Chromatography and Rf Values

How can you find out whether a food colouring is a single dye or a blend of several? How does a forensic scientist compare the ink in a forged signature with the ink from a suspect's pen? The answer to both questions is paper chromatography — a separation technique that spreads the components of a coloured mixture out along a strip of paper so you can count them, compare them and even identify them. This lesson, part of Topic C2 of your OCR Gateway Combined Science course, explains how chromatography works, the steps of the required practical, and how to calculate and use the Rf value to identify a substance.

By the end of this lesson you should be able to describe how paper chromatography separates a mixture, explain the roles of the stationary and mobile phases, carry out the required practical correctly, calculate an Rf value, and use a chromatogram to identify substances and judge purity.

This lesson develops AO2 (carrying out the chromatography required practical and calculating an Rf value), alongside AO1 (explaining the roles of the stationary and mobile phases) and AO3 (interpreting a chromatogram to identify substances and judge purity).

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How Chromatography Separates a Mixture

Paper chromatography separates substances that are dissolved in a solvent. It involves two phases:

• the stationary phase — the chromatography paper, which stays still;
• the mobile phase — the solvent (such as water or ethanol), which moves up through the paper, carrying the dissolved substances with it.

As the solvent moves up the paper it carries the components of the mixture along. They separate because each component has its own balance between two competing tendencies: how strongly it is attracted to the paper (which holds it back) and how soluble it is in the solvent (which carries it forward).

• A component that is more soluble in the solvent and less attracted to the paper is carried further up.
• A component that is less soluble and more attracted to the paper stays nearer the start.

Because the components travel different distances, they end up as separate spots — and the pattern of spots is the chromatogram.

Exam Tip: The separation depends on two factors: each substance's solubility in the solvent and its attraction to the paper. A substance that travels far is more soluble (and less attracted to the paper) — a common misconception is that it is "lighter". Avoid talking about weight.

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The Required Practical: Method

Carrying out chromatography correctly is a required practical, and the details earn marks.

Method:

1. Draw a start line (baseline) near the bottom of the paper in pencil — about $1\,\text{cm}$1cm up.
2. Use a capillary tube to put a small spot of each sample on the start line; let it dry, then add more to keep the spot small and concentrated.
3. Pour a shallow depth of solvent into the container so that the level is below the start line.
4. Stand the paper in the solvent (with the spots above the solvent), put a lid on, and leave it. The solvent rises up the paper, carrying the components.
5. Remove the paper before the solvent reaches the top, and immediately mark the solvent front (the highest point the solvent reached) in pencil.
6. Leave the chromatogram to dry, then measure the distances.

Two details are essential and are frequently tested:

• The start line is drawn in pencil, not ink. Pencil is insoluble — if you used ink, the ink itself would dissolve and rise up the paper with the samples, ruining the result.
• The solvent must start below the start line. If the solvent level were above the line, the spots would dissolve straight into the solvent in the tank instead of travelling up the paper.

Exam Tip: Two marks examiners love: start line in pencil (ink would run and separate and spoil the result) and solvent below the start line (or the samples wash off into the solvent). Learn both reasons, not just the rules.

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Pure Substances and Mixtures on a Chromatogram

A chromatogram tells you at a glance whether a sample is pure:

• a pure substance produces only one spot, in all solvents (there is only one thing to separate);
• a mixture produces two or more spots, because each component travels its own distance.

So if a sample of "green" food colouring separates into a blue spot and a yellow spot, it was a mixture of two dyes; if it gives a single spot, it was a single pure dye. Testing in more than one solvent makes this more reliable, because two different substances can occasionally travel the same distance in one particular solvent but rarely in two.

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Rf Values

To compare results properly, chemists use a number called the Rf value (retention factor). It is the fraction of the way up the paper a spot has travelled, compared with the solvent:

$R_f = \dfrac{\text{distance moved by the substance}}{\text{distance moved by the solvent front}}$Rf​=distance moved by the solvent frontdistance moved by the substance​

Both distances are measured from the start line: the substance distance to the centre of the spot, the solvent distance to the solvent front. Because it is a ratio of two distances, the Rf value has no units and is always between 0 and 1 (a spot can never travel further than the solvent that carries it).

The power of the Rf value is that, in a given solvent, a particular substance always gives the same Rf. So you can identify an unknown by comparing its Rf with the Rf values of known reference substances run under the same conditions: a match strongly suggests they are the same substance.

Worked Example 1 — Calculating an Rf value

A spot of dye travels $4.5\,\text{cm}$4.5cm from the start line. The solvent front travels $6.0\,\text{cm}$6.0cm. Calculate the Rf value.

Step 1 — write the formula: $R_f = \dfrac{\text{distance moved by substance}}{\text{distance moved by solvent}}$Rf​=distance moved by solventdistance moved by substance​.

Step 2 — substitute: $R_f = \dfrac{4.5}{6.0}$Rf​=6.04.5​.

Step 3 — calculate: $R_f = 0.75$Rf​=0.75.

Answer: $R_f = 0.75$Rf​=0.75 (no units).

Worked Example 2 — A second Rf value

On the same chromatogram, a different spot has moved $2.4\,\text{cm}$2.4cm while the solvent front moved $6.0\,\text{cm}$6.0cm. Calculate its Rf value.

$R_f = \dfrac{2.4}{6.0} = 0.40$Rf​=6.02.4​=0.40

Answer: $R_f = 0.40$Rf​=0.40. This spot has a smaller Rf than the first, so it is more strongly attracted to the paper (and less soluble in the solvent), which is why it travelled less far.

Worked Example 3 — Identifying a substance from Rf

A mixture is run alongside three reference dyes in the same solvent. The unknown spot has $R_f = 0.62$Rf​=0.62. The references give: dye A, $R_f = 0.31$Rf​=0.31; dye B, $R_f = 0.62$Rf​=0.62; dye C, $R_f = 0.88$Rf​=0.88. Which dye is present?

Step 1 — compare the unknown's Rf with each reference, in the same solvent.

Step 2 — the unknown's $R_f = 0.62$Rf​=0.62 matches dye B exactly.

Answer: the mixture contains dye B, because it has the same Rf value as the unknown spot in the same solvent.

Worked Example 4 — Comparing two substances by Rf

On a chromatogram the solvent front moved $8.0\,\text{cm}$8.0cm. Substance X moved $6.0\,\text{cm}$6.0cm and substance Y moved $2.0\,\text{cm}$2.0cm. Calculate both Rf values and state which substance is more soluble in the solvent.

Step 1 — Rf of X: $R_f = \dfrac{6.0}{8.0} = 0.75$Rf​=8.06.0​=0.75.

Step 2 — Rf of Y: $R_f = \dfrac{2.0}{8.0} = 0.25$Rf​=8.02.0​=0.25.

Step 3 — interpret: X has the larger Rf, so it travelled further. A substance travels further when it is more soluble in the solvent and less attracted to the paper.

Answer: $R_f(\text{X}) = 0.75$Rf​(X)=0.75 and $R_f(\text{Y}) = 0.25$Rf​(Y)=0.25; X is more soluble in the solvent (and less strongly held by the paper), which is why it moved further up the paper.