Chromatography

What is it?

Chromatography separates the substances in a mixture so that each one can be identified and measured. Every method has two phases:

a stationary phase, which stays in place (paper, a thin layer of silica on a plate, or the inside of a column);
a mobile phase, which moves through or over it (a solvent, or a gas).

As the mobile phase moves, each substance in the mixture is constantly shared between the two phases. A substance that is attracted more strongly to the stationary phase spends more time stuck there and moves slowly; one that prefers the mobile phase moves quickly. Different substances therefore travel different distances (or take different times) and come apart.

Key idea

Separation depends on the balance of attractions: stronger attraction to the stationary phase means slower movement. A pure substance gives one spot or peak; a mixture gives several.

Why does it matter?

Identification. Forensic scientists identify inks, drugs and explosives; food chemists check for banned dyes.
Purity. One spot on a plate is a quick check that a product is pure; chemists use columns to purify the compounds they make.
Measurement. Gas and liquid chromatography measure pesticides in food, alcohol in blood and drugs in urine, often down to billionths of a gram.

How does it work?

1. Paper and thin-layer chromatography (TLC)

Draw a baseline in pencil (ink would itself separate) about 1 cm from the bottom of the paper or plate.
Put small spots of the mixture and of known standards on the baseline.
Stand the plate in a little solvent, below the baseline (otherwise the spots would dissolve into the solvent), and cover the container so the air stays saturated with solvent vapour.
Remove the plate before the solvent reaches the top, and mark the solvent front at once.
Make colourless spots visible (for example under UV light) and measure the distances.

2. The retention factor, Rf

R_f = \frac{\text{distance moved by spot}}{\text{distance moved by solvent}}

Both distances are measured from the baseline (to the centre of the spot) in the same unit, so $R_f$ has no unit and lies between 0 and 1. Under the same conditions (same stationary phase, solvent and temperature), a substance always has the same $R_f$ , so a spot can be identified by matching it to a standard run on the same plate.

3. The role of polarity

On a TLC plate of silica (very polar), polar compounds, such as those with –OH or –COOH groups, are held strongly and have low $R_f$ values; non-polar compounds, such as hydrocarbons, have high $R_f$ values. Using a more polar solvent pulls all the compounds further and raises every $R_f$ .

4. Gas chromatography (GC)

In GC the sample is vaporized and carried by an unreactive gas (such as helium) through a long, thin column coated with the stationary phase. A detector at the end records a peak as each substance comes out.

The retention time, the time from injection to the peak, identifies a substance (compare with standards run under the same conditions).
The area under a peak is proportional to the amount of that substance. For quantities, a calibration with standards is used, as in spectrophotometry.

Liquid chromatography (HPLC) works the same way, with a liquid mobile phase pumped through a packed column.

Think of it like this

Picture a crowd walking through a shopping street. Window-shoppers (strongly attracted to the shops, the stationary phase) stop often and move slowly; people hurrying to work (who prefer the moving crowd, the mobile phase) get through quickly. After an hour, the two groups are far apart, even though everyone set off together.

More precisely

A substance distributes between the phases according to its partition (or adsorption) equilibrium, which depends on intermolecular forces: hydrogen bonding, dipole–dipole forces and dispersion forces. In GC, volatility matters too: compounds with low boiling points spend more time in the gas and come out first. Two substances with the same $R_f$ or retention time under one set of conditions are not necessarily the same compound; confirming identity needs a second method, such as a different solvent or column, or mass spectrometry.

Visualise it

A thin-layer chromatography plate standing in solvent below a dashed pencil baseline. Two lanes are spotted: a mixture and dye A. The solvent front is a dashed line 7.80 cm above the baseline. The mixture lane shows a red spot 3.20 cm above the baseline, level with the single red spot of dye A, and a blue spot higher up. Red spot: Rf = 3.20 cm ÷ 7.80 cm = 0.410, the same as dye A. The blue spot, with Rf 0.782, is a second, different dye. — Distances are measured from the baseline. The red spot matches dye A run on the same plate.

A gas chromatogram of detector signal against retention time from 0 to 10 minutes, with three peaks: ethanol at 2.4 minutes, propan-1-ol at 4.1 minutes (the tallest peak) and butan-1-ol at 6.8 minutes. Here, the lower the boiling point, the sooner the peak. — Retention time identifies each compound; peak area tells how much is present.

Worked example

Worked example: Calculating an Rf value

Question: On a TLC plate, the solvent front is 7.80 cm above the baseline and a red spot is 3.20 cm above it. Find $R_f$ .

$R_f = \dfrac{3.20\ \text{cm}}{7.80\ \text{cm}} =$ 0.410 (the cm cancel)

Worked example: Identifying a dye with replicate plates

Question: A food dye is run on three plates beside standards. Its $R_f$ values are 0.42, 0.44 and 0.43. Standards on the same plates: dye P 0.43, dye Q 0.52. Which dye is it?

Mean: $\bar{R}_f = \dfrac{0.42 + 0.44 + 0.43}{3} = 0.43$
Standard deviation (sample, $n - 1$ ): $s = 0.01$
Report $R_f = 0.43 \pm 0.01$ ( $n = 3$ ). Dye P (0.43) lies within this spread; dye Q (0.52) is 9 standard deviations away.
The dye is consistent with dye P. Running replicates shows the result is reproducible, not a one-off reading.

Worked example: Composition from GC peak areas

Question: A GC run of a solvent mixture gives peak areas of 1250 (ethanol), 3400 (propan-1-ol) and 850 (butan-1-ol), in the same arbitrary units. Assuming the detector responds equally to each, find the percentage of each component.

Total area $= 1250 + 3400 + 850 = 5500$
Ethanol: $\dfrac{1250}{5500} \times 100\ \% =$ 22.7 %
Propan-1-ol: $\dfrac{3400}{5500} \times 100\ \% =$ 61.8 %
Butan-1-ol: $\dfrac{850}{5500} \times 100\ \% =$ 15.5 %
Check: 22.7 % + 61.8 % + 15.5 % = 100.0 %. ✓

Common mistake

Common mistake: Measuring from the bottom of the plate

Both distances in $R_f$ are measured from the baseline, not from the edge of the plate. Measuring from the edge makes every $R_f$ too large.

Common mistake: Comparing Rf values from different conditions

$R_f$ depends on the solvent, the stationary phase and the temperature. Only compare with standards run on the same plate, or under identical conditions.

Common mistake: Drawing the baseline in ink or below the solvent

Ink contains dyes that separate too; a baseline below the solvent level lets the sample dissolve away into the solvent instead of travelling up the plate.

Notation note

$R_f$ is the retention factor (sometimes called the retardation factor); it is written without a unit.
TLC = thin-layer chromatography; GC = gas chromatography; HPLC = high-performance liquid chromatography.
The substance being analysed is often called the analyte; the mobile phase in GC is the carrier gas.

Remember this

Stationary phase stays put; mobile phase moves. Stronger attraction to the stationary phase = slower.
$R_f$ = distance moved by spot ÷ distance moved by solvent front, both from the baseline; no unit; compare with standards on the same plate.
On polar silica: polar compounds have low $R_f$ ; a more polar solvent raises all $R_f$ values.
GC: retention time identifies; peak area measures amount. Measure in replicate and report mean ± standard deviation.

Practise this topic with flashcards and a quiz at chemistryclarity.com/chemistry/chromatography/

Chromatography

What is it?

Why does it matter?

How does it work?

1. Paper and thin-layer chromatography (TLC)

2. The retention factor, Rf

3. The role of polarity

4. Gas chromatography (GC)

Visualise it

Worked example

Common mistake

Remember this

Test yourself

Flashcards

Quiz

Notes and downloads

Chromatography Worksheet

References

What is it?

Why does it matter?

How does it work?

1. Paper and thin-layer chromatography (TLC)

2. The retention factor, Rf

3. The role of polarity

4. Gas chromatography (GC)

Visualise it

Worked example

Common mistake

Remember this

Test yourself

Flashcards

Chromatography: Flashcards

Quiz

Chromatography: Quiz

Notes and downloads

Chromatography Worksheet

References