What you'll learn
Chromatography and electrophoresis are separation techniques that allow chemists to identify and analyse the components of mixtures — from the dyes in food colourings and inks to the amino acids in proteins, the pesticides in drinking water, and the DNA profiles used in forensic science. This guide covers the general principles of chromatography, including the stationary and mobile phases, partition and adsorption, paper and thin-layer chromatography (TLC) and calculating R_f values, column chromatography, gas–liquid chromatography (GLC) and high-performance liquid chromatography (HPLC), including retention time and peak area, and electrophoresis for separating charged species such as amino acids, proteins and DNA. Applications in Caribbean contexts include testing food safety and monitoring agricultural chemicals in water.
CAPE questions ask you to explain how separation occurs, calculate R_f values, interpret chromatograms and electropherograms, and choose an appropriate technique for a given analysis.
Key terms and definitions
Chromatography — A technique for separating the components of a mixture based on their different distribution between a stationary phase and a mobile phase.
Stationary phase — The phase that does not move (paper, silica gel, alumina, or a liquid coating on a solid).
Mobile phase — The phase that moves through the stationary phase (a solvent or a carrier gas).
Partition — The distribution of a solute between two immiscible phases, according to its relative solubility in each.
Adsorption — The attraction of molecules to the surface of a solid.
R_f value — Distance moved by a component ÷ distance moved by the solvent front.
Retention time — The time taken for a component to pass through a chromatography column to the detector.
Locating agent — A substance used to make colourless spots visible (e.g. ninhydrin for amino acids).
Electrophoresis — The separation of charged particles by their movement in an electric field.
Isoelectric point — The pH at which an amino acid or protein has no overall charge.
Core concepts
How chromatography separates
Components of a mixture are carried by the mobile phase over or through the stationary phase. Each component distributes itself between the two phases to a different extent:
- a component that is more soluble in the mobile phase (or less strongly adsorbed) moves faster and further;
- a component that is more strongly attracted to the stationary phase moves more slowly.
The separation mechanism may be mainly partition (paper chromatography, GLC) or mainly adsorption (TLC, column chromatography with silica or alumina).
Paper chromatography
The stationary phase is water held in the cellulose fibres of the paper; the mobile phase is a solvent. Separation is mainly by partition.
Method:
- Draw a pencil baseline (ink would itself separate) about 1–2 cm from the bottom.
- Spot the samples on the baseline; allow to dry.
- Stand the paper in solvent with the baseline above the solvent level (so spots do not dissolve into the solvent).
- Cover the container to keep the atmosphere saturated with solvent vapour.
- Remove when the solvent has nearly reached the top; mark the solvent front.
- Dry and, if necessary, develop with a locating agent (ninhydrin for amino acids turns them purple; UV light for some compounds).
Two-way chromatography: if spots overlap, the paper is turned 90° and run again in a different solvent, separating components with similar R_f values in the first solvent.
R_f values
R_f = distance moved by the spot ÷ distance moved by the solvent front
R_f is always between 0 and 1. Under the same conditions (same solvent, stationary phase, temperature), each substance has a characteristic R_f, so unknowns can be identified by comparison with known standards run on the same plate.
Thin-layer chromatography (TLC)
The stationary phase is a thin layer of silica gel or alumina on a glass, metal or plastic plate; separation is mainly by adsorption. TLC is faster than paper chromatography, gives sharper separations, and needs smaller samples. It is widely used to monitor the progress of organic reactions and to check purity.
Column chromatography
A glass column is packed with silica or alumina; the mixture is added at the top and solvent (the eluent) flows down. Components separate into bands and are collected as separate fractions at the bottom. This is used to purify larger quantities of a compound.
Gas–liquid chromatography (GLC)
Stationary phase: a high-boiling liquid coated on a solid support inside a long, coiled column in an oven. Mobile phase: an unreactive carrier gas such as nitrogen or helium.
Volatile samples are injected and vaporised; components separate according to their boiling points and their solubility in the liquid stationary phase. A detector records each component as it emerges.
- Retention time identifies a component (compared with known standards under identical conditions).
- Peak area is proportional to the amount of each component, so GLC is used for quantitative analysis.
Uses: testing blood alcohol, detecting drugs in athletes, measuring pesticide residues in food, and checking the purity and composition of rum, fragrances and fuels. GLC is often combined with mass spectrometry (GC–MS) to identify each separated component.
HPLC
High-performance liquid chromatography forces a liquid mobile phase at high pressure through a column packed with very small particles. It separates non-volatile or heat-sensitive compounds such as drugs, vitamins and food additives, with retention times and peak areas used as in GLC.
Electrophoresis
In electrophoresis, a mixture is placed on a gel or paper soaked in a buffer solution, and a potential difference is applied. Charged particles migrate:
- positive ions move towards the cathode (negative electrode);
- negative ions move towards the anode (positive electrode);
- species with no overall charge do not move.
The rate of movement depends on:
- the charge (greater charge → faster);
- the size and mass (smaller → faster);
- the pH of the buffer, which determines the charge on amino acids and proteins;
- the applied voltage.
Amino acids: at a pH below its isoelectric point, an amino acid is protonated and positive (moves to the cathode); above its isoelectric point, it is negative (moves to the anode); at its isoelectric point, it does not move.
DNA profiling: DNA is cut into fragments by restriction enzymes; the fragments, which are negatively charged because of their phosphate groups, are separated by gel electrophoresis according to size, with smaller fragments travelling further. The pattern of bands is used in forensic science, paternity testing and identifying disease-causing mutations.
Worked examples
Example 1: Calculating R_f
A spot moved 3.6 cm while the solvent front moved 8.0 cm. Calculate R_f.
R_f = 3.6 ÷ 8.0 = 0.45.
Example 2: Identifying a dye
A food colouring gives three spots with R_f values 0.20, 0.45 and 0.78. A permitted dye has R_f 0.45 and a banned dye has R_f 0.78 under the same conditions. What can be concluded?
The food colouring contains the permitted dye and also appears to contain the banned dye (R_f 0.78), as well as an unidentified component. Confirmation would require further tests, such as running the samples alongside standards in a different solvent.
Example 3: Interpreting GLC
A GLC trace of a fuel shows peaks at retention times of 2.1 min and 4.5 min with areas in the ratio 3 : 1. What does this show?
The fuel contains two components, identifiable by comparing retention times with standards. The peak areas show they are present in an approximate ratio of 3 : 1 by amount (assuming similar detector responses).
Example 4: Electrophoresis of amino acids
An amino acid with an isoelectric point of 6.0 is placed in a buffer at pH 9.0. In which direction will it move?
At pH 9.0 (above its isoelectric point), the amino acid loses a proton and is negatively charged, so it moves towards the anode (positive electrode).
Example 5: Two-way chromatography
Two amino acids give a single spot with R_f 0.38 in solvent A. Explain how they could be separated and identified.
Run the chromatogram in solvent A, then dry the paper, turn it through 90° and run it in a second solvent B. Because the two amino acids have different R_f values in solvent B, the single spot separates into two. After spraying with ninhydrin, each spot's R_f values in both solvents are compared with those of known amino acids run under the same conditions.
Example 6: Choosing a technique
Suggest the most suitable technique for each task and justify your choice: (a) checking the purity of a newly synthesised organic solid, (b) measuring the ethanol content of rum, (c) detecting a banned dye in a food colouring, (d) comparing DNA samples from a crime scene.
(a) TLC — quick, needs very little sample, and a single spot suggests purity. (b) GLC — ethanol and water are volatile; peak areas give quantitative composition. (c) Paper chromatography or TLC — dyes are coloured and can be compared with standards by R_f. (d) Gel electrophoresis — DNA fragments are charged and separate by size.
Example 7: Quantitative GLC
A GLC trace of a fragrance mixture shows three peaks with areas of 60, 30 and 10 units. Assuming the detector responds equally to each component, calculate the percentage composition.
Total area = 100 units, so the components make up 60%, 30% and 10% of the mixture. In practice, detector response differs between compounds, so calibration with standards of known concentration is needed for accurate results.
Example 8: Factors affecting R_f
Explain why R_f values must be compared under identical conditions.
R_f depends on the relative affinity of a substance for the stationary and mobile phases. Changing the solvent, the type of paper or plate, the temperature or the saturation of the tank with solvent vapour changes how far substances travel, so R_f values measured under different conditions cannot be compared reliably.
Techniques compared
| Technique | Stationary phase | Mobile phase | Main separation basis | Typical use |
|---|---|---|---|---|
| Paper | water in cellulose | liquid solvent | partition | dyes, amino acids |
| TLC | silica or alumina | liquid solvent | adsorption | purity checks, reaction monitoring |
| Column | silica or alumina | liquid solvent | adsorption | purifying larger samples |
| GLC | liquid on solid support | inert gas | partition / boiling point | volatile mixtures, quantitative analysis |
| HPLC | fine solid particles | liquid under pressure | partition / adsorption | drugs, vitamins, non-volatile compounds |
| Electrophoresis | gel or paper with buffer | (no mobile phase; electric field) | charge and size | amino acids, proteins, DNA |
Common mistakes and how to avoid them
- Drawing the baseline in ink, or placing it below the solvent level.
- Measuring R_f to the top of the spot instead of its centre.
- Forgetting to mark the solvent front immediately.
- Comparing R_f values obtained under different conditions.
- Confusing retention time (identity) with peak area (amount) in GLC.
- Getting electrophoresis directions wrong — negative species move to the positive anode.
How this links to your Internal Assessment
Chromatography is ideal for SBAs: separating the dyes in local sweets, drinks or markers; identifying amino acids from a hydrolysed protein using ninhydrin; comparing the pigments in leaves from different plants (such as hibiscus, mango and sorrel); or investigating how the choice of solvent affects R_f values. Strong reports describe the method precisely, measure R_f values to the centre of spots, compare with standards, discuss the effect of conditions, and evaluate limitations such as spot spreading and overlapping components.
Exam technique for Chromatography and electrophoresis
- Explain separation in terms of relative affinity for the stationary and mobile phases.
- Show R_f calculations with both distances clearly stated.
- For GLC, mention retention time, peak area and comparison with standards under identical conditions.
- For electrophoresis, link pH, charge and direction of movement, and mention size.
Quick revision summary
- Chromatography separates by distribution between stationary and mobile phases (partition or adsorption).
- Paper: water in cellulose (partition); pencil baseline above solvent; locating agents such as ninhydrin.
- R_f = spot distance ÷ solvent front distance; compare with standards under the same conditions.
- TLC (silica/alumina, adsorption) is faster and sharper; column chromatography purifies larger amounts.
- GLC: carrier gas, liquid stationary phase; retention time identifies, peak area quantifies; GC–MS for identification.
- HPLC for non-volatile compounds at high pressure.
- Electrophoresis separates charged species by charge, size and pH; used for amino acids, proteins and DNA profiling.