Paper Chromatography

How paper chromatography works

Paper chromatography exploits the difference in how strongly each component of a mixture partitions between two phases: a mobile liquid solvent and a stationary water-rich layer bound to cellulose paper. Capillary forces wick the mobile phase up through the paper. As the moving solvent passes over a deposited mixture, each component shuttles between the two phases — dissolving into the moving solvent, then reattaching to the paper, then dissolving again — and the time-averaged forward velocity depends on its preference. Components that strongly favour the mobile phase ride along with it; components that favour the stationary phase get held back. The result, after the solvent front has travelled some distance up the paper, is a column of spots stratified by partition coefficient.

The classic teaching procedure:

1. Cut a strip of chromatography paper (Whatman No. 1 is the standard, fast-flowing grade).
2. Draw a pencil line ~1.5 cm from one end — the baseline.
3. Spot the mixture in a small dot, < 5 mm wide, on the baseline. Allow to dry.
4. Pour solvent into a tank to a depth of 1 cm — below the baseline.
5. Hang the paper so the bottom edge dips into solvent and the lid seals the tank to saturate with vapour.
6. Let the solvent climb until it reaches a marked endpoint near the top of the paper (typically 80 – 90% of the strip length).
7. Remove, immediately mark the solvent front with pencil before it evaporates, dry the strip, visualise spots if colourless.
8. Measure d_spot from the baseline to the centre of each spot, and d_solvent from the baseline to the front. Compute Rf.

Rf calculation: a worked example

You run an unknown ink mixture in butan-1-ol / acetic acid / water (4:1:5 v/v) for two hours. After drying:

• Solvent front is 8.0 cm above the baseline.
• Yellow spot at 5.6 cm.
• Red spot at 2.4 cm.
• Blue spot at 6.8 cm.

Compute each Rf as d_spot / d_solvent:

Spot	d_spot (cm)	d_solvent (cm)	Rf
Yellow	5.6	8.0	0.70
Red	2.4	8.0	0.30
Blue	6.8	8.0	0.85

Compare against a reference table (or run authentic standards alongside the unknown on the same plate). The blue dye at Rf 0.85 is consistent with brilliant blue FCF; the yellow at 0.70 with tartrazine; the red at 0.30 with an azo dye that hydrogen-bonds heavily to the paper. To turn an Rf into an identification, run a known standard in the same solvent on the same paper at the same temperature. Rf values are reproducible only within roughly ±0.05 even with care — direct co-spotting beats library matching.

Choosing the solvent system

The solvent's polarity sets the entire separation. Too polar a solvent and everything moves to the front (Rf ≈ 1, no separation). Too non-polar and nothing moves (Rf ≈ 0). The art is to dial the eluent strength so the components of interest spread between Rf 0.2 and 0.8.

Solvent system	Polarity	Best for
Water	Most polar	Sugars, very polar amino acids
n-Butanol / acetic acid / water (4:1:5)	High	Amino acids — the classic Consden-Gordon-Martin system
Phenol / water (75:25)	High, hydrogen-bonding	2D amino-acid separations (orthogonal to BAW)
Methanol / water (80:20)	Medium-high	Plant pigments, anthocyanins
Ethyl acetate / methanol / water (60:30:10)	Medium	Flavonoids
Petroleum ether / acetone (90:10)	Low	Chlorophyll, carotenoids on impregnated paper

Mixed solvents give a tunable polarity scale: a stronger eluent increases Rf for everything; a more polar additive increases it more for polar components than non-polar ones. Acid (acetic acid) added to a solvent suppresses ionisation of carboxylic acids and ammonium groups so they stay protonated and chromatograph as a single neutral spot, rather than streaking across pH-dependent equilibria.

Paper vs TLC vs HPLC vs GC

Technique	Stationary phase	Mobile phase	Run time	Sample (typical)	Strength
Paper chromatography	Water on cellulose	Liquid (capillary)	30 min – 4 h	1 – 50 µg per spot	Cheapest; teaching; polar biologicals
Thin-layer chromatography (TLC)	Silica or alumina on plate	Liquid (capillary)	10 – 30 min	0.1 – 10 µg per spot	Faster; sharper; reaction monitoring
High-performance LC (HPLC)	5–10 µm silica/polymer in column	Liquid (high-pressure pump)	5 – 60 min	1 ng – 1 mg injection	Quantitative; pharma; coupled to MS
Ultra-HPLC (UHPLC)	Sub-2 µm particles	Liquid (1000+ bar)	1 – 10 min	1 ng – 1 mg injection	Speed and resolution gains over HPLC
Gas chromatography (GC)	Liquid film in capillary column	Gas (He, H₂)	5 – 60 min	1 pg – 1 µg	Volatiles; petroleum; environmental
Ion exchange / size exclusion	Charged or porous resin	Aqueous buffer	30 min – several h	µg – mg protein	Protein purification; desalting

The whole family runs the same physics — partition or adsorption between two phases — and the same retention quantity (Rf for plates, retention time for columns). What differs is the engineering: thinner stationary phases give sharper bands; high pressure gives faster flow; columns let you quantify with a spectrometer at the outlet. Paper sits at the bottom of the engineering ladder and yet, exactly because it's so simple, it's still the right tool for visualising a sugar mix in a teaching lab or fingerprinting an unknown ink at a forensic scene.

Practical considerations

• Saturate the tank. Line the inside of the tank with solvent-soaked filter paper and equilibrate for 15 minutes before introducing the chromatogram. An unsaturated tank evaporates solvent off the paper edges, making the front rise higher in the centre than at the sides — the famous "edge effect" that ruins symmetric spots.
• Spot small. A 5 mm starting spot is the upper limit; aim for 2 mm. Larger spots smear.
• Don't overload. Pile too much sample on a spot and Rf shifts up (apparent — the spot is so wide that its centre sits closer to the leading edge). Run a serial dilution if quantitation matters.
• Use a pencil, never a pen. Ink streaks in the solvent.
• Record temperature and humidity. Both shift Rf slightly. Standards run on the same paper and tank correct for daily drift.
• Mark the front before drying. The solvent edge is invisible once the paper dries; without that pencil mark you can't compute Rf.
• Sandwich your unknown between standards. Spot a standard, the unknown, then the standard again on the same baseline. This catches paper-curl effects and confirms identity by direct alignment.

Real-world applications

• Forensic ink analysis. Disputed-document examiners run paper chromatography of ink samples to compare a questioned signature with reference inks. The dye blends are subtle and Rf patterns differ between manufacturers and even between batches; a 1990s paper-chromatography fingerprint can establish the date a pen was made.
• Food dye testing. Regulatory labs separate food-grade colour additives — tartrazine, sunset yellow, allura red, indigo carmine, brilliant blue — from a sweet's coating in 30 minutes to verify the declared ingredient list.
• Plant pigment teaching. The classic spinach-extract experiment separates chlorophyll a (blue-green), chlorophyll b (yellow-green), xanthophylls (yellow) and β-carotene (orange) on a single strip — visible without staining, and a foundational demo of partition theory.
• Amino acid identification (historical). Frederick Sanger's 1950s work on insulin sequence used 2D paper chromatography of dansyl-amino-acid mixtures. Two solvents in perpendicular directions resolved 20 amino acids on a single sheet — work that won him a 1958 Nobel.
• Sugar separations. The classic Consden-Gordon-Martin butanol/acetic-acid/water system still separates glucose, fructose, sucrose, lactose, maltose for dairy or honey QC, with detection by aniline-diphenylamine spray.
• Field testing. Paper chromatography needs no electricity, no compressed gas, no detector. Aid agencies and field hospitals carry paper kits for confirming pharmaceutical authenticity in low-resource settings — a counterfeit antimalarial pill spots and runs in 20 minutes.

Common mistakes and pitfalls

• Letting the solvent run off the top. Once it does, you can't measure d_solvent; the entire run is invalid. Stop the chromatogram before the front reaches the top edge.
• Spot below the solvent level. If the spot dips into solvent at the start, the analyte dissolves into the bulk and is lost. Always spot at least 1 cm above the solvent line.
• Touching the paper bare-handed. Skin oils contaminate cellulose with stearic acid, ninhydrin-positive amino acids, and a faint UV-active mess. Use forceps or wear gloves.
• Ignoring tank saturation. An unsaturated tank gives curved solvent fronts and asymmetric spots. Pre-equilibrate.
• Quoting Rf to three decimal places. Day-to-day variation in temperature, humidity and paper batch means Rf is good to ±0.02 at best. Quote to two decimals; use authentic standards for ID.
• Streaking with too much sample. Overloaded spots resemble bananas, not circles. Decrease loading or pre-concentrate.
• Using ink for the baseline. Pen ink runs with the solvent and contaminates everything. Pencil only.
• Mistaking a co-spot for identity. Two compounds with the same Rf are not necessarily the same compound — they just happen to partition similarly in this solvent. Run a second solvent or follow up with TLC, HPLC or MS for confident identification.