Experiment Overview

Chromatography is a separation technique used to separate the components of a mixture based on their different affinities towards a stationary phase and a mobile phase.

What This Experiment Demonstrates

• Separation of mixture components
• Distribution of substances between two phases
• Identification of substances using retention factors

Apparatus Required

• Chromatography paper or TLC plate
• Capillary tube
• Beaker or chromatography chamber
• Watch glass or lid
• Pencil and ruler

Chemicals Required

• Sample mixture (e.g. ink, dyes, amino acids)
• Solvent (mobile phase)
• Distilled water (if required)

Theory

In chromatography, we often talk about mobile and stationary phases. The stationary phase is an absorbant solid (silica on glass, chromatography paper etc.), while the mobile phase is a suitable solvent/mixture of solvents.

Chromatography works on the principle that different substances travel at different rates depending on their relative attractions to the stationary and mobile phases.

Components that have a stronger attraction to the stationary phase move more slowly, while those with a greater affinity for the mobile phase move faster.

To determine the strength of the affinity of a substance, we use the concept of intermolecular forces. The stationary and mobile phases are chosen to have opposite polarities. For example, when we use very polar silica as the stationary phase, the mobile phase is a non-polar solvent, such as hexane or ethyl acetate. This way, polar compounds, will move slower, because they are "held back" by their affinity for the stationary silica, while non-polar compounds will move with the mobile phase.

The movement of a component is often expressed using the retention factor (\(R_f\) value), which helps in identifying substances.

\[R_f = \frac{\text{Distance travelled by substance}}{\text{Distance travelled by solvent front}}\]

Method

1. Draw a pencil baseline near the bottom of the chromatography paper/TLC plate.
2. Place a small spot of the sample mixture on the baseline, using the capillary.
3. Wash the capillary with a solvent, such as acetone, before making another spot.
4. Pour solvent into the chromatography chamber (can be a covered beaker, to saturate the atmosphere in solvent vapours).
5. Place the paper into the chamber without immersing the spot (the distance from the base of the chromatography paper/TLC plate to the baseline should be higher than the level of the solvent).
6. Allow the solvent to rise up the paper, by absorbtion.
7. Remove the paper when the solvent front is near the top.
8. Mark the solvent front immediately and allow the paper to dry.

Observations

The mixture separates into distinct spots at different heights. Each spot corresponds to a different component of the mixture.

Calculations

• Measure the distance travelled by each component.
• Measure the distance travelled by the solvent front.
• Calculate the \(R_f\) value for each component.

Observation: revealing spots

In chemistry we deal with a lot of colorless compounds. That is, of course a problem when trying to identify spots in the case of a chromatography. When dealing with colorless compounds, there is a number of ways to see the spots:

• Using a UV lamp: the TLC plate/chromatography paper is viewed under UV light. A lot of organic compounds, colorless to visible light, actually absorb UV light. While our sample is under the UV light, we can use a pencil to circle the spots, so that measurmenets can be done later.
• Revealing by reaction with a specific substance: some colorless substances react to give colorful compounds. After the paper/TLC plate has dried, we can spray it with a substance to reveal some of the spots. For example, phenlophtalein can be used to reveal relatively strong bases (or bases can be used to reveal phenlophtalein) or ninhydrin is used for revealing amino acids.

Result

The components of the mixture are separated and identified using their \(R_f\) values. Because \(R_f\) values are too dependent on conditions (exact mobile phase composition, temperature etc.), they are usually not tabulated. It is common practice to put reference spots from samples of known composition, to basically create our own reference \(R_f\) values.

Video Demonstration

Video courtesy of Professor Dave Explains (YouTube). Video used for educational purposes under YouTube’s embedding policy.