Chromatography Revision Notes

Paper chromatography is a method used to separate mixtures of soluble coloured substances based on their different affinities for a stationary phase and a mobile phase. The stationary phase is chromatography paper, which is made of cellulose fibres that adsorb substances. The mobile phase is a solvent that moves through the paper by capillary action, carrying dissolved substances with it. Different substances travel different distances depending on their solubility in the solvent and their adsorption to the paper, resulting in separation.

• Stationary phase: chromatography paper (cellulose)
• Mobile phase: suitable solvent (e.g., water, ethanol)
• Separation depends on solubility and adsorption

To perform paper chromatography, the following apparatus is needed: chromatography paper, a solvent in a suitable container (e.g., a beaker or a chromatography tank), a pencil, a capillary tube or a fine pipette, and a lid or watch glass to cover the container and reduce solvent evaporation.

• Cut a strip of chromatography paper longer than the solvent depth
• Draw a light pencil line about 2 cm from the bottom (baseline)
• Use a capillary tube to place a small, concentrated spot of the mixture on the baseline
• Pour solvent into the container to a depth less than the baseline to avoid dissolving the spot directly
• Place the paper in the container so the baseline is above the solvent level
• Cover the container to prevent solvent evaporation

When the paper is placed in the solvent, the solvent rises up the paper by capillary action. As it moves, it dissolves the substances in the spot and carries them upwards. Substances with higher solubility in the solvent travel further, while those with stronger adsorption to the paper travel less. Over time, the substances separate into distinct coloured spots at different heights.

The process is stopped before the solvent front reaches the top of the paper. The distance travelled by the solvent (solvent front) and each substance can be measured to calculate Rf values if needed.

• Solvent moves up paper by capillary action
• Substances dissolve and move at different rates
• Distinct coloured spots appear separated along the paper
• Stop before solvent front reaches paper top
• Measure distances for analysis

Separation in paper chromatography depends on two main factors: solubility in the solvent and adsorption to the paper. A substance more soluble in the solvent spends more time dissolved and moves further. A substance more strongly adsorbed to the paper spends more time attached and moves less.

This balance causes substances in a mixture to separate as the solvent moves. The stationary phase (paper) adsorbs substances, while the mobile phase (solvent) dissolves and transports them.

• Higher solubility → greater distance travelled
• Stronger adsorption → shorter distance travelled
• Separation due to differing interactions with paper and solvent

The solvent must dissolve the substances in the mixture but not dissolve the paper. Common solvents include water, ethanol, or mixtures of solvents. The choice depends on the substances’ solubility to achieve good separation.

Safety precautions include working in a well-ventilated area if using volatile solvents, avoiding ingestion or inhalation, and handling glassware carefully to prevent breakage.

• Solvent must dissolve substances but not paper
• Choose solvent to optimise separation
• Use appropriate safety measures with solvents and apparatus

Chromatography separates components of a mixture based on their differing affinities between 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, components dissolve and 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 visible pattern of spots formed after the solvent has moved a certain distance. The position of each spot is characteristic of the substance it represents, allowing identification by comparison with known substances.

• Stationary phase: paper
• Mobile phase: solvent
• Separation based on solubility and adsorption
• Chromatogram shows separated spots representing substances

To identify an unknown substance, run a chromatogram of the unknown alongside chromatograms of known substances under identical conditions (same solvent, paper, and time).

Compare the positions of the spots by measuring the distance travelled by each spot relative to the solvent front. Substances with matching spot positions and colours are likely the same.

The retention factor (Rf) value can be calculated to quantify this comparison: Rf = (distance travelled by substance) ÷ (distance travelled by solvent front). Although calculation of Rf is excluded here, understanding spot position comparison is essential.

• Run unknown and known substances side by side
• Compare spot positions and colours
• Matching spots indicate identical substances

A pure substance produces a single spot on the chromatogram because it consists of only one component.

An impure substance produces two or more spots, indicating the presence of multiple components or impurities.

This visual evidence helps assess the purity of samples quickly and effectively without complex instrumentation.

• One spot = pure substance
• Multiple spots = impure substance or mixture

Use a pencil to draw the baseline on the chromatography paper to avoid ink running with the solvent.

Apply small, concentrated spots of the sample to the baseline to prevent spot spreading and overlapping.

Use a suitable solvent that dissolves the substances but does not dissolve the paper or react chemically with the samples or paper chemicals (if any). Allow the solvent front to travel a sufficient distance before removing the paper to ensure clear separation of spots, then mark the solvent front immediately to avoid evaporation errors during measurement.

• Draw baseline in pencil
• Apply small, concentrated spots
• Use appropriate solvent
• Mark solvent front before drying

An unknown dye is analysed by paper chromatography alongside three known dyes: A, B, and C. The solvent front travels 12.0 cm. The unknown dye spot travels 7.2 cm. Dye A travels 7.2 cm, Dye B travels 5.0 cm, and Dye C travels 9.0 cm.

Since the unknown dye spot travels the same distance as Dye A, it is likely the same substance.

If the unknown dye chromatogram shows only one spot matching Dye A, the sample is pure. If multiple spots appear, the sample is impure or a mixture.

• Distance travelled by unknown = 7.2 cm
• Distance travelled by Dye A = 7.2 cm
• Unknown matches Dye A
• Single spot indicates purity

Paper chromatography separates components of a mixture based on their differing solubilities in a solvent and their differing affinities for the stationary phase, which is chromatography paper made of cellulose fibres. The paper is polar and can form hydrogen bonds with polar substances, affecting how far substances travel.

When a spot of the mixture is placed near the bottom of the paper and the paper is placed in a solvent, the solvent moves up the paper by capillary action. Components of the mixture dissolve in the solvent to different extents and interact differently with the paper, causing them to move at different rates and separate along the paper.

• Stationary phase: chromatography paper (polar cellulose fibres).
• Mobile phase: suitable solvent (chosen for solubility and polarity).
• Separation depends on solubility in solvent and affinity for paper.

The solvent must dissolve the substances in the mixture and be able to move up the paper by capillary action. It should not dissolve the paper or react with the substances.

The choice of solvent affects the separation: a solvent in which substances are more soluble will carry them further up the paper. A solvent too polar or non-polar may not separate the substances effectively.

Common solvents include water, ethanol, or mixtures such as ethanol-water. The solvent front is the furthest point reached by the solvent on the paper and must be marked for calculations.

• Solvent must dissolve all components of the mixture.
• Solvent must travel up the paper by capillary action.
• Solvent polarity affects separation efficiency.
• Mark solvent front immediately after removal from solvent.

Many substances separated by paper chromatography are colourless and invisible on the paper after solvent evaporation. A locating agent is used to make these spots visible.

Locating agents react with the separated substances to produce coloured spots or fluorescence. Common locating agents include ninhydrin for amino acids or iodine vapour for many organic compounds.

The locating agent is applied after the chromatogram has dried. It must be chosen to react with the substances present but does not require prior knowledge of specific agents for this objective.

• Colourless substances are invisible on chromatogram without treatment.
• Locate spots by spraying or exposing to locating agent.
• Locating agents produce coloured or fluorescent spots.
• Apply locating agent after chromatogram is dry.

1. Draw a pencil line near the bottom of chromatography paper and place a small spot of the mixture on this line.

2. Place the paper in a container with a shallow layer of solvent, ensuring the spot is above the solvent level.

3. Allow the solvent to rise up the paper by capillary action until near the top; mark the solvent front immediately and remove the paper to dry it.

• Use pencil to avoid ink dissolving.
• Spot size should be small and concentrated.
• Ensure solvent level is below the spot.
• Dry chromatogram before applying locating agent.

After visualising the spots, measure the distance travelled by each substance from the baseline and the distance travelled by the solvent front.

The retention factor (Rf) is calculated as: Rf = (distance travelled by substance) / (distance travelled by solvent front).

Different substances have characteristic Rf values under the same conditions, aiding identification and comparison.

• Measure distances accurately in millimetres.
• Calculate Rf values to compare substances.
• Rf values are unitless and always less than or equal to 1.
• Repeat experiments to ensure reliability.

Chromatography is a technique used to separate and identify components in a mixture based on their different affinities for a stationary phase and a mobile phase (solvent). When a mixture is spotted near the base of a chromatography paper and placed in a solvent, the solvent moves up the paper by capillary action, carrying the substances with it.

The Rf value, or retention factor, is a numerical value that expresses how far a particular substance travels relative to the solvent front during chromatography. It is a characteristic property under fixed conditions and helps in identifying substances by comparison with known standards.

• Chromatography separates substances based on differential solubility and adsorption.
• Rf value quantifies the relative movement of a substance on the chromatogram.
• Rf values are reproducible under consistent experimental conditions.

The Rf value is defined as the ratio of the distance travelled by the substance (solute) to the distance travelled by the solvent front from the baseline (origin). It is a dimensionless number between 0 and 1.

The formula is: \n\nRf = (distance travelled by substance) / (distance travelled by solvent front)\n\nwhere distances are measured from the baseline to the centre of the substance spot and to the solvent front respectively.

• Distance measurements must be in the same units (e.g., cm).
• The solvent front is the furthest point reached by the solvent on the chromatography paper.
• The substance spot distance is measured to the centre of the spot for accuracy.

To determine Rf values in practice, a small spot of the mixture is placed near the bottom of chromatography paper and the paper is suspended in a solvent so that the spot is above the solvent level. The solvent moves up the paper, carrying the components at different rates.

After the solvent has travelled a suitable distance, the paper is removed and dried. The distances from the baseline to the solvent front and to each substance spot are measured carefully using a ruler.

Multiple repeats improve accuracy and help identify anomalies. Safety precautions include handling solvents in a well-ventilated area and avoiding skin contact.

• Use a pencil to mark the baseline and solvent front to avoid smudging.
• Measure distances in centimetres to two decimal places for precision.
• Repeat chromatography to confirm consistent Rf values.
• Use appropriate solvents that dissolve the substances without reacting chemically.

Suppose a chromatogram shows the solvent front has travelled 8.0 cm from the baseline, and a particular substance spot has travelled 5.2 cm from the baseline.

Using the formula: \nRf = distance travelled by substance / distance travelled by solvent front\nRf = 5.2 cm / 8.0 cm = 0.65

This means the substance travelled 65% of the distance the solvent travelled under these conditions.

• Always include units in measurements but the Rf value itself is unitless.
• Rf values are always less than or equal to 1 because the substance cannot travel further than the solvent front.
• Common error: measuring distances from different baselines or to the edge of the spot instead of the centre.

Rf values are useful for identifying substances by comparing experimental values with known reference values obtained under the same conditions.

However, Rf values depend on experimental factors such as solvent type, temperature, and paper type. Therefore, Rf values are only comparable when these conditions are consistent.

Different substances have characteristic Rf values, but some substances may have similar Rf values, so chromatography is often combined with other analytical techniques for confirmation.

• Rf values provide a fingerprint for substances in chromatography.
• Consistency in experimental conditions is essential for reliable comparison.
• Rf values alone do not confirm identity; they support other evidence.