Chemistry

2.5 - Chromatography & Rf Values

10 min

Intro & method

Mobile & stationary phases

Rf values

Pure substances & solvents

note 1 of 4

Paper chromatography

Chromatography separates the substances in a mixture to count, compare, or identify them.
Paper chromatography works on any substance that dissolves in the solvent used, such as food colourings and ink dyes.
It uses two phases:
1. Mobile phase - The phase the substances move in (a solvent, such as water or ethanol).
2. Stationary phase - The phase that does not move (the paper).

Method

Draw a pencil line near the bottom of the paper and add a spot of the mixture. This line is the baseline. It must be drawn in pencil because pencil doesn't dissolve in the solvent, whereas ink would travel up the paper.
Stand the paper in a shallow layer of solvent with the solvent level below the baseline. If the solvent touches the spot directly, the mixture dissolves into the solvent instead of travelling up the sheet.
Cover the container with a lid to stop the solvent evaporating. The solvent moves up the paper, carrying the substances in the mixture with it.
Take the paper out before the solvent reaches the top, mark the solvent front, and leave it to dry. The solvent front is the furthest point the solvent reaches. It must be marked immediately in pencil because it cannot be seen once the paper dries. The dried sheet is a chromatogram.

Three stages: a lidded beaker with paper standing in solvent, a mixture spot on the pencil baseline; the solvent risen, splitting the spot into six colours; the dried sheet labelled chromatogram, with one solvent front label pointing to the last two panels.

note 2 of 4

Reading a chromatogram

A chromatogram is the finished, dried sheet with its pattern of separated spots.

Why substances separate

As the solvent moves up the paper, each substance is distributed between the two phases: dissolving in the solvent (mobile phase) and sticking to the paper (stationary phase).
Two properties determine how far a substance travels:
1. How soluble it is in the solvent (more soluble substances spend more time in the mobile phase and travel further).
2. How strongly it is attracted to the paper (substances more strongly attracted spend more time in the stationary phase and travel a shorter distance).
Different substances differ in both properties, so they travel different distances and separate into distinct spots.

Interpreting the chromatogram

Number of substances - Each spot represents at least one substance. A spot that stays on the baseline didn't dissolve in that solvent.
Purity - A pure substance produces a single spot; a mixture produces more than one spot.
Identifying substances - A pure reference sample of a known substance is run alongside the mixture. A spot in the mixture level with the reference may be that substance. If no spot aligns with the reference, that substance is not present.

A chromatogram with baseline and solvent front marked, four lanes labelled mixture, reference A, reference B and reference C, each reference starting from a pencil cross; A and C sit level with two of the mixture's three spots, B matches none.

Testing with a second solvent

Two different substances can travel the same distance in one solvent and appear as a single spot.
Repeating the run with a different solvent settles this, because the substances will distribute differently in a new solvent.

note 3 of 4

R_f values

How far a spot travels depends on how long the chromatogram is run for.
The $R_{f}$ value is the ratio of the distance travelled by the substance to the distance travelled by the solvent. It is constant for a given substance under the same conditions.

A chromatogram sheet, baseline and solvent front marked, with one spot whose middle is crossed and labelled centre of the spot, and two arrows: distance travelled by spot, and distance travelled by solvent.

Formula for R_f value

$R_{f} = \frac{distance travelled by substance}{distance travelled by solvent}$

Distance travelled by the substance - Measured from the baseline to the centre of the spot.
Distance travelled by the solvent - Measured from the baseline to the solvent front.

Interpreting the value

An $R_{f}$ value is always between 0 and 1.
An $R_{f}$ value of 0 means the substance didn't dissolve in the solvent and stayed on the baseline.
An $R_{f}$ value has no units because the units of distance cancel out.

Using R_f values for identification

The same substance has different $R_{f}$ values in different solvents or with different stationary phases.
An $R_{f}$ value can only identify a substance when compared against a known reference run under the exact same conditions, or a published standard value for that solvent and paper.

note 4 of 4

Worked example - An R_f value

On a chromatogram, one spot has moved 3.2 cm from the baseline (measured to the centre of the spot), and the solvent front is 7.5 cm from the baseline. Calculate the R_f value of that substance.

A chromatogram sheet with baseline and solvent front marked, one spot, its centre crossed, and two arrows from the baseline reading 3.2 cm to the spot's centre and 7.5 cm to the solvent front.

Step 1 - Identify the values

Distance travelled by the substance = 3.2 cm (baseline to the centre of the spot)
Distance travelled by the solvent = 7.5 cm (baseline to the solvent front)

Step 2 - Formula

$R_{f} = \frac{distance travelled by substance}{distance travelled by solvent}$

Step 3 - Substitution and calculation

$R_{f} = \frac{3.2 cm}{7.5 cm} = 0.42666...$

Both distances are in cm, so the units cancel and a plain number is left.

Step 4 - Round the answer

Each distance was measured to two significant figures, so give the answer to two:

$R_{f} = 0.43$

The value sits between 0 and 1, as it must, and 3.2 cm is a little under half of 7.5 cm - so a value a little under 0.5 is the right size.

key terms

TermDefinition

Mobile phaseThe phase the substances move in during chromatography, usually a liquid or a gas.

Stationary phaseThe phase that does not move during chromatography, such as the paper or silica gel.

Solvent frontThe furthest point the solvent reaches as it moves up the paper.

ChromatogramThe record of a chromatography run that shows the separated substances, such as the spots on dried paper or the peaks from gas chromatography.

R_f valueThe ratio of the distance travelled by the substance to the distance travelled by the solvent.

end of notes

Key points to remember

Paper chromatography

Chromatography separates the substances in a mixture to count, compare, or identify them.

Paper chromatography works on any substance that dissolves in the solvent used, such as food colourings and ink dyes.

It uses two phases:

Mobile phase - The phase the substances move in (a solvent, such as water or ethanol).
Stationary phase - The phase that does not move (the paper).

Method

Draw a pencil line near the bottom of the paper and add a spot of the mixture. This line is the baseline. It must be drawn in pencil because pencil doesn't dissolve in the solvent, whereas ink would travel up the paper.

Stand the paper in a shallow layer of solvent with the solvent level below the baseline. If the solvent touches the spot directly, the mixture dissolves into the solvent instead of travelling up the sheet.

Cover the container with a lid to stop the solvent evaporating. The solvent moves up the paper, carrying the substances in the mixture with it.

Take the paper out before the solvent reaches the top, mark the solvent front, and leave it to dry. The solvent front is the furthest point the solvent reaches. It must be marked immediately in pencil because it cannot be seen once the paper dries. The dried sheet is a chromatogram.

Reading a chromatogram

A chromatogram is the finished, dried sheet with its pattern of separated spots.

Why substances separate

As the solvent moves up the paper, each substance is distributed between the two phases: dissolving in the solvent (mobile phase) and sticking to the paper (stationary phase).

Two properties determine how far a substance travels:

How soluble it is in the solvent (more soluble substances spend more time in the mobile phase and travel further).
How strongly it is attracted to the paper (substances more strongly attracted spend more time in the stationary phase and travel a shorter distance).

Different substances differ in both properties, so they travel different distances and separate into distinct spots.

Interpreting the chromatogram

Number of substances - Each spot represents at least one substance. A spot that stays on the baseline didn't dissolve in that solvent.

Purity - A pure substance produces a single spot; a mixture produces more than one spot.

Identifying substances - A pure reference sample of a known substance is run alongside the mixture. A spot in the mixture level with the reference may be that substance. If no spot aligns with the reference, that substance is not present.

2.5 - Chromatography & Rf Values

Paper chromatography

Method

Reading a chromatogram

Why substances separate

Interpreting the chromatogram

Testing with a second solvent

Rf values

Formula for Rf value

Interpreting the value

Using Rf values for identification

Worked example - An Rf value

Step 1 - Identify the values

Step 2 - Formula

Step 3 - Substitution and calculation

Step 4 - Round the answer

Key points to remember

2.5 - Chromatography & Rf Values

Paper chromatography

Method

Reading a chromatogram

Why substances separate

Interpreting the chromatogram

Testing with a second solvent

Rf values

Formula for Rf value

Interpreting the value

Using Rf values for identification

Worked example - An Rf value

Step 1 - Identify the values

Step 2 - Formula

Step 3 - Substitution and calculation

Step 4 - Round the answer

Key points to remember

R_f values

Formula for R_f value

Using R_f values for identification

Worked example - An R_f value

R_f values

Formula for R_f value

Using R_f values for identification

Worked example - An R_f value