Paper Chromatography: Principles, Applications, and Calculations

Exam Tip: Always remember that the solvent must be below the starting line. If the spots are submerged, they will wash away into the solvent reservoir instead of traveling up the paper.

Separating Coloured and Colourless Substances

Chromatography can be used to separate two different types of mixtures depending on whether the components are visible to the naked eye.

Soluble Coloured Substances

If the mixture consists of soluble coloured substances (such as food dyes or plant pigments), the components will appear as distinct coloured spots at different heights on the paper once the process is complete.

Soluble Colourless Substances

Some substances, such as amino acids from proteins or sugars from carbohydrates, are colourless. These will not be visible on the paper even after the solvent has moved. To see these, a locating agent must be used. After the chromatogram has dried, it is sprayed with this agent, which reacts with the invisible spots to turn them into visible coloured patches.

Interpreting Results: Purity and Identification

Once the chromatogram is developed, you can interpret the results to learn about the nature of your sample.

• Testing Purity: The number of spots indicates how many substances are in the mixture. A pure substance will produce only one single spot. An impure substance (a mixture) will produce two or more spots.
• Identifying Components: To identify an unknown substance, you should run "known" reference substances on the same piece of paper at the same time. If a spot from your unknown sample travels the exact same distance as a spot from a known reference, they are likely the same substance.

Calculating $R_f$ Values

To provide a mathematical basis for identification, scientists use the $R_f$ value (Retention Factor). This is a ratio that describes how far a substance traveled compared to how far the solvent traveled.

The formula for calculating the $R_f$ value is:

$R_f = \frac{\text{distance travelled by substance}}{\text{distance travelled by solvent}}$

When performing this calculation, both distances must be measured from the original pencil baseline to the center of the substance spot and to the solvent front, respectively.

Important Characteristics of $R_f$:

• It has no units because it is a ratio of two lengths.
• The value is always less than 1.

Worked Example: If a spot moves $3.6\text{ cm}$ from the baseline and the solvent front moves $8.0\text{ cm}$ from the baseline, the calculation is:

$\begin{aligned} R_f &= \frac{3.6}{8.0} \\ R_f &= 0.45 \end{aligned}$

Practical Application: Chlorophyll Extraction

A common way to observe chromatography in action is by extracting pigments from green leaves.

1. Crushing: Grind small pieces of green leaves with sand to increase friction and help release the pigments.
2. Extraction: Add ethanol to dissolve the chlorophyll, then filter the mixture to remove solid leaf particles.
3. Concentration: Gently heat the solution over a water bath to concentrate it.
   • Safety Note: Ethanol is flammable; never use an open flame.
4. Analysis: Run the concentrated solution on chromatography paper. Typically, you will observe two distinct spots, revealing that chlorophyll is actually composed of different pigment components.

Beyond the classroom, chromatography is vital in medical laboratories for analyzing biological samples like blood and urine to detect drugs, hormones, or other chemical compounds.