Paper Chromatography

Isopropyl alcohol is flammable and its vapour is unpleasant in a closed space. Open a window, keep it away from flames, and wear splash goggles when pouring.

Cut a strip about 20 mm wide and 20 mm longer than the height of your jar. Filter paper or a coffee filter works if you have no chromatography paper; ordinary printer paper does not — it is too dense and the solvent crawls.

Pencil, never pen. Graphite does not dissolve and stays put; ink would separate along with your sample and ruin the result.

Touch a black felt-tip pen to the centre of the baseline to make a spot 2-3 mm across. Let it dry, then spot the same place again 2-3 times. A small concentrated spot resolves; a big wet blot smears.

Use isopropyl alcohol for felt-tip inks, or plain water for washable markers and food colouring. The depth matters only in that it must stay below the baseline.

Tape the top of the strip to a pencil laid across the jar mouth, so the bottom edge dips in but the pencil line stays clear of the liquid. If the spot is submerged it dissolves off into the solvent and nothing separates — this is the single commonest failure.

Put a lid or foil over the top. A saturated atmosphere stops the solvent evaporating off the paper as it climbs, which would otherwise distort the front and give inconsistent Rf values.

Watch the bands separate over 10-20 minutes. Do not move, tilt or reposition the jar — the solvent front must stay level.

Take it out when the solvent front is about 10 mm from the top edge. Immediately mark the front with a pencil line — it is invisible once the paper dries and you cannot calculate anything without it.

Lay it on a paper towel and let the solvent evaporate. The bands stay where they are; only the solvent leaves.

From the baseline, measure in millimetres to the centre of each coloured band, and to the solvent front line. Measure to the centre, not the leading edge — bands are diffuse and the centre is the reproducible point.

Rf = (distance moved by the band) ÷ (distance moved by the solvent front). The result is always between 0 and 1 and has no units. Record every value with the solvent and paper you used — an Rf is meaningless without them.

Spot two different black pens on the same baseline, 10 mm apart, and run them together. Matching Rf values mean the same dye is present in both — this is chromatography used as identification rather than demonstration.

Crush a green leaf with a little isopropyl alcohol, spot the extract repeatedly on a fresh baseline, and run it. Expect a yellow-green band, a blue-green band and a faint yellow one travelling furthest — chlorophyll b, chlorophyll a and the carotenoids, in that order of increasing distance.

Two phases, one competition. The paper is cellulose, and it holds a thin layer of water bound to it — that water is the stationary phase. The solvent climbing by capillary action is the mobile phase. Every molecule in your spot is constantly partitioning between the two. A molecule that prefers the bound water spends most of its time stuck and moves slowly; one that prefers the solvent rides along and moves fast. Repeated over thousands of cycles up the strip, a tiny difference in preference becomes centimetres of separation. Nothing is filtering by size — this is a difference in solubility, amplified.

Why Rf is constant, and when it is not. Because both distances scale together, the ratio cancels out how long you ran it and how far the front got. What it does not cancel is the solvent, the paper, the temperature, or how saturated the jar atmosphere was. That is why published Rf values always come with their conditions attached, and why the honest way to identify something is to run a known standard on the same strip rather than trust a number from a book.

Who actually invented this. Chromatography was created by the botanist Mikhail Tsvet, who separated leaf pigments on a column of calcium carbonate around 1903-1906 and named it from the Greek for colour-writing. His work was largely ignored for decades. Paper chromatography specifically is much later — Archer Martin and Richard Synge developed partition chromatography in the early 1940s, and the paper method was published by Consden, Gordon and Martin in 1944. Martin and Synge received the 1952 Nobel Prize in Chemistry for it. It is a common and wrong shorthand to credit Tsvet with the paper technique; he invented the principle, not this implementation.

What it grew into. The same partition logic, run through a packed column under pressure, is HPLC; run through a heated capillary with a gas as the mobile phase, it is GC. Those instruments dominate analytical chemistry, drug testing and forensics. The physics you measured with a ruler is the physics they run on.