Pigment Chromatography Lab

In this laboratory, the degree of absorbance for the pigments in a leaf sample were observed using mechanisms that involved pigment isolation from a leaf extract, obtaining wavelength measurements, and displaying the measurements on an absorption spectra.

Separation of Pigments by Paper Chromatography

The pigment line of the sample leaf was extracted by repeatedly rolling a coin along a ruler edge that held the leaf 1.5-2cm from the bottom of Whatman #1 chromatography paper. Subsequently, a saturated environment was created to ensure that the solvent was separated by placing the beaker containing the rolled Whatman paper with the sample line on the outside into a mason jar containing the separation solvent, and sealing both compartments.

After 15-40 minutes, the solvent front reached 2.5-3cm of the top of the cylinder, thus displaying many pigment bands. The solvent front was then marked to determine the RF (relative to front).

Absorption Spectrum of Each Pigment
Once the chromatography paper was dry, the pigment bands were cut out, and another leaf sample extraction line was drawn on the bottom 2cm of the chromatography paper, following the same coin and ruler method initially described.

The cutouts were then placed into large test tubes containing 4ml of isopropyl alcohol for each pigment band, total pigment sample 1, and total pigment sample 2. They were then sealed, until the pigments from the paper transferred onto the isopropyl alcohol. The same amounts of smaller test tubes were obtained, plus an additional small test tube, which was filled with isopropyl alcohol and acted as a blank. The eluted pigment solution lacking the paper was transferred into the rest of their respective smaller test tubes.

Using the spectrophotometer, the absorption of each sample was measured by scanning the wavelengths. After calibrating the spectrophotometer with the blank test tube, each sample was placed into the spectrophotometer and read at 360nm. Observations were continued for each pigment sample increasing the wavelength by 20nm increments. Once these absorbance values were recorded, absorption spectra for each pigment were graphed.

Measuring the RF

The distance of the initial extract line to a pigment band was divided by the distance of the marked solvent front to the initial extract line both were measured in cm. The RF (relative to front) was calculated for each pigment band, indicating the travelled distance between the pigment and the front (solvent line) on the chromatography

paper.