Thursday, December 5, 2013

Chromatography and Photosynthesis/Light Reactions Lab (12/5)


Purpose: 

Photosynthesis Lab
    The purpose of this lab was to test how boiled and unboiled chloroplasts and the presence of light affect the transmission and absorption of light in a solution of phosphate buffer, distilled water, DPIP, and chloroplasts.

Chromatography Lab
    The purpose of the paper chromatography lab was to calculate the Rf values of the different pigments in spinach. The purpose of this lab was to separate the different chlorophyll that exists in spinach leaves. This would separate the individual chlorophyll according to their size. Also, we used a color coder to reveal which specific chlorophyll we were actually seeing.

Procedure: 

Photosynthesis Lab
    Set up an incubation area with a light, water flask and test tube rack, respectively. Keep the containers containing the unboiled and boiled chloroplasts solutions on ice. Label cuvettes 1,2,3,4,5, respectively. Cover cuvette 2 with tin foil. Label test tubes 1,2,3,4,5, respectively. Add 20 drops of phosphate buffer to all test tubes. Add 80 drops of H2O to test tube 1. Add 60 drops of H2O to test tubes 2,3 and 4. Add 63 drops f H2O to test tube 5. Now add 40 drops of DPIP to all test tubes with the exception of test tube 1. Transfer all solutions in the test tubes to their corresponding cuvettes, fill cuvettes 3/4 full. Now with respect to the spectrophotometer adjust the amplifier control knob until the meter reads 0% transmittence. Add 3 drops of unboiled chloroplasts to cuvette 1 and place it in the sample holder, adjust knob to read 100% transmittence by turning knob to the red light. You will now add a drop of unboiled chloroplast solution to cuvette 2, and immediately take a reading using spectrophotometer, removing tin foil to do so. After the reading place the cuvette in the test tube rack in the incubation area covering it with its tin foil. Let it sit for 5 minutes and then take another reading. Don't forget to recalibrate the spectrophotometer before every reading. You will take reading in intervals of 5 minutes up until 15 minutes. In respect to cuvette 3 also add a drop of unboiled chloroplasts. Cuvette 4 add a drop of boiled chloroplasts. Cuvette 5 will be used as is. Follow the same guidelines for readings and place cuvettes in incubation area in same intervals.

Setting up the individual cuvettes

Here is the device in which we placed the cuvettes in. It shone red light through the sample and then recorded it. 

This is our set up. Light is being separated via water and enters the samples. 

Chromatography Lab
    Obtain a long test tube. Add 1 cm of the provided solvent. Cut a piece of paper in a manner that it is long enough to reach the solvent but short enough to stopper the test tube. The paper will also be cut with a point, this end will touch the solvent. Draw a line 1.5 cm above the point. Place spinach leaf on top of pencil line. Rub the leaf with the edge of a coin. Repeat several times using a different part of the spinach leaf. Place the pigmented paper in the test tube now with the point barely touching the solvent and stopper the tube tightly. When solvent is 1 cm from the top mark the front. Remove the paper from the tube and mark the bottom of each pigment. Measure the distance the pigment migrated from the origin pigment line.

Extracting the pigments 

Isolating the paper with the pigment. Note the color. 


After about 10 minutes or so, there is already noticeable color separation of pigment. 


Measuring how far the pigment traveled. Cork was used to keep the paper from bending. 




Methods:
 
Photosynthesis Lab
  
    The light in the flask will absorb most of the infrared radiation from the light and transmit most of the visible radiation. Wavelengths of light within the visible light spectrum power photosynthesis. Cuvette 2 will be covered with tin foil to prevent light from coming in it is a control group. The test tubes will provide room in which substances can become diluted. There is more H2O added to test tube 1 and 5 because test tube 1 will be used for calibration, and the extra H2O in test tube 5 replaces the chloroplasts. Cuvette 1 will be used to recalibrate between readings it demonstrates the measurement of a 0 and 100% transmittence, this is the scale on which the other cuvettes will be measured by. Recalibration between readings will reset the spectrophotometer.
 
Chromatography Lab
 
    In order to calculate the Rf values for the different pigments we observed, we used the equatiom for Rf values. Rf = (distance of pigment migration) / (distance solvent migrated). 

Discussion: 

Photosynthesis Lab
 
    The phosphate buffer is used in the expirement in attempt to slow the light reactions, because in reality the reactions take place extremely fast. H2O in the experiment provides the electrons for the light reactions to take place in the chloroplast. DPIP is our form of NADP in the chloroplast which will be reduced by accepting electrons that are passed down the electron transport chain. So the light will hit the cuvettes and power photosynthesis. Each cuvette will contain chloroplast to a different degree. Depending on the contents of each cuvette and its conditions whether covered by tin foil or with boiled chloroplasts, different results will be obtained. In general H2O will donate electrons which through a series of steps including the passing through PS1, ETC, and PS2 in the chloroplasts; DPIP will be the last acceptor and there will be a change in color in the cuvettes from blue to colorless as DPIP is used. 
    As you can see in our graph the highest rate of absorption happened when the chloroplasts were unboiled and were exposed to light. This makes sense because the chloroplasts were not altered or boiled, and they were exposed to light which provided the partial fuel it needed to  carry out its reaction. 
    DPIP, as stated before, is an electron acceptor that takes place of NADPH. In our first attempt at this experiment, we used too much DPiP and not enough chloroplasts to yield good results. The fast acting DPiP already finished reacting with the chloroplasts, thus finishing the reaction by the time we got to our 2nd run. As a result, our data was unreliable past the second and sometimes third run.  In order to improve this, we decided to increase the amount of chloroplast while keeping the amount of DPiP the same. This would allow the reaction to go at a more gradual rate as DPiP has more chloroplasts to react with.  This made our data more gradual and reliable. We also calibrated our device in between every run to ensure that our data was as accurate as possible. 

Chromatography Lab
 
    For the paper chromatography lab, we were able to get 3 distinct bands of pigment. The first pigment we observed had an olive green color. An olive green colored pigment corresponds to chlorophyll b. The second pigment we were able to distinguish had a bright green color; bright green colored pigment indicates chlorophyll a. The last pigment we were able to see had a yellow color, indicating the pigment xanthophyll. It is worth noting that chlorophyll b and chlorophyll a were located closer to the marked line and did not travel that far up the filter paper. This is most likely due to the fact that the oxygen and nitrogen in chlorophyll make it cling to the paper more, preventing it from traveling farther up. Another interesting observation we made was that the xanthophyll pigment was located farther down the paper; the explanation we had for this phenomenon is that xanthophyll is not very soluble in the solvent we used.
    The solvent moves up the paper mimicked capillary action. This this is a result of the attraction of the solvent molecules to the paper (adhesion), and the attraction of the solvent molecules to one another (cohesion). As the solvent moves up it carries with it the pigments dissolved in it. The pigments travel different distances up the paper because they are soluble to the solvent unequally. Also each pigment attracts to the paper fibers differently. A pigment can bond to the paper fibers through H-bonds. In this manner certain pigments can near the solvent front more than other pigments when they are more soluble in the solvent and when they don't form H-bonds with the paper fiber. On the other hand other pigments will travel less and not near the solvent front when they aren't very soluble in the solvent and create strong H-bonds with the paper fiber.

Graphs & Charts:

 
Photosynthesis Lab

In this graph we have the rate of transmittence in percentage with the amount of time it took to transmit that amount. 
Run 1: Unboiled with light
Run 2: Unboiled chloroplasts with no light
Run 3: Boiled Chloroplasts with Light
Run 4: No chloroplast with light 







Here we have the absorption rate of the different runs. 

Run 1: Unboiled with light
Run 2: Unboiled chloroplasts with no light
Run 3: Boiled Chloroplasts with Light
Run 4: No chloroplast with light 

Chromatography Lab


Calculated Rf for each color that we saw. 




Conclusion: 

Photosynthesis Lab 
    In this lab we concluded that chloroplasts will react at the highest rate when they’re exposed to light and unboiled (not denatured). In contrast, the slower rate of light reactions occurred when the chloroplast is denatured and/or in the dar. That way the chloroplasts have no way of carrying out a light reaction. 

Chromatography Lab
    In this lab we found out the chlorophyll presence in the cells of a spinach plant. Using the chromatography paper, we concluded that the most prevailing cholorphylls were xanthophyll, cholorphyll a and chlorophyll b. In that order, from most present to least, the chlorophyll traveled up the chromatography paper separating the layers.

References:
Lab

2 comments:

  1. For the discussion and conclusion, you will want to reference actual data you captured to substantiate your claims. Good Information.

    ReplyDelete
  2. Cuvettes are perfect for measuring a research lab samples and points you made is good, did get good information.
    spectrophotometer cuvettes
    http://www.precisioncells.com/categories/Spectrophotometer-Cuvettes/1/1

    ReplyDelete