Plant Pigments and Photosynthesis Lab

PURPOSE

4A
The purpose of this experiment was to use chromatography to separate and identify the various pigments in chlorophyll.

4B
The purpose of this experiment was to determine the change in the rate of photosynthesis of chloroplasts when boiled or removed from light by using DPIP to determine the chloroplasts' electron output.

INTRODUCTIONS

4A Paper chromatography separates pigments by dissolving them in a solvent that moves up a strip of paper by capillary action. Different pigments are carried different distances by the solvent because of their varying solubility and attraction to the paper. Pigments like beta carotene that are more soluble and less attracted to the paper will be carried further than less soluble, more attractive pigments like xanthophyll. The distance traveled by the pigments is called Rf and is always the same for a certain pigment. It can be calculated as the quotient of the distance the pigment moved divided by the distant the solvent front moved. There are many light-harvesting pigments in chloroplasts. Chlorophyll a is the main photosynthetic pigment, absorbing blue-violet and red light best. Chlorophyll b and carotenoids are accessory pigments that are used to extend the plant's light absorption and to protect the chlorophyll a from too much harmful high-frequency light. 4B
Chloroplasts are found in plants and absorb light energy to "excite" electrons removed from water to produce ATP and NADPH, which are then used to fixate carbon into sugars in the Calvin cycle. However, the high-energy electrons produced that reduce NADP+ to NADPH can also reduce other electron acceptors, like the dye DPIP. DPIP is blue before reduction, but becomes colorless as it is reduced. DPIP, when blue, only transmits blue light, while colorless substances transmit all wavelengths of light on the visible spectrum, so as DPIP becomes colorless, it transmits more and more light. This transmittance can be measured using spectrophotometer.

METHODS

4A

Using a quarter to scratch off pigment off a spinach leave and placed that on a pencil line on the paper.  We took the chromotography paper and placed it into a cylinder with solvent. We waited time to see the pigments spread out over the paper. We mark where the bottom of the pigment band is. We measured the distance the pigment band moved. 

4B

In this experiment we set up a flood light, heat sink, and cuvettes. There were boiled and unboiled chloroplasts. We had five cuvettes the first was our control group. We put phosphate buffer and Distilled water to test tubes then DPIP to certain test tubes. We transferred the solutions to the correct cuvettes then added chloroplast to the cuvettes.  Immediately after adding the necessary chloroplasts we placed them behind the heat sink. We waited to place each in the colorimeter to read the rate of photosynthesis. Then tracked it on the logger pro. After taking them out of the colorimeter the cuvettes were placed behind the heat sink again.

GRAPHS

4A:

4B:

DISCUSSIONS

4A

From our trail, it appeared that the spinach sample separated into five different pigments. Due to the capillary action habits of plant pigments discussed in the introduction, we can guess that the top segment outlined in the corresponding picture above is beta carotene and the lowest segment is possibly xanthopyll. 

4B

To be completely honest, our experiment ended up being a complete flop due to incorrect usage of the colorimeter as can be even seen by our rather pointless data. However, I would like to ask the reader to focus on line 3 in Table 4.4 above. After realizing the mistake that had been made, we added a few more drops of DPIP into the cuvette containing the unboiled chlorophyl that was exposed to light. As can be seen from this set of data, the longer it was exposed to the light, the more transmittance was detected. This shows that over time, the DPIP was being used up in photosynthetic like reactions at a moderate rate. However, we don't have any other reliable data to compare it with but we can assume that transmittance in this cuvette was increasing at a faster rate than cuvette #2 yet slower than the boiled samples. 

CONCLUSION

4A 

From our trial, chlorophyll divided into five pigments.

4B

...I don't know what to say other than if time had been permitted we would have probably redone the experiment.