Chromatography Lab

Chromatography Lab

Purpose

In the process of photosynthesis, a producer’s pigment(s) in their chloroplasts determine what wavelengths of light it can use to produce sugars. Depending on the pigment or pigments’ absorption spectrum/spectra, the usable light wavelengths will differ - some will be totally absorbed by the pigment, making them usable for photosynthesis, while others are reflected and cannot be used. In this experiment, we use the process of chromatography to separate the various pigments within spinach and compare the variety of colors of the pigments that broaden the plant’s absorption spectrum. The pigments will all travel different distances up the chromatography paper, so we compare pigments using Rf values that are calculated using the individual pigment’s traveled distance and the overall distance traveled by the solvent.

Introduction

Chromatography is a common lab procedure used to determine the pigments present in a liquid mixture of molecules. The results of chromatography changes depending on the liquid being tested, but they appear similarly; bands of color are seen on chromatography paper as pigments are pulled up by capillary action, the ability of liquids to be pulled up within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The distance each color travels up the paper from the surface of the liquid depends on the polarity of the pigment molecules. When using a non-polar solvent, pigment molecules that are polar will not travel as high up the polar cellulose of the paper because there is a stronger attraction between the two materials. These molecules are more resistant to being pushed up the paper.

  In the context of this lab, it is also important to examine the pigments that commonly compose plant material. The most common pigments in plants are chlorophyll a, chlorophyll b, and carotene. The distance each pigment travels on the chromatography paper depends on its ability to form hydrogen bonds with the chromatography paper. Carotene is known to have weaker polar molecules, and so it is more likely to be moved along with the solvent up the length of the paper. Chlorophyll a and chlorophyll b have polar molecules that are more attracted to the chromatography paper.

Methods

In order to separate and identify the different pigments in a spinach leaf using paper chromatography, a coin was used to crush spinach leaf cells onto a piece of chromatography paper in one area well above where the point of the paper reaches the solvent. The intended center of the sample was marked with a blue line from a grease pencil.

With the cork in place, a hook running through the top of the paper, and the tip of the paper submerged, time was allowed for the solvent to make its way up to the top of the paper and leave 4 different pigment bands. The bands of colors seen were a dark green, a bluish green, a light yellow, and a dark yellow.

The peak of each band was marked in order for distinct separations between them to be seen. This allowed measurements to be taken more easily. The location of the solvent front from which all measurements had to be made was also marked.

Data

Discussion

The bands had distinct colors because as the solvent moves up the paper, the pigments go with it but at different rates because each has a different degree of attraction to the polar paper fibers. This result might also lend to the solubility of the pigments in the solvent; carotene is more soluble in the solvent that the other pigments, and thus, it is the first yellow color seen and it moves with the solvent up the length of the paper. The calculated Rf-factor, or the relationship of the distance moved by a pigment to the distance moved by the solvent, is .93 for this pigment, which is significantly larger than that of the three other pigments. Xanthophyll specifically does not travel as far as carotene because it is contains hydrogen bonds that have oxygen. The Rf-factor for this was .484. It corresponded with a darker yellow color. The two types of chlorophyll, a and b, had Rf-factors of .446 and .376, respectively. These bands of olive green and blue-green, respectively, had the smallest distance between them.

Conclusion

The calculated Rf values of the plant pigments within the spinach range differ significantly because the pigments possess different molecular characteristics. The pigments with the higher Rf values were more attracted to the mobile phase (the solvent), while the lower-Rf pigments were more attracted to the stationary phase (the chromatography paper). The spot of spinach at the pigment front was larger than it should've been, so the pigments started from slightly below the marked line. Despite this, our end data was not significantly skewed.

References

• http://www.chemguide.co.uk/analysis/chromatography/paper.html
• http://water.usgs.gov/edu/capillaryaction.html
• The procedures, graphs, and charts used here were taken from "Lab Four: Plant Pigments and Photosynthesis" by CollegeBoard.