Paper Chromatography
Summary
Letting solvent wick up a paper strip pulls a “black” ink apart into its hidden dyes, because each dye is tugged between the paper and the moving solvent by a different amount. You’ll be able to explain what a stationary and mobile phase are, what an Rf value measures, and why the same principle powers instruments costing tens of thousands of dollars.
History
Chromatography was invented by the Russian botanist Mikhail Tsvet in 1900. He was separating plant pigments by passing solutions through a glass column packed with chalk, and noticed that different pigments traveled at different speeds, creating distinct colored bands. He named the technique chromatography from the Greek chroma (color) and graphein (to write) — though today chromatography separates colorless compounds far more often than colored ones.
Paper chromatography was developed in 1944 by Martin and Synge, who later received the Nobel Prize in Chemistry for the work. It was instrumental in the identification of amino acids, sugars, and peptides in the postwar era, before modern instrumental methods were available. Today it is used primarily for teaching because it requires no instrumentation — but the same underlying principle governs high-performance liquid chromatography (HPLC) systems that cost tens of thousands of dollars.
Hazards & preparation
PPE: none needed for water; light care with alcohol.
- If you use isopropanol (rubbing alcohol) as the solvent, it’s flammable — keep it from flames and work with ventilation.
- Cover the jar to slow evaporation; the fumes are mild but best not inhaled.
Disposal: water-based runs go down the drain; small amounts of alcohol solvent can be flushed with plenty of water or left to evaporate in a vented spot. See the Safety page.
Materials
- Coffee filter or chromatography paper — cut into strips 3 cm × 15 cm
- Water-soluble markers (black or brown reveal the most colors) or food coloring
- Solvent: water, or 70% isopropanol (rubbing alcohol) for a different separation
- Tall jar, beaker, or glass
- Pencil and ruler
Procedure
- Cut strips from the coffee filter.
- Using pencil (pen ink smears), draw a line 2 cm from the bottom edge.
- Make a small dot or short line of marker on the pencil line — keep it small.
- Pour 0.5–1 cm of solvent into the jar (water for water-based inks; isopropanol for permanent markers).
- Hang the strip so its bottom edge touches the solvent but the ink dot sits above the liquid surface.
- Watch the solvent front climb, carrying the pigments at different rates.
- Remove the strip when the front is 1–2 cm from the top; let it dry.
- Observe the separated bands.
What you should see
The solvent creeps up the paper and, as it passes the ink dot, drags the dyes upward at different speeds — a single “black” mark fans out into a ladder of blues, reds, and yellows. Fast, weakly-held dyes ride near the top; sticky ones lag near the start line.
| Symptom | Likely cause | Fix |
|---|---|---|
| Ink dot dissolves into the pool | Dot was below the solvent level | Keep the solvent shallow so the dot starts above it |
| Bands smear together | Dot too big, or run too long | Use a smaller dot; remove the strip before the front reaches the top |
| Nothing separates | Permanent/insoluble ink with water | Switch to isopropanol, or use washable markers |
The Science
Chromatography separates compounds based on how strongly they interact with two competing phases: a stationary phase (the paper, made of cellulose fibers with water molecules clinging to them) and a mobile phase (the solvent traveling up the paper).
Each dye molecule is constantly partitioning between the two phases:
- A molecule that prefers the stationary phase (more polar, more attracted to water on the paper) moves slowly
- A molecule that prefers the mobile phase (more soluble in the solvent) moves quickly
After the solvent has traveled a fixed distance, each component ends up at a reproducible position. This position is expressed as the Rf value (retardation factor):
\[R_f = \frac{\text{distance traveled by compound}}{\text{distance traveled by solvent front}}\]
Rf values range from 0 (stayed at the origin) to 1 (traveled with the solvent front). Each compound has a characteristic Rf for a given stationary phase and solvent — making Rf a simple identification tool.
Black marker ink typically contains 3–5 distinct dyes: blue, yellow, red, and sometimes orange, which the eye integrates into apparent black. Chromatography unmixes them. Food colorings labeled as a single color are often mixtures as well.
Questions to Explore
What is a molecule’s Rf value actually measuring? An Rf of 0.5 means a compound traveled half as far as the solvent front. What does this say about how much time the molecule spent in the moving solvent versus stuck to the paper?
Hint / answer
Rf reflects the fraction of time a molecule spends dissolved and moving versus parked on the paper. An Rf of 0.5 means it was, on balance, carried along about half the time — a molecule equally attracted to paper and solvent. Higher Rf means it prefers the solvent; lower means it clings to the paper.
Why does changing the solvent change the separation pattern? The same ink with water versus isopropanol often separates different bands. What property of the solvent decides which dyes it pulls off the paper?
Hint / answer
Mainly the solvent’s polarity. A polar solvent (water) lifts polar dyes easily but leaves nonpolar ones behind; a less polar solvent (alcohol) carries the nonpolar dyes. Swapping solvents changes which dyes prefer the mobile phase, so the pattern shifts.
Why does black marker ink separate into multiple colors? Black is perceived as a mixture of several dyes. Why use a mixture rather than a single black dye?
Hint / answer
A true single black dye is hard and costly to make, and each dye fades differently. Blending cheaper coloured dyes to look black is easier to manufacture and tune — at the cost that chromatography (or fading in sunlight) reveals the mixture.
How would you identify an unknown compound using Rf? Given an unknown dye and a library of known dyes, how would you determine a match — and what controls make the comparison valid?
Hint / answer
Run the unknown alongside the knowns on the same paper in the same solvent and compare Rf values, ideally spotting the unknown next to a suspected match to see if they co-migrate. Same paper, solvent, temperature, and run distance are the controls that make Rf comparable.
What does modern HPLC have in common with a coffee filter and a ruler? HPLC separates at parts-per-billion using the same principle. What makes it so much more sensitive and precise?
Hint / answer
The separating idea is identical — compounds partition between a stationary and a mobile phase. HPLC just does it with tiny, uniform packing, high pressure to push solvent through, and sensitive detectors, giving far sharper bands and the ability to see minute amounts. The principle does the separating; the engineering sharpens it.
Going further
- Measure Rf values. Mark the start line and the solvent front in pencil, then measure each band to compute its Rf and compare different markers.
- Two solvents, one ink. Run the same marker in water and in isopropanol side by side and compare how the pattern changes.
- Another way to separate: pull apart a salt mixture by Fractional Crystallization — separation by solubility instead of wicking.