Isolation of Plant Pigments
Introduction The outward physical appearance of plants can be deceiving for what lies within. Our eyes respond to the wavelengths of light reflecting from the plant surface. The wavelengths which are absorbed vs. reflected are a function of the various pigments found within the plant cell. These pigments are concentrated in plastids (chloroplasts, chromoplasts, amyloplasts, etc.) and/or vacuoles and absorb different wavelengths (and thus energies) of light. The pigments represent a wide range of polarity from the chlorophylls and carotenoids which are very water insoluble (hydrophobic or nonpolar) and found embedded in membranes to the anthocyanins which are very water soluble (hydrophilic or polar). We can exploit these different chemical properties to extract and separate the pigments from each other by varying the solvents used with thin-layer chromatography (TLC).
Below are listed four solvents in increasing order of polarity that can be mixed in varying proportions in an attempt to separate chlorophylls, carotenoids and anthocyanins from a number of plant species and organs:
- Solvent
- hexanes
- 1-propanol
- acetone
- water
- hexanes
Experimental Background: Frequently, 80% acetone is used to
extract chlorophyll from plant samples and because this contains 20%
water, anthocyanins are also extracted during the process. When an
aliquot of this extract is spotted onto a silica TLC plate and allowed
to chromatogram in a solvent system of hexanes:acetone (60:40 v/v),
it is noted that the chlorophylls migrate up the chromatogram, but the
anthocyanins do not migrate from the origin. Your assignment is to
come up with the correct proportion of three different solvents (1-propanol:acetone:water) which will allow separation of all three
pigments (chlorophyll, carotenoids, anthocyanins). The one
requirement is that acetone must remain constant at 10% v/v in all of
your solvent combinations.
hexane:acetone (60:40 v/v)
Laboratory Procedures:
We have made up 8 combinations of 1-propanol (P), water (W) and 10% v/v acetone for your use. These solvents are labeled 1-8. These combinations of PAW (propanol:acetone:water) range from 80:10:10 to 10:10:80.
You will work in groups of three. One student from each group can proceed with putting 0.75 mls of each solvent in a test tube, and labeling with the appropriate solvent number. The other two students will proceed with preparing the samples. You will be extracting pigments from 3 sources, the "Margarite" sweet potato, spinach and the "Beta Sweet" carrot. Weigh out about 1 g of each of the 3 plant materials. For each one, place the tissue in a motor, add 10 ml 80% acetone, and grind thoroughly with a pestle.
Pipet about 1 ml each of your plant extracts into microfuge tubes. Place the tubes in the microfuge in a balanced position and centrifuge for 3 min. at maximum speed.
Pre-cut TLC strips will be provided. You will need 8 strips for each plant extract, for a total of 24 strips. Label each strip at the top with the solvent number and extract. Using a micropipette, apply a total of about 6 to 10 µl of your sample in 2 µl aliquots about 1 cm up from the bottom of your strip (your spot should not come in direct contact with the solvent in the tube). Wait between each 2 µl aliquot to allow the sample to dry before adding more. The object is to keep your spot small and not scratch the silica. The sample will come out of the tip by just touching it to the surface of the TLC strip.
BE SURE YOUR SPOT IS DRY BEFORE PLACING YOUR TLC STRIPS IN THE TUBES. Cover the tubes and allow the chromatogram to develop. This may take from 10 to 30 min. Immediate results, however, can be visually observed. After development, remove the TLC strips from the tubes and quickly mark the solvent front, origin, pigments.
Finally, observe your dried chromatograms under a UV light.
- Data from the lab of 17 February 2003
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1. Record your results by drawing and labeling your chromatograms.
Chromatogram outlines for recording data.
A. What solvent system worked best for separating all three pigments? Why?
2. Explain what you observed when you exposed your chromatogram to the UV light and why it appeared as it did?
3. Observe the absorption spectra of the 3 different plant extracts obtained with the scanning spectrophotometer. Determine the different peak wavelengths for your sample and speculate as to their origin. Explain what was meant by the first sentence of this report, "The outward physical appearance of plants can be deceiving for what lies within."