Chromatography Experiment

TLC_black_inkChromatography is a process used for separating mixtures by differentiating between absorbency levels. Chromatography is important because it separates different solvents by their molar masses. One can separate the different pigments within a solution and measure how much of each pigment is present. When a solvent diffuses, the solvent carries the dissolved substance along with it. The further the solvent diffuses, the slower the solvent goes. The larger molecules fall out first, meaning that they are the lowest molecules on the paper, then the smaller molecules fall out towards the middle, and the smallest fall out at the top. The different dissolved materials sort out as different stripes or peaks and the banding can identify the material dissolved in the solution. In paper chromatography there is what is known as the stationary phase which is the absorbent Chromatography paper and the mobile phase which is a liquid solvent (or mixture of solvents) used to carry the sample solutes under analysis along the paper. Usually, leavesChromatography is used to find out the components of a sample which are seperated depending how much soluble these are in particular solvents and hence how far they travel along the chromatography paper. Chromatography paper allows one to separate different organic compounds from a mixture that changes its solubility as well as the size of the molecules and the retention by the paper being used to separate the compounds. The solvent moves the pigments up the chromatography paper using capillary action so that the pigments can be separated. The Rf value stands for the Retention value. The Retention value of particular compound will travel the same distance along the stationary phase by a specific solvent given that other experimental conditions are kept constant.  Rf values are important for identification because one can compare Rf values of the unknown sample (or its consituents) with Rf Values of known compounds. Each substance has a specific Rf value, and is useful for scientists to identify them using their unique Rf values. This allows us to study what is contained in a mixture.

Rf = (distance of pigment)/(distance of solvent)

The  Dunknown is the distance of pigment from where the original pigment was spread to the top of the chromatography paper where the pigment ends. The Dsolvent is the distance of solvent from the bottom of the chromatography paper where the paper dips into the solvent to the top of the chromatography paper where the solvent has stopped traveling upwards.

big roundWe were able to observe three different pigments from the green leaf (labeled big round above); these included a mustard yellow/orange color, then a lighter green/yellow color immediately followed by a darker green color.

 floppy redWe also observed three different pigments from the red leaf chromatogram (labeled floppy red above). The original pigment was a dark purple-ish red, followed by a lighter yellow/green immediately followed by a dark green pigment at the very top.

From this Chromatography experiment I learned that there a wide variety of pigments in leaves, as well as other plants and species in general. Although we can only see three different pigments in each experiments documented, they are an infinite range that are blended with each other to create the broader, vivid colors we see on the chromatography paper. After completing this experiment, I began to elaborate on my main question about photosynthesis; “why is green the primary pigment in photosynthetic organisms?” I am still curious about the answer of this question, since the chromatography experiment supported the hypothesis that even red or purple leaves with no hints of green still include green as a main pigment.

For more cool information on chromatography check out this link!


1 Comment

  1. NickSchnabel

    Being a student in AP chemistry, learning about chromatography is very important and we use it for the separation of mixtures. It was very interesting to see how chromatography is used in an experiment for AP Biology. This experiment was very informative and will help me as I use chromatography in my chemistry class.

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