitive. A confirmatory blood sample is sent to a forensic laboratory, where the alcohols are separated by GC. Ethanol has a characteristic retention time of about 3.2 minutes on a standard GC column under typical method conditions; an internal standard (e.g. n-propanol) is added at known concentration and shows up at about 4.1 minutes. The ratio of the ethanol peak area to the n-propanol peak area gives the unknown ethanol concentration when read off a calibration curve. This is the legally defensible result used in UK courts.

3.4 Identification and quantification

Task	How GC does it
Identify a component	Compare retention time with a known standard run under identical conditions
Quantify a component	Measure the peak area — area is proportional to the amount of substance

The peak area (not peak height) is used for quantification because peaks can be sharp or broad depending on the column conditions, but the integrated area still equals the total amount of analyte detected. A calibration curve — plotting peak area against known concentration of a series of standards — lets you read off the unknown concentration from its peak area.

Worked example — ethanol calibration: A forensic chemist runs five ethanol standards through a GC and measures the peak area:

Ethanol concentration / g per 100 mL	Peak area / arbitrary units
0.00	0
0.05	240
0.10	485
0.15	730
0.20	975

A plot of area vs concentration is linear with gradient ~4850 area-units per (g per 100 mL). A blood sample gives a peak area of 380. Read off: concentration = 380 / 4850 = 0.078 g per 100 mL, which is below the 0.08 g per 100 mL legal driving limit in England and Wales but above the lower 0.05 g per 100 mL Scottish limit (refer to the official Road Traffic Act for current statutory limits). This is the kind of quantitative argument GC supports.

3.5 Limitations of GC