7.11.5 Interpreting Chromatograms

Interpreting Chromatograms in Thin Layer Chromatography (TLC)

When analysing TLC chromatograms, the movement of different components up the plate provides insights into their polarity and interactions with the mobile and stationary phases.

Key Observations on Polarity and Movement

1. Less Polar Components:

• Position: These components travel further up the TLC plate, away from the baseline.
• Rf Values: They have higher Rf values due to their greater movement with the solvent.
• Solubility: Less polar molecules are more soluble in the mobile phase (solvent) and are carried forward as the solvent rises up the plate.

2. More Polar Components:

• Position: These components stay closer to the baseline, not moving far up the plate.
• Attraction to Stationary Phase: Polar molecules are more strongly attracted to the stationary phase (often a polar substance like silica or alumina), which limits their movement.
• Rf Values: As they travel less, their Rf values are lower compared to less polar components.

Separation and Solubility

• The extent of separation in a TLC experiment depends on the relative solubilities of the components in the mobile and stationary phases.
  • Components more soluble in the mobile phase travel further.
  • Components more strongly attracted to the stationary phase travel less.

Using Rf Values to Analyze Polarity

• Rf (Retention Factor): The Rf value of a compound in TLC is calculated as:

$${Rf} = \frac{\text{Distance travelled by the component}}{\text{Distance travelled by the solvent front}}$$

• By comparing the Rf values of various compounds, you can infer their relative polarities. Lower Rf values generally indicate higher polarity (more attraction to the stationary phase), while higher Rf values indicate lower polarity (more affinity for the mobile phase).

Table of Amino Acids and $R_f$ Values in TLC Analysis

Amino Acid	$R_f$ Value
Alanine	0.33
Aspartic acid	0.24
Valine	0.44
Leucine	0.61
Cysteine	0.37
Isoleucine	0.53

This analysis allows for the identification of amino acids in the sample based on their $R_f$ values.