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.

Enzyme Catalyst Lab

Purpose:
The purpose of this lab to analyze how enzyme affects the conversion of hydrogen peroxide to water and oxygen gas. In other word, how does the enzyme affect the rate of hydrogen peroxide breaking down into water and oxygen. We wanted to test how much enzyme we need to really start affecting the solution to change. The independent variable was how much of the enzyme solution we put into the hydrogen peroxide solution. The dependent variable was how soon the hydrogen peroxide changed to pink/brown and how much enzyme is needed to do so.

Introduction:

This experiment is all about enzymes and how they react to different situations. So what exactly is an enzyme? An enzyme is a substance produced by a living organism that acts as a catalyst to bring about a specific biochemical reaction. A catalyst is something that affects the rate at which the chemical reaction takes place. In this experiment, we will deal with enzyme-catalyzed reactions which means that the substance to be acted upon, the substrate represented in an equation as S, binds to an active site of the enzyme, represented as E. One advantage of enzyme-catalyzed reactions is that it makes the reaction require less activation energy so that products (P) of the reaction form. This is expressed with the following equation:

E + S -> ES -> E + P

It is important to know that each enzyme is specific for a particular reaction because of the amino acid sequence, which has an effect on its structure. Active sites in an enzyme interact with the substrate so that any substrate that changes the shape or blocks the active site will have a completely different purpose/shape than it did before. How can this happen? Well, salt concentration, pH, temperature, and activations & Inhibitors are four ways that enzyme activity can be affected. Depending on the amount of salt concentration, the proteins in the enzyme will denature and in turn become inactive such as with low concentration. On the other hand, concentrations that are too high will cause blockage. Denaturing of proteins also applies to pH and temperature. When the pH & temperature is too high or too low, it causes enzymes to become inactive, since most enzymes perform optimally at about neutral pH and temperature. Lastly, activations and inhibitors regulate how fast the enzyme acts.

In this lab, the enzyme used was catalase. It takes part in some of the many oxidation reactions that occur in cells, but the primary reaction catalyzed by catalase is decomposing H2O2 to form water and oxygen as shown in the following equation:

2 H2O2 -> 2 H2O + O2 (GAS)

Without catalase, a reaction would still in fact occur, but at an obviously slower rate. 

Methods: 

We started out this experiment by putting 10 ml of H2O2  into 7 separate beakers. Each beaker was labeled with a number ranging from 10 to 360 seconds (depending on how much time we would have to swirl it). Then, we added 1 ml of the catalyst. Now we actually got to "mix" the H2O2 by slightly swirling it. We also set a timer to keep track of how long we needed to mix it. Once the timer hit zero, we quickly added 10 ml of H2SO4. Then we put it under the burette and added one drop at a time of the KMnO4. We waited until the the mixture turned permanently pink/brown . This allowed us to see how well the catalyst and enzymes work and how it varies depending on the amount if time it has been in the H2O2 mixture.

Data:

Graph:

Discussion:

For the lab we filled out a chart with our results. The charts explain how long it took our base line. The intial reading was what the tube was filled at before we used it and the final reading was what it was after the reading. We took the base line and added the enzyme and kept on stirring for 10, 30, 60, 90, 180, and 360 seconds. After the time is down we would check and check how much KMnO4 it takes for it to give it a pinkish color. Our results are valid because we compared our results with the class and our data is very close to other groups. In the graph the blue line shows the H2O2 used and the red line shows the KMnO4 consumed.The longer we waited on the H2O2 the more of it was being used in the chemical process and the longer we waited on the KMnO4, less of it was being released because all the enzymes in the chemical reaction are being used.

Initially, we anticipated that, with more time passing between our addition of catalase into our solution of H2O2, the quicker we would be able to see a presence of color indicating the efficiency of the reacting enzyme that was catalase. This proved to be correct as our data began to show a decrease of used catalase from our burette as we began mixing it with our beakers that had been swirled with catalase for a longer period of time.

For example, the beaker that we added and swirled catalase with for a period a six minutes only required .5 mL of KMnO4 to be added until a pink coloration was seen to indicate a reaction whereas our beaker that was swirled for a shortened period of only 10 seconds required a larger quantity of KMnO4 to be added, a difference of 3 mL to our six minute beaker's .5 mL, in order for a pink color hue was seen in the solution. This showed that the more time that catalase was given with the H2O2 solution, the quicker a visual reaction would occur with KMnO4.

Additionally, our collected data showed an inverse relationship with the amount of KMnO4 consumed versus the amount of H2O2 used. Our data showed that when looking at the amount of KMnO4 used (here we are going to describe the amounts in terms of first, the beakers that had a shorter period of time to mix with catalase on to those who had longer time) began high at 3 mL for our 10 second beaker and then dropped down to a smaller amount of .5 mL used, as seen with our 6 minute beaker. However, the amount of H2O2 that was used in our 10 second beaker, the one which had used 3 mL of KMnO4, only .5 mL of H2O2 was used. This inverse relationship between KMnO4 and H2O2 became more evident as we observed the dropping amount of KMnO4 used in regards to the raising amount of H2O2 used as we increased the the time given for catalase to react with our beakers. The 6 minute beaker of H2O2 ended up using .5 mL of KMnO4 to a higher 3 mL of H2O2 used. These two beakers were our extremes and more clearly showed the contrast in use of the two kinds of solutions used in this lab.

We have to acknowledge that for some of our beakers, the time we spent swirling in catalase might have differed by some few seconds or so by either some means of loss of time or distraction of some kind and that may have altered results. For future tests, we should keep close attention to the time spent swirling the catalase with our various beakers for that specified time the lab calls for.

Conclusion: 

Once all data was accumulated from the previous procedure, the results showed an inverse relationship with the amount of KMnO4 consumed and the amount of H2O2 used. As the time that passed increased, KMnO4 increased from its initial 3mL down to a final .5 mL whereas H2O2 decreased from its initial .5mL up to 3mL. This shows that the increased time spent adding H2O2 lowered the amount of substrate needed to catalyze a reaction when in use of KMnO4. The trials exemplified the very aspect of how a present enzyme of catalase sped up the reaction of our KMnO4 solution. As the time increased where our catalase was mixed in with our solution supported the anticipated result that the presence of color in the initial colorless solution would occur more quickly, which shows that less KMnO4 is need as the amount of H2O2 is used. Because the addition of KMnO4 was done by hand, the precise amount of minimal substance that could be added until a color change was seen could be off by a, minute but still present, amount.

Cellular Respiration Lab

Purpose:

The purpose of this experiment was to see whether germination and temperature affect the amount of CO2 released. We were testing to see if germination on (barley) seeds would affect their cellular respiration process and eventually cause them to not produce CO2. The independent variable was whether or not the seeds were germinated and if the temperature changed. The dependent variable was how much CO2 was released. We wanted to see how much of a difference there would be between the four groups we tested out.

Introduction:

The lab we were about to conduct dealt with the process of cell respiration. Cell respiration is the series of metabolic processes by which living cells produce energy through the oxidation of organic substances. This conversion is done to benefit organisms because it converts chemical energy into a form that can be readily used. This is shown through the following equation:

C6H12O6  +  6O2 →  6CO2  +  6 H2O  +  Energy (ATP)

It is a fact that all animals and plants go through this process of oxidizing glucose for energy. Therefore, barley seeds with go through this process as well. We know that different variables may alter or affect carbon dioxide product during respiration (such as germination and temperature).

Methods:

For this lab, we would be performing the same practical procedure a multitude of four times, each with a different barley seed. We would begin with germinated barley seeds, twenty five of them that had been wrapped in moist paper towel for a period of time. Those seeds would then be blotted dry and the temperature of the room would be taken for comparison of a later trail that would compare the effect that temperature could have on the CO2 release of the seeds. With the room temperature recorded and the germinated seeds blotted dry, all twenty five would be placed into a carbon dioxide reading chamber which would then be connected with our LabQuest to graphically show the amount of CO2 being released from within the chamber. In order to get an accurate reading of the carbon dioxide being released from the germinated seeds, they would be left in the chamber to collect data for a minimum ten minutes. Once the time had passed and data for the graph on the LabQuest was present, the seeds were taken out and put into a beaker of cold water that had ice in it as well. Whilst the germinated seeds cooled in the cold beaker water, the CO2 probe that was taking carbon dioxide readings in the chamber was to be fanned with paper for a minute total and the chamber itself was washed out with water and then thoroughly dried with paper towels. Once the chamber was dried, we were ready to begin the alteration to our testing trials which would then provide us with data to see the effects that temperature has on the release of carbon dioxide from our seeds. The germinated seeds that we had put in the ice beaker would then be taken out and blotted dry with paper towel and then placed in the carbon chamber to begin collecting data on our LabQuest for ten minutes just as we had done before with the room temperature germinated seeds. The very same processes would be again repeated for our twenty five non germinated seeds as well as for our control group of glass beads, the only exception being that the glass beads would not be chilled in a beaker of cold water. The graph that we collected from all trials would be stored on our LabQuest and we would calculate the slope of the graph for each trial run for comparison. Once all the runs were completed we compiled the graphs together so that the differences between all of them would be visually easier to see.

Non-Germinated barley seeds

Germinated barley seeds

Testing for CO2 release

Checking temperature change

Data:

Graphs:

Discussion:

Our graphs show that for the non-germinated barley seeds, as time went on, the release of CO2  decreased. For the germinated seeds, as time went on, the release of CO2 increased. For the germinated seeds in cold water, as time went on, the release of CO2  decreased. The control group (glass beads) had no change in CO2 release, and it's slope was 0.

The data for germinated seeds in cold water was very similar to the non-germinated seeds and for both the release of CO2  decreased. However, the germinated seeds increased in the release of CO2  and the control group remained constant.

The amount of CO2 produced by the germinated seeds in cold water and the non-germinated seeds was about the same. This showed that both germination of the seeds and temperature change have an effect on the amount of  of CO2 released.

What we did well in this lab was that we had an accurate indication of both variables (germination and temperature change). However, what we did badly in this lab was that our control group data was not helpful. It was probably tampered with, so it's information is almost useless to compare to the other test groups.

The validity of our data is not very good. Our data was too low compared to other in the class who also did barley seeds. It was off by a good 0.12-0.24 ppm/s. It was double what we had. However, our data was at a reasonable range, so it wasn't completely off. Also, when compared to the average at room temperature Mr.Filipek gave us, our data wasn't too far off.

Going off what we talked in class, we thought that temperature and germination would affect the barley somehow. Obviously the groan shows that these variables do affect the amount of CO2 released, so we were able to support this hypothesis.

Conclusion:

The question we were trying to answer was does germination and temperature affect the amount of CO2 released?

Obviously they do affect the outcome, and our data supports it. For instance, the slopes between the germinated seeds in cold water and the non-germinated seeds were very similar. The amount of CO2 produced was also very similar between the two. Also, based on the control group (the glass beads) given to us, the germinated seeds at room temperature data was very close to that average.  

What we did wrong in the lab was that we messed up the outcome of the control group. We believe the glass beads test was tampered with, whether someone knocked it over or it was left out too long. If we were to do this experiment again, we would fix that part and do multiple trials to make sure the first trial was not faulty.