You are viewing a free preview of this lesson.
Subscribe to unlock all 10 lessons in this course and every other course on LearningBro.
Chromatography is not a spectroscopic technique — it does not probe molecular structure directly. Instead, it is a separation technique that divides a mixture into its individual components. However, when combined with spectroscopic methods (particularly mass spectrometry in GC-MS and HPLC-MS), chromatography becomes an extraordinarily powerful identification tool.
All forms of chromatography work on the same basic principle. A mixture is dissolved in a mobile phase (a liquid or gas) and passed through or over a stationary phase (a solid or a liquid adsorbed onto a solid). The components of the mixture interact differently with the stationary phase:
The result is that different components travel at different rates and become separated.
The separation depends on partition (the distribution of a substance between two phases). Each component has a different partition coefficient — the ratio of its concentration in the stationary phase to its concentration in the mobile phase. The greater the difference in partition coefficients between components, the better the separation.
flowchart LR
A["Mixture injected"] --> B["Mobile phase\ncarries components"]
B --> C{"Interaction with\nstationary phase"}
C -->|"Weak affinity"| D["Moves quickly\n(elutes first)"]
C -->|"Strong affinity"| E["Moves slowly\n(elutes later)"]
D --> F["Separated\ncomponents\ndetected"]
E --> F
TLC is a simple, fast technique widely used for monitoring reactions, checking purity, and identifying compounds.
A thin layer of stationary phase (usually silica gel or alumina) is coated onto a glass, plastic, or aluminium plate. A small spot of the sample is applied near the bottom of the plate, and the plate is placed upright in a shallow pool of mobile phase (solvent). The solvent rises up the plate by capillary action, carrying the components of the mixture with it.
The distance each component travels relative to the solvent front is expressed as the Rf value:
Rf = distance travelled by component / distance travelled by solvent front
Rf values range from 0 (component stays at the origin) to 1 (component moves with the solvent front). Each compound has a characteristic Rf value under specific conditions (stationary phase, mobile phase, temperature).
Column chromatography uses the same principles as TLC but on a larger scale, allowing the physical separation and collection of individual components.
The stationary phase (silica gel or alumina) is packed into a vertical glass column. The mixture is loaded onto the top, and the mobile phase (solvent) is passed through the column under gravity or gentle pressure. Components with lower affinity for the stationary phase elute (come out) first, and fractions are collected as they emerge from the bottom of the column.
Column chromatography is the standard laboratory technique for purifying organic compounds after synthesis.
Gas chromatography separates volatile compounds using a gaseous mobile phase.
Each component has a characteristic retention time — the time between injection and detection. Under constant conditions (flow rate, temperature programme, column type), retention time is reproducible and can be used for identification by comparison with known standards.
The area under each peak in the chromatogram is proportional to the amount of that component in the mixture, enabling quantitative analysis.
GC-MS couples a gas chromatograph to a mass spectrometer. As each component elutes from the GC column, it immediately enters the ionisation chamber of the mass spectrometer.
This gives two pieces of information for each component:
GC-MS is used in forensic science (drug detection, arson investigation), environmental monitoring (pollutant analysis), food science, and clinical chemistry.
HPLC is used for compounds that are not volatile enough for GC — including large biomolecules, ionic compounds, and thermally unstable substances.
Reverse-phase HPLC is by far the most commonly used form.
| Technique | Best for | Advantages | Limitations |
|---|---|---|---|
| TLC | Quick checks, monitoring reactions | Fast, cheap, simple | Qualitative only |
| Column chromatography | Purifying products (mg to g scale) | Collects pure fractions | Slow, large solvent volumes |
| GC | Volatile/semi-volatile compounds | Excellent resolution, quantitative | Sample must be volatile |
| GC-MS | Identifying volatile unknowns | Separation + identification | Volatile compounds only |
| HPLC | Non-volatile, thermally sensitive compounds | Versatile, high resolution | Expensive, complex |
flowchart TD
A["Need to analyse\na sample?"] --> B{"Is it a mixture\nor pure compound?"}
B -->|"Pure"| C["Use spectroscopy\n(MS, IR, NMR)"]
B -->|"Mixture"| D{"Quick check\nor full separation?"}
D -->|"Quick check"| E["TLC\n(Rf values, purity)"]
D -->|"Full separation"| F{"Is the sample\nvolatile?"}
F -->|"Yes"| G{"Need to identify\ncomponents?"}
F -->|"No"| H{"Preparative or\nanalytical?"}
G -->|"Just separate"| I["GC\n(retention times)"]
G -->|"Identify too"| J["GC-MS\n(retention + mass spectra)"]
H -->|"Preparative"| K["Column chromatography\n(collect fractions)"]
H -->|"Analytical"| L["HPLC or HPLC-MS"]
A GC-MS analysis of a perfume sample produces a chromatogram with peaks at retention times of 3.2, 5.8, 8.1, and 12.4 minutes. The mass spectra of each peak are:
| Retention time (min) | M⁺ (m/z) | Key fragments | Identification |
|---|---|---|---|
| 3.2 | 78 | 78 (base peak) | Benzene (C₆H₆) |
| 5.8 | 92 | 91, 65 | Toluene (C₆H₅CH₃) — 91 = tropylium |
| 8.1 | 136 | 95, 81, 43 | Likely a terpene (monoterpene) |
| 12.4 | 204 | 161, 105, 43 | Likely a sesquiterpene or larger compound |
The GC separates the mixture components by volatility (lower boiling point elutes first), and the MS provides the mass spectrum for identification of each separated component.
Mistake 1: Assuming Rf values are constant regardless of conditions. Rf depends on the specific stationary phase, mobile phase composition, and temperature. You can only compare Rf values obtained under identical conditions.
Mistake 2: Confusing retention time (GC/HPLC) with Rf value (TLC). Retention time is measured in minutes; Rf is a dimensionless ratio between 0 and 1. Both are used for identification but by different mechanisms.
Mistake 3: Thinking chromatography alone can identify a compound. GC or HPLC retention times allow tentative identification by comparison with standards, but they cannot definitively identify an unknown. GC-MS or HPLC-MS is needed for definitive identification.
Mistake 4: Using GC for non-volatile compounds. Proteins, sugars, and many pharmaceuticals would decompose at the temperatures needed for GC. HPLC must be used for these compounds.
Chromatography separates mixtures based on differential affinity for stationary and mobile phases. TLC provides quick qualitative information with Rf values. GC separates volatile compounds and gives retention times. GC-MS combines separation with mass spectral identification — the gold standard for analysing mixtures of volatile organic compounds. HPLC extends chromatographic separation to non-volatile and thermally sensitive compounds. Understanding which technique to use and how to interpret the results is essential for the combined analytical problems you will face in exams.
Edexcel 9CH0 specification, Topic 18 — Modern Analytical Techniques I, sub-strand 18.3 covers the principles of chromatography: separation by partition between a stationary phase and a mobile phase; thin-layer chromatography (TLC) on silica or alumina with non-polar mobile phases; gas chromatography (GC) with an inert mobile gas and stationary liquid film; high-performance liquid chromatography (HPLC) with high-pressure liquid mobile phases; calculation of retention factor (Rf) for TLC; comparison with reference standards for identification; and the role of polarity and intermolecular forces in determining retention (refer to the official specification document for exact wording). Examined on Paper 2 (Core Organic and Physical Chemistry) and Paper 3 (General and Practical Principles), with synoptic links to intermolecular forces (Topic 2), purification (Topic 6), and forensic applications. Rf calculations and TLC procedures are explicitly assessable.
Question (8 marks):
A student carries out a TLC of a mixture of three amino acids (alanine, valine, leucine) on a silica plate using a non-polar solvent system (butan-1-ol/ethanoic acid/water 4:1:1). After development and visualisation with ninhydrin, the spots have moved as follows from the baseline:
(a) Calculate the Rf for each amino acid. (3)
(b) Suggest why leucine has the highest Rf. (2)
(c) Why must the solvent line be marked on the plate before the plate dries? (1)
(d) State two precautions when handling the developed plate. (2)
Solution with mark scheme:
(a) Rf = distance moved by spot / distance moved by solvent front.
(b) Leucine has the longest non-polar (isobutyl) side chain of the three amino acids. The mobile phase is relatively non-polar (mostly butan-1-ol), so leucine partitions more strongly into the mobile phase and travels further. M1. Valine has a shorter isopropyl side chain, alanine has only a methyl side chain — so the order of Rf reflects increasing alkyl-chain length and decreasing polarity. A1.
(c) Once the plate dries, the solvent front cannot be located accurately, and Rf cannot be calculated reliably. A1.
(d) Two of: wear gloves (ninhydrin is irritant; some solvents are toxic); work in a fume hood (volatile solvents); avoid touching the plate surface (fingerprint contamination introduces amino-acid residues from skin). M1 A1 (one mark per precaution, two needed).
Total: 8 marks (M2 A6).
Question (6 marks): A forensic chemist uses GC to identify the components of a vehicle paint sample.
(a) State the role of the mobile phase and the stationary phase in GC, and one suitable mobile phase. (2)
(b) The chromatogram shows three peaks at retention times 2.4, 3.7, and 6.1 minutes. Suggest how the chemist could confirm that one of these peaks corresponds to a particular pigment using a reference sample. (2)
(c) Explain why GC alone may be insufficient for confident identification, and suggest a combined technique used in forensic laboratories. (2)
Mark scheme decomposition by AO:
(a)
Subscribe to continue reading
Get full access to this lesson and all 10 lessons in this course.