Be sure to use 95 percent EtOH and not drugstore isopropyl 70 percent , though some drugstores carry 91 percent EtOH, which will work.
I just dip them in boiling water for about one minute Tear them into pieces and place in methanol or isopropyl or whatever organic solvent is cheap and available. The spectrophotometer measures the percentage of light transmittance through the cuvette due to DPIP reduction. It is not necessary to be 'very exact'—the setting could be nm or nm but, again, it just makes things easier if you make an effort to set it at the best wavelength when you begin. I would not, however, add any food coloring.
If the sucrose solution isn't absolutely cold, the heat of the blender will deactivate the chloroplasts.
Place cuvette 1 in the sample holder of the spectrophotometer. All Rights Reserved Figure 1! Chlorophyll's contain oxygen and nitrogen and are bound more tightly to the paper than the other pigments. In the dye-reduction technique, the compound DPIP 2,6-dichlorophenol-indophenol is substituted for NADP nicotinamide adenine dinucleotide phosphate , the primary electronaccepting compound of photosynthesis. But it just makes the whole procedure so much easier if you choose the best wavelength to begin with. I have never seen a blender motor that did not make sparks.
Carotenoids absorb light in the blue and blue-green regions. If unboiled chloroplasts are not in the blank cuvette, all the other readings will be nonsense. Be sure to cover and mix the cuvette before each reading. I am a little unsure what you mean about calibrating with water versus ethanol.
Once the strip has dried, mark the middle of each pigment band on your chromatography strip with a pencil. This wavelength is nm. The cylinder is tightly stopped because this solvent is volatile, be careful to keep the stopper on as much as possible. If you choose to use the prepared buffer and DPIP solutions available for order from Carolina, you just need to keep the ratio of drops or mL. I would not, however, add any food coloring.
If each student in a class of 20 makes a chromatogram, there is plenty of pigment to extract. The preset wavelengths can be used for a variety of labs, including bacterial growth by turbidity that I recently worked on. Extract the chlorophyll from any leaves. Now, one political tip: If after reading all the postings and you're interested in both, you could get the expensive specs now while the money is available, and gradually get the less expensive colorimeters over a period of years if you want them for other uses.
What are some other ways chromatography is used to separate plant pigments? I had each group of three students do a separate part of the lab and plan an assembly line.
I already had familiarity with their Serial Box interface and the probes are very reliable. Another good thing is that it only takes a couple of leaves for the entire class. Boiling denatures the protein molecules and stops the reduction. Acetone is also very volatile, so if you are using acetone, you almost certainly have acetone fumes in the area.
Refer to the MSDS for proper disposal of chromatography solvent. Chlorophyll a occurs in all photosynthetic eukaryotes and in prokaryotic blue-green algae. Of course, in making this choice, you must pick a wavelength that is strongly absorbed by reduced DCPIP. Putting the chlorophyll in front of the overhead or any other strong white light will also work, but the effect is not as striking as the black-light version. You can buy about colorimeters that work with graphing calculators and data collection devices CBLs, etc.