Chromatography Revision Notes

Paper chromatography requires simple apparatus: a strip of chromatography paper, a suitable solvent (mobile phase), a container (such as a beaker or a glass jar) to hold the solvent, and a pencil to mark the paper. The chromatography paper acts as the stationary phase. The solvent chosen must dissolve the substances in the mixture but not dissolve the paper itself.

Before starting, a small, concentrated spot of the mixture is carefully applied near one end of the chromatography paper using a capillary tube or a fine pipette. The spot should be above the solvent level to prevent it dissolving directly into the solvent pool. The paper is then suspended vertically in the container so that the solvent can rise up the paper by capillary action.

• Chromatography paper (stationary phase)
• Suitable solvent (mobile phase)
• Container to hold solvent
• Pencil for marking start line
• Capillary tube or pipette for spotting

The chromatography paper is marked with a pencil line about 2 cm from the bottom. A small spot of the mixture is placed on this line. The paper is then placed upright in the container with the solvent level below the spot. As the solvent moves up the paper, it carries the components of the mixture with it.

Different substances in the mixture dissolve in the solvent to different extents and interact differently with the paper fibres. These differences cause each substance to travel at a unique rate, separating them along the paper. When the solvent front nears the top, the paper is removed and dried to reveal separated spots of coloured substances.

• Mark start line with pencil ~2 cm from bottom
• Apply small spot of mixture on start line
• Place paper upright with solvent below spot
• Allow solvent to rise by capillary action
• Remove paper before solvent reaches top and dry

Separation occurs because each substance has a different solubility in the solvent and a different affinity for the paper. Substances more soluble in the solvent move further with it, while those with stronger attraction to the paper move less.

The stationary phase (paper) is polar cellulose fibres, which can adsorb substances. The mobile phase (solvent) dissolves substances and carries them upwards. The balance between these interactions determines how far each substance travels.

For example, a dye that dissolves well in the solvent but binds weakly to the paper will travel further than one that is less soluble or binds strongly.

• Stationary phase: polar cellulose paper
• Mobile phase: solvent dissolving substances
• Substances separate based on solubility and affinity
• Higher solubility = travel further
• Stronger attraction to paper = travel less

After drying, the paper shows distinct spots representing separated substances. The distance each spot travels from the start line can be measured and compared.

The retention factor (Rf) value is calculated as the distance travelled by the substance divided by the distance travelled by the solvent front. Rf values help identify substances by comparison with known standards.

For example, if the solvent front moves 10 cm and a spot moves 6 cm, Rf = 6 cm / 10 cm = 0.6. Different substances have characteristic Rf values in a given solvent.

• Measure distance from start line to spot centre
• Measure distance from start line to solvent front
• Calculate Rf = (distance travelled by substance) / (distance travelled by solvent)
• Use Rf values to identify substances

Use a pencil to mark the start line because ink may dissolve and interfere with results. Apply small, concentrated spots to avoid overlapping spots.

Choose a solvent that dissolves the substances but does not damage the paper. Perform the experiment in a well-ventilated area if using volatile solvents.

Repeat the experiment to check consistency of results. Avoid touching the paper with fingers to prevent contamination. Dispose of solvents safely according to guidelines.

• Use pencil, not ink, for markings
• Apply small, concentrated spots
• Select appropriate solvent
• Work in ventilated area if solvent is volatile
• Repeat for reliability
• Avoid contamination

Chromatography is a technique used to separate mixtures into individual components based on their different affinities for a stationary phase and a mobile phase. In paper chromatography, the stationary phase is the paper, and the mobile phase is a solvent that moves through the paper by capillary action.

As the solvent moves, different substances in the mixture travel at different rates depending on their solubility in the solvent and their attraction to the paper. This results in separated spots on the chromatogram, each representing a different substance.

A chromatogram is the pattern of spots formed on the paper after the solvent has moved a certain distance. The position and number of spots provide information about the components of the mixture.

• Chromatography separates substances based on movement through stationary and mobile phases.
• Different substances travel different distances, forming distinct spots.
• Chromatograms visually represent separated substances.

To identify an unknown substance using chromatography, its chromatogram is compared with chromatograms of known substances run under the same conditions (same solvent, paper, and time).

The key comparison is the distance each spot travels relative to the solvent front, often expressed as the Rf value (distance travelled by substance ÷ distance travelled by solvent). Although calculating Rf is outside this objective, the relative position of spots can be compared visually.

If the unknown sample produces a spot at the same position as a known substance, it suggests the unknown contains that substance. For example, if an unknown ink sample shows a spot at the same height as a known blue dye, it likely contains that dye.

• Run known substances and unknown under identical conditions.
• Compare spot positions to identify matching substances.
• Matching spot positions indicate the presence of the same substance.

A pure substance typically produces a single spot on a chromatogram because it consists of only one component that moves uniformly with the solvent.

An impure substance or mixture produces two or more spots, indicating multiple components with different affinities for the solvent and paper.

For example, a pure food colour dye would show one spot, while a mixture of dyes in a coloured drink would show multiple spots.

• Single spot indicates a pure substance.
• Multiple spots indicate impurities or mixtures.
• Number of spots reveals composition complexity.

When analyzing chromatograms, ensure that the solvent front is clearly marked and that spots are distinct and not overlapping, as this affects accuracy.

Use the same solvent and paper type for both unknown and known samples to ensure valid comparisons.

Repeat chromatography runs if spots are unclear or if results are inconsistent to improve reliability and confidence in identification and purity assessment.

• Mark solvent front clearly for measurement consistency.
• Use identical conditions for unknown and known samples.
• Repeat tests to confirm results and reduce errors.

Suppose an unknown ink sample produces two spots on a chromatogram: one at 3 cm and another at 6 cm from the baseline. Known inks A and B produce spots at 3 cm and 6 cm respectively under the same conditions.

By comparing, the unknown ink contains components matching inks A and B, indicating it is a mixture of these two substances.

Since there are two spots, the unknown ink is impure or a mixture, not a single pure substance.

• Measure spot distances from baseline.
• Compare with known samples' spot distances.
• Multiple matching spots indicate mixture and impurity.

Chromatography is a technique used to separate components of a mixture based on their different affinities for a stationary phase and a mobile phase (solvent). The Rf value, or retention factor, is a numerical measure that expresses how far a particular substance travels relative to the solvent front during chromatography. It provides a standardised way to compare the movement of substances under the same experimental conditions, aiding in their identification.

• Rf stands for retention factor.
• It is a ratio comparing distances travelled on the chromatogram.
• Rf values are characteristic for substances under specific conditions.

To calculate the Rf value, measure two distances on the chromatogram: the distance from the baseline (where the sample was initially spotted) to the centre of the spot for the substance, and the distance from the baseline to the solvent front (the furthest point reached by the solvent). The Rf value is the ratio of these two distances, a dimensionless number between 0 and 1.

Mathematically, the formula is: Rf = (distance travelled by substance) ÷ (distance travelled by solvent front).

For example, if the solvent front moves 8.0 cm from the baseline, and a substance spot moves 5.0 cm, then: Rf = 5.0 cm ÷ 8.0 cm = 0.625. This means the substance travelled 62.5% of the distance the solvent travelled.

• Measure distances from baseline to spot centre and solvent front.
• Use consistent units (usually centimetres).
• Calculate Rf as a ratio, no units.
• Example: substance 5.0 cm, solvent 8.0 cm, Rf = 0.625.

Rf values help identify substances by comparing their chromatographic behaviour to known standards under identical conditions. Each substance has a characteristic Rf value in a given solvent and stationary phase, reflecting its relative affinity for the mobile and stationary phases.

A higher Rf value indicates the substance is less attracted to the stationary phase and moves further with the solvent. Conversely, a lower Rf value means stronger interaction with the stationary phase, so the substance travels less.

In practical use, chromatograms of unknown mixtures can be analysed by calculating Rf values of spots and comparing them to Rf values of known substances to identify components.

• Rf values are characteristic under fixed conditions.
• Higher Rf means weaker attraction to stationary phase.
• Used to identify substances by comparison.
• Conditions must be consistent for valid comparison.

Accurate measurement of distances is crucial. Use a ruler to measure from the baseline to the centre of each spot and to the solvent front. The baseline should be drawn lightly in pencil to avoid interference.

Repeat the chromatography experiment to check consistency of Rf values, as slight variations can occur due to experimental factors such as solvent composition, temperature, or paper quality.

Safety precautions include working in a well-ventilated area if using volatile solvents and avoiding skin contact with chemicals. Dispose of solvents properly after use.

• Draw baseline with pencil to avoid ink running.
• Measure distances carefully to spot centre and solvent front.
• Repeat experiments to confirm Rf values.
• Maintain consistent experimental conditions.
• Follow safety procedures with solvents.

Rf values depend on experimental conditions; changing the solvent or stationary phase alters Rf values, so comparisons must be made only when conditions are identical.

Impurities or overlapping spots can affect accuracy. Using more refined techniques or different solvents can improve separation.

Digital imaging and software can enhance measurement precision and reduce human error in determining distances.

• Rf values are condition-dependent.
• Impurities can distort results.
• Improved solvents or techniques enhance separation.
• Digital tools can improve measurement accuracy.