Establishing Purity using Chromatography for the ESAT

The Principles of Chromatography

Chromatography is a physical method of separation where components of a mixture are distributed between two phases: a stationary phase and a mobile phase. The stationary phase (such as chromatography paper or a thin layer of silica on a plate) remains fixed, while the mobile phase (a solvent) moves through it.

Substances separate because they have different levels of attraction to the stationary phase and different solubilities in the mobile phase. A component with a high affinity for the mobile phase will travel further up the plate, whereas a component with a higher affinity for the stationary phase will move more slowly and stay closer to the origin.

Assessing Purity and Identity

The most direct way to establish purity using chromatography is to examine the number of spots produced by a sample.

1. Pure Substances: A pure substance consists of only one type of element or compound. When subjected to chromatography, it will produce exactly one spot on the chromatogram, regardless of the solvent used, provided the substance is soluble.
2. Impure Substances: An impure substance is a mixture. Chromatography will separate the different components of the mixture into two or more distinct spots at different heights from the baseline.

To confirm the identity of a substance and its purity, chemists compare the position of the sample's spot with the positions of known pure reference standards run on the same plate under identical conditions.

The Chromatography Procedure

To ensure accurate results when checking for purity, the experimental setup must follow specific protocols:

• The Baseline: A starting line is drawn in pencil near the bottom of the stationary phase. Pencil is used because graphite is insoluble and will not dissolve or travel with the solvent. If ink were used, the dyes in the ink would separate and interfere with the sample results.
• Sample Application: Small, concentrated spots of the sample are placed on the pencil baseline.
• Solvent Level: The paper or plate is placed in a beaker containing the solvent. The depth of the solvent must be below the pencil baseline. If the solvent level is too high, the samples will wash off the paper into the bulk solvent rather than travelling up the stationary phase.
• The Solvent Front: The experiment is stopped before the solvent reaches the very top of the paper. The furthest point reached by the solvent is immediately marked with a pencil: this is the solvent front.

Calculating Retardation Factor ($R_f$) Values

The Retardation Factor ($R_f$) is a quantitative measure used to identify substances. It is a ratio and therefore has no units. The value always falls between 0 and 1. It is calculated using the following formula:

$R_f = \frac{\text{distance moved by the substance}}{\text{distance moved by the solvent front}}$

Both distances must be measured from the pencil baseline to the centre of the spot. Because a specific substance will always have the same $R_f$ value in a specific solvent and stationary phase, these values can be compared against databases to establish the identity of a pure sample.

Worked Example: Testing a Drug Sample for Purity

A student is testing a sample of aspirin to check for impurities. They perform thin layer chromatography (TLC) and obtain the following measurements:

• Distance from the baseline to the solvent front: $12.0\text{ cm}$.
• The sample produces two spots.
• Spot A travelled $4.8\text{ cm}$ from the baseline.
• Spot B travelled $9.6\text{ cm}$ from the baseline.

Step 1: Determine the purity. Because the sample produced two distinct spots (A and B), it is not pure. It is a mixture containing at least two different substances.

Step 2: Calculate the $R_f$ values. For Spot A: $R_f = \frac{4.8}{12.0} = 0.40$ For Spot B: $R_f = \frac{9.6}{12.0} = 0.80$

Step 3: Identify the substances. If pure aspirin is known to have an $R_f$ value of $0.80$ under these specific conditions, the student can conclude that Spot B is aspirin and Spot A represents an impurity in the sample.