Chromatography/Cellular Respiration Lab

Chromotagraphy

Purpose: The purpose of this lab is to see the separation of plant pigments using chromatography. We tested this to be able to calculate the Rf values. We also wanted to measure the separation of the colors which represented the different kind of chlorophyll which were present in the spinach leaf. 

Introduction: Chromatography is the technique of separating and identifying pigments that contain a mixture of molecules. Adhesion is when dissimilar molecules stick to each other. Cohesion is similar to adhesion in the sense that the same molecules stick to each other.  The higher the color, the more adhesive properties it has. Solvents goes up through capillary action through which it carries any substance dissolved in it. Present inside the spinach we tested were different types of chlorophyll that emerged when we submerged in the solvent.  Carotene protects it from ultra-violet rays. Chlorophyll A absorbs most energy from wavelengths of violet-blue and orange-red light. Chlorophyll B absorbs light energy and blue light. Xanthophylls are oxygenated carotenoids that are synthesized within the plastids.

Methods: In this lab, we took a 12 cm strip of filter paper and cut it to a point at the tip. We filled a graduated cylinder with 1 cm of solvent. We then had to cut a piece of the paper and mark a line 1.5 centimeters from the bottom. We took a portion of spinach and (using a coin) extracted spinach pigment by smearing it on the paper. The smeared spinach pigment was done above the 1.5 cm line making sure it was secured with a stopper. We then barley submerged the tip of the filter paper into the solvent, enough so that the point of the paper took in solvent but not enough so that the solvent wouldn't make contact with the smeared spinach pigment. We waited for half an hour and marked the location of the solvent front before it evaporated. We marked the bottom of each band measured the distance. 

Data:

Discussion: For the light green to the pale yellow pigment, there was a consistent rate of distance between each band. Band 1 came in at 45 cm, band 2 was a distance of 48 cm, band 3 was 63 cm, and band 4 was 65 cm. band 5 varied the most in distance, measuring almost equidistant to the combined distances of bands 1-4. Band 5 measured at 96 cm. Band 1 and 3 were identified as chlorophyll a. Band 2 was identified as chlorophyll b. Band 4 was identified as xanthrophyll and band 5 was carotene. There was reasoning for as to why the different colored bands  came in the order that they did. Because chlorophyll a and b and xanthophyll were located near the bottom of the filter paper, we can safely say that it's adhesive properties are weaker than the band of carotene that was located higher up the strip of paper. This means that chlorophyll a and b along with xanthophyll cannot grab onto other surfaces or substances as well as carotene. Xanthophyll specifically is also less soluble and is slowed down by hydrogen bonding to the paper strip. We can also infer that their cohesive properties that allow their similar molecules to stick to one another are likely also as weak as their poor cohesive abilities. The band measurements did come out to match the predicted results and band order than we'd initially assumed meaning that the experiment results were successfully accurate. The experimental procedure as a whole was mostly successful. 

Conclusion:This experiment tested the relationship of the distance moved by a pigment to the distance moved by the solvent which was referred to as Rf. We used this calculation to test our hypothesis which is supported by our data table. We found out that different pigments migrated at different rates due to cohesion  and adhesion ability. The ones that traveled higher up the paper had greater adhesion property (Chlorophyll A, B, and xanthrophyll). We could have improved the experiment by leaving the strip submerged longer so that we may see more bands appear and this would have given us more results to give added accuracy. Also, more trials could help us compare our results and make sure the trends are consistent. 

Cellular Respiration

Purpose: The purpose of this lab was to test to see if light and chloroplast are required for light reactions to occur. This allowed us to see the effects of light and light deprivation. 

Introduction: When light is absorbed by leaf pigments, electrons are boosted to a higher energy level. Then ATP is produced. For this to happen, there needs to be a reduction of NADP to NADPH. However, in this experiment, DPIP is used as a substitute for NADP (electron accepter). When light strikes the chloroplast, the electrons are excited and changes the DPIP from blue to colorless. Then, ATP and NADPH combine with CO2 into organic molecules (carbon fixation). 

Methods: While the spectrophotometer was warming up, we set up an incubation area with a water flask in front of a light and test tube rack. We were provided with 2 bottles: one with boiled and one with unboiled chloroplast which were kept in ice the whole time. Different additions of phosphate buffer and distilled water were added to each test tube. We also added DPIP to test tubes 2-5. We put cuvette 1 (the control group) into the spectrophotometer and calibrated the lab quest. This allowed us to have a comparison to the other cuvette. However, cuvette 2 was covered in foil to keep light out. We added 3 drops of chloroplast to test tubes1-4 and immediately took a transmittance reading from the spectrophotometer. Then we put them in the light behind the flask and recorded a new transmittance reading every 5 minutes for 20 minutes. Additionally, the second attempt at the experiment was meant to allow us to come up with a way to show a more steady increase in our graphical data instead of the spikes in data we had acquired in our first attempt. 

Data:

Attempt 1 Graph:

Attempt 2 Graph:

Discussion: Our control group for this experiment was our cuvette the that was unsoiled and exposed to light because this sample would be the only beaker that would record and show the normal light transmittance that a common chloroplast would have. (Cuvette 1 was used to calibrate our spectrophotometer, so it is not in our data table, meaning our data results would begin with cuvette 2). The trends we found in the second attempt were that cuvettes 1,2, and 4 increased in transmittance percentage as time went on and decreased in absorbance. However, cuvette 3 decreased in transmittance percentage as time went on and increased in absorbance. This shows that regardless of light transmittance increasing or decreasing, there was an inverse relation with the absorbance of light. One thing that we found inconsistent with the collected data was that cuvette 4, which contained no chloroplast at all, showed an increase then decrease in light transmittance and an increase then decrease in light absorbance. What is boggling about this is that, because this cuvette did not contain any chloroplast, there really shouldn't have been any absorbance or transmittance of any kind because there was no medium for it light to travel through. The first attempt was littered with errors. We calibrated correctly, but we added the chloroplast directly to the cuvettes instead of the test tubes. We also waited too long to put the cuvettes in front of the light causing a delayed reaction. All the cuvettes we're not put it together which didn't maximize the time they could've all spent behind the light. This limited time resulted in only being able to perform two trials whereas there should've been four. However, the one of the trials did not fulfill the required time. The second attempt was a lot more successful because we completed all four timed runs for the required 5 minutes each, added the chloroplast to the test tubes, and instead of adding the chloroplast directly into the four cuvettes, we added the chloroplast into the beaker so that the chloroplast would distill in with the other substances in its respectable beaker instead of possibly overpowering the solution before when the chloroplast was added directly into its cuvette. This proved successful because after we distilled the chloroplast into the beaker as opposed to adding straight to the cuvette, the graphical data increased steadily and did not spike. Our data did not support our hypothesis because cuvette 3 decreased then increased when it should've done the opposite. 

Conclusion: This experiment tested how chloroplasts in different situations absorb and transmits light.  By using the blank cuvette with water to calibrate the spectrophotometer, we were able to individually calculate the amount of transmittance in each cuvette. Since cuvette 2, unboiled and light (originally in test tube 3) acted as the control group, we were able to compare the others to that cuvette. By looking at our data tables, we were able to conclude that transmittance and absorbance acted as reciprocals to one another. As transmittance went up, absorbance went down and vice versa.  After attempt one, we realized we needed to decrease the amount of chloroplast used so the graph would not spike up.  As a result of this, in attempt two we put the chloroplast solution directly into the test tubes instead of the cuvettes. This made our results more accurate and helped prove our hypothesis.