Coffee Filter Chromatography of Plant Pigments
Chlorophyll, Carotenoids Flavanoids Revealed by Paper Chromatography
Oct 4, 2009
Plant pigments, chlorophylls, carotenoids, xanthophylls and flavanoids, are useful for simple, paper chromatography experiments. Leaves, flower petals, fruits (berries) and vegetables (beets, squash) are good pigment sources for scientific inquiry and chromatography.
Principles of Extraction of Plant Pigments for Paper Chromatography
The principles of chromatography are helpful to review before and after this experiment.
Leaf pigments are fat-soluble molecules, embedded in chloroplast membrane lipids, and include: two main chlorophyll molecules (a and b), and one or two types of carotenoids.
These pigments are extractable with fat-solubilizing solvents such as isopropanol (rubbing alcohol). Caution and adult-supervision are necessary since solvents can irritate the skin, eyes, or respiratory tract. Typically, pigment extraction requires a well-ventilated room, or chemistry fume hood. Instructors should exercise these cautions and precautions and issue clear instructions about all aspects of the experiment.
Chlorophyll Pigment Extraction onto Filter Paper
Plant pigments may be extracted from leaves (or, flowers) by:
- crushing the plant tissue directly onto the filter paper. Use the edge of a plastic knife or spoon. Take care not to cut the paper.
- crushing small cut leaf or flower petals with mortar and pestle in minimal solvent, or
- cutting plant tissue into tiny pieces in a glass or jar and add a small amount of isopropanol solvent and crush tissue with an ice cream stick or related object . Use as little solvent as possible, for best pigment concentration. Safety goggles are recommended. Crushed leaves and pigment may be filtered through a funnel-shaped coffee filter to obtain tissue-free extracts.
Preparation of Paper Chromatography Filters and Solvents, Materials and Methods
Check all photos below for aspects of this experiment. Coffee filters may be used whole, halved or quartered, for economy and efficiency.
Materials needed:
- coffee filters halved, quartered or whole.
- wood applicators or pencil pointed ends for extract applications. Plastic dinner knife for direct, gentle, pressured application onto the filter paper.
- clear container (glass jar or bowl) to hold filters for the experiment.
- clear stretch wrap to cover the chromatography container.
Methodology:
- Apply pigment to narrow, defined spots or lines, by one of three methods indicated. Non-solvent, direct application may work – try it first! When using solvent extracts, allow to air dry before applying next drop. A hair dryer permits multiple layer applications to narrow defined places on filter paper.
- Use only a small amount of solvent (isopropanol, or nail polish remover) in the container to just cover the bottom and wet the paper.
- Next, insert pigment-impregnated filter paper in a upright manner, and not touching the outer walls. The walls of the container should not be wet. The paper may be taped to a pencil and the pencil may be placed across the top of the jar
- Cover the jar with clear, flex wrap, sealing film.
- Incubate for a minimum of 35-45 minutes. Some specimens may be permitted to run longer if a class or instruction period must be stopped.
Chlorophyll, Carotenoid and Flavoprotein Results, Detection and Separation by Paper Chromatography
Air dry the paper at the end of the run. Observe chlorophyll and carotenoid bands. Carotenoids typically move faster, and are found above chlorophyll. Depending on the length of the filter paper 1 or 2 bands of chlorophyll should be obvious, and 1 or 2 bands of carotenoids.
The Knockout Rose red pigment migrated quickly as a single band in either solvent as shown in the photo below.
Paper chromatography is a useful, research tool to test and discover the properties of compounds. Other compounds can be detected using spray detector reagents that can reveal amino acids, nucleic acids, carbohydrates and lipids, drugs and other compounds.
Resources
Bioinstrumentation, Tools for Understanding Life. 1996. Wandersee, J.H., et al. eds. National Assoc. Biol. Teachers, Reston, Va. 329 pp
Paper Chromatography. 1976. 15th ed., Micropaedia of Encyclopedia Brittanica. Chicago, Illinois
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