Analytical Techniques – Chromatography (Cambridge IGCSE/A‑Level Chemistry 9701, Topic 22)
0. Context within the Syllabus
Chromatography is one of the major analytical families studied alongside:
- Spectroscopic methods – UV‑Vis, IR, NMR, atomic absorption.
- Electro‑chemical techniques – potentiometry, voltammetry.
- Classical quantitative methods – gravimetry, titrimetry.
Examination objectives (AO1–AO3) require candidates to:
- Select the most appropriate technique for a given sample.
- Explain the underlying principles (partition, selectivity, resolution, etc.).
- Interpret qualitative data (Rf, retention time) and perform quantitative calculations (calibration curves, % recovery).
- Design, carry out and critically evaluate a chromatographic experiment.
Decision‑making flow‑chart (sample → technique)
| Sample description | Preferred technique | Rationale |
|---|
| Non‑volatile, solid mixture; only a few mg available |
Paper chromatography or TLC |
Simple, inexpensive, works with small amounts; gives Rf values for identification. |
| Non‑volatile, larger quantity, need to isolate product |
Column chromatography (gravity or flash) |
Scalable, can collect purified fractions. |
| Volatile, thermally stable compounds (e.g., hydrocarbons, alcohols) |
Gas‑liquid chromatography (GC) |
Fast, highly sensitive, provides quantitative data via peak areas. |
| Mixture of polar & non‑polar compounds, need high resolution |
High‑performance liquid chromatography (HPLC) – not covered in depth for IGCSE but relevant for A‑Level. |
Reverse‑phase column gives excellent separation of a wide polarity range. |
1. Fundamental Principles of Chromatography
- Stationary phase: solid (silica, alumina, cellulose) or a liquid film on a solid.
- Mobile phase: liquid (paper, TLC, column) or gas (GC).
- Partition coefficient (K) – equilibrium distribution of a solute between stationary and mobile phases:
$$K=\frac{C_{\text{stationary}}}{C_{\text{mobile}}}$$
- Selectivity (α) – ability of the system to distinguish two components:
$$\alpha=\frac{K_2}{K_1}\qquad(K_2>K_1)$$
- Retention factor (Rf) – planar chromatography**:
$$R_f=\frac{\text{distance travelled by component}}{\text{distance travelled by solvent front}}$$
- Retention time (tR) – column chromatography**: time taken for a component to elute from the column.
- Resolution (Rs)** – separation between two adjacent peaks:
$$R_s=\frac{2\,(t_{R2}-t_{R1})}{w_1+w_2}$$
where $w$ = peak width at base.
2. Paper Chromatography
2.1 Principle & Set‑up
- Stationary phase: cellulose paper (highly polar).
- Mobile phase: organic solvent (commonly butanol : acetone = 4 : 1).
- Separation is based on differential adsorption of components onto the polar paper versus dissolution in the mobile solvent.
2.2 Procedure
- Draw a faint pencil line ≈2 mm from the bottom edge of a strip of chromatography paper.
- Apply a ≤2 µL spot of the sample solution onto the line; allow it to dry.
- Place the strip in a sealed developing chamber containing a shallow pool of solvent (solvent level < spot).
- Seal the chamber; the solvent rises by capillary action. When the front reaches ~80 % of the strip length, remove the paper.
- Immediately mark the solvent front with a pencil.
- Visualise the spots (UV lamp, iodine vapour, or a specific staining reagent).
- Measure distances (to 0.1 cm) and calculate Rf values.
2.3 Interpretation (Qualitative Identification)
Compare the measured Rf values with a reference table obtained under identical conditions.
| Compound | Solvent system (butanol : acetone = 4 : 1) | Typical Rf |
|---|
| Glucose | – | 0.45 |
| Fructose | – | 0.62 |
| Sucrose | – | 0.78 |
Worked example (exam style):
A paper chromatogram of an unknown sugar gives Rf = 0.46. Which sugar is most likely present?
Solution: The value matches glucose (Rf ≈ 0.45). Hence the unknown is identified as glucose.
3. Thin‑Layer Chromatography (TLC)
3.1 Principle & Materials
- Stationary phase: a thin layer (≈0.2–0.3 mm) of silica gel or alumina coated on a glass, aluminium or plastic plate.
- Mobile phase: an organic solvent or mixture (e.g., ethyl acetate : hexane = 1 : 3).
- Separation occurs by differential adsorption of the analytes onto the polar adsorbent versus their solubility in the mobile solvent.
3.2 Procedure
- Draw a faint line 1 cm from the bottom edge of the plate.
- Spot ≤2 µL of the sample onto the line using a capillary tube; allow to dry.
- Place the plate in a sealed developing chamber containing a shallow pool of solvent (solvent level < spot).
- Develop until the solvent front is ~8 cm from the origin.
- Remove, dry, and visualise under UV (254 nm) or by iodine vapour, ninhydrin, etc.
- Measure distances and calculate Rf values.
3.3 Advantages over Paper Chromatography
- Higher resolution – thinner stationary layer reduces band broadening.
- Rapid development (typically 5–10 min).
- Wider choice of solvents, including non‑polar mixtures.
- Quantitative analysis possible via densitometry (spot‑intensity measurement).
3.4 Example
Separate a mixture of benzoic acid, acetophenone and phenol using silica TLC with ethyl acetate : hexane = 1 : 3. Measured Rf values: 0.21 (benzoic acid), 0.45 (acetophenone), 0.78 (phenol). The values are within the optimal 0.2–0.8 range, confirming a suitable solvent system.
4. Column Chromatography (Preparative)
4.1 Types of Columns
| Feature | Normal‑phase column | Reverse‑phase column |
|---|
| Stationary phase | Polar silica gel or alumina | Non‑polar C18‑bonded silica |
| Mobile phase | Non‑polar solvents (hexane, petroleum ether) | Polar solvents (water, methanol, acetonitrile) |
| Typical use | Separation of non‑polar to moderately polar compounds | Separation of polar to moderately non‑polar compounds; basis of HPLC |
4.2 Column Packing (wet‑packing method)
- Choose column dimensions (e.g., 2 cm × 30 cm). Approx. 30 g silica per 10 cm column length.
- Slurry the silica in the chosen solvent (e.g., hexane) to form a uniform suspension.
- Pour the slurry into the column, allowing it to settle without forming air bubbles.
- Tap gently to settle the bed; add more slurry until the column is filled to the desired height.
- Condition the packed column by passing 5–10 column volumes of the mobile phase.
4.3 Running the Column
- Apply the sample on top of the stationary phase (dry‑load for solid samples or dissolve in a minimal amount of solvent).
- Elute with the mobile phase at a steady flow (gravity or low‑pressure pump).
- Collect fractions of a convenient volume (e.g., 10 mL).
- Analyse each fraction by TLC or GC to locate the target component.
- Combine the appropriate fractions and remove solvent (rotary evaporator).
4.4 Quantitative Example – % Recovery
A 0.500 g mixture contains 0.120 g of component A. After column chromatography 0.108 g of A is isolated.
$$\%\,\text{recovery}= \frac{0.108\;\text{g}}{0.120\;\text{g}}\times100 = 90\%$$
5. Gas‑Liquid Chromatography (GC)
5.1 Principle & Main Components
- Stationary phase: a high‑boiling liquid (e.g., 5 % phenyl‑methylpolysiloxane) coated on the inner wall of a fused‑silica capillary (10–30 m long, 0.1–0.53 mm i.d.).
- Mobile phase: inert carrier gas (helium or nitrogen) at a constant flow (≈ 1 mL min⁻¹).
- Detector: flame ionisation detector (FID), thermal conductivity detector (TCD) or mass spectrometer (GC‑MS).
- Separation arises from repeated partition of vapourised analytes between the gas phase and the liquid film.
5.2 Typical Run (step‑by‑step)
- Inject ~1 µL of liquid sample into a heated injector (220–250 °C for most organics).
- The sample instantly vapourises and is carried onto the column by the carrier gas.
- Components interact with the stationary liquid film; stronger interaction → longer retention time.
- Each component elutes at a characteristic tR and produces a peak on the chromatogram.
- Peak area is proportional to the amount of component present.
5.3 Key Parameters
- Column dimensions – longer columns give higher efficiency; smaller i.d. improves resolution but increases back‑pressure.
- Temperature programming – isothermal for narrow‑boiling‑range mixtures; ramped (e.g., 40 °C → 250 °C at 10 °C min⁻¹) for complex samples.
- Resolution (Rs)** – see equation in Section 1.
5.4 Quantitative Example – Calibration Curve
- Prepare standards of a volatile compound (e.g., n‑butanol) at 0.1, 0.5, 1.0 and 2.0 mg mL⁻¹.
- Inject each under identical GC conditions; record peak areas (A).
- Plot A (y‑axis) against concentration (C, x‑axis). A straight line is obtained:
$$A = 1.23\times10^{5}\,C + 2.1\times10^{3}$$
- Inject an unknown; measured area = 6.18 × 10⁴.
$$C_{\text{unk}} = \frac{6.18\times10^{4} - 2.1\times10^{3}}{1.23\times10^{5}} = 0.49\;\text{mg mL}^{-1}$$
6. Quantitative Chromatography – General Approach
6.1 Calibration Curves (any technique)
- Prepare a series of standards covering the expected concentration range.
- Analyse each standard under identical conditions; record the analytical response (peak area, intensity, etc.).
- Plot response (y) vs. concentration (x) and obtain a linear equation $y = mx + c$.
- Measure the response of the unknown and calculate its concentration using the equation.
6.2 % Recovery (Preparative work)
$$\%\,\text{recovery}= \frac{m_{\text{recovered}}}{m_{\text{theoretical}}}\times100$$
Useful for evaluating the efficiency of a column or extraction procedure.
7. Practical Skills, Experimental Design & Safety
7.1 Designing a Chromatographic Experiment (AO3)
- Define the aim – e.g., “Separate and identify the components of a mixed food‑colour sample”.
- Choose the technique – start with TLC for rapid screening; if components are volatile, move to GC.
- Select a solvent system – test at least two ratios; target Rf (or tR) values between 0.2 and 0.8 (or spacing > 1 min in GC).
- Plan controls – run known standards alongside the unknown.
- Determine data handling – record distances to 0.1 cm, calculate Rf to two decimal places; for GC, record tR to 0.01 min and integrate peaks with software.
- Evaluate results – discuss possible sources of error (solvent purity, temperature fluctuations, column packing) and suggest improvements.
7.2 Safety Considerations
| Hazard | Precaution |
|---|
| Organic solvents (hexane, acetone, butanol) |
Use a fume hood, wear gloves and safety goggles, keep away from ignition sources. |
| High‑temperature injector & column (GC) |
Allow equipment to cool before servicing; use heat‑resistant gloves when handling liners. |
| Silica/alumina dust (column packing) |
Wear a dust mask, avoid inhalation, and clean spills immediately. |
| Pressurised systems (flash column, HPLC) |
Never seal a column completely; use pressure‑relief valves and check fittings before use. |
8. Summary Comparison of Chromatographic Techniques
| Technique |
Stationary Phase |
Mobile Phase |
Typical Sample Type |
Key Advantages |
Key Limitations |
| Paper Chromatography |
Cellulose (polar) |
Organic liquid |
Small, soluble mixtures (pigments, sugars) |
Very simple, inexpensive, visual |
Low resolution; limited to soluble, non‑volatile samples |
| Thin‑Layer Chromatography (TLC) |
Silica gel / alumina (adsorbent) |
Organic solvent or mixture |
Organic compounds, pharmaceuticals, pigments |
Fast, high resolution, quantitative (densitometry) |
Limited sample load; requires careful spotting |
| Column Chromatography |
Silica, alumina or bonded phases (normal or reverse) |
Liquid solvent (gravity or pressure) |
Mixtures needing preparative separation |
Scalable, can be automated (flash, HPLC) |
Longer run times, higher solvent consumption |
| Gas‑Liquid Chromatography (GC) |
Liquid film on fused‑silica capillary |
Inert gas (He, N₂) |
Volatile, thermally stable compounds |
High sensitivity, rapid, excellent quantification |
Unsuitable for non‑volatile or thermally labile substances |
9. Practical Tips for A‑Level Examinations
- Always mark the solvent front immediately after removal and calculate Rf to two decimal places.
- Select a solvent system that gives Rf values between 0.2 and 0.8 for all components of interest.
- When packing a column, ensure the bed is uniform and free of air bubbles – tap gently and allow the slurry to settle.
- For GC, set the injector temperature just above the boiling point of the sample; avoid excessive heating that can cause decomposition.
- In data interpretation, compare Rf or tR values with those of standards run under identical conditions.
- When constructing a calibration curve, use at least four concentration points and check that the correlation coefficient (R²) is ≥ 0.99.
- Discuss sources of error (e.g., solvent evaporation, incomplete elution, detector drift) and suggest realistic improvements.