Friday, December 6, 2013

Photosynthesis and Chromatography Lab


     Photosynthesis is a process that converts light energy into chemical energy that can be used to fuel the organisms. Below is the formula for photosynthesis.
                                       6H20 + 6CO2   -->  C6H12O6 + 6O2
         Photosynthesis has two parts, the light dependent reactions (light reactions) and the light independent reactions (dark reactions). The light reaction happens in the thylakoid and convert light energy into chemical energy. This chemical reaction must take place in the light. The dark reaction takes place in the stroma within the chloroplast, and converts CO2 to sugar. Although called dark reactions this reaction needs the energy  produced in the light reactions in order to trigger the reaction. Organisms that carry out photosynthesis and make their own organic molecules are called autotrophs.
         Chloroplasts are organelles in plants that conduct photosynthesis.  Chloroplasts capture light energy from the sun to produce the energy stored in ATP and NADPH photosynthesis.  Under certain circumstances the proteins in chloroplasts can be denatured or unable to conduct photosynthesis, such as high temperature or lack of light.  In this experiment we will test these circumstances and see for ourselves the affects that can harm photosynthesis.
         This experiment tests the light reactions. The light reactions are the first step of photosynthesis. The light reaction have 2 photosystems. There is photosystem I and II, they can be abbreviated PS I and PS II . PS II comes before PS I. PS II is also called P680, because it absorbed a wavelength of 680 nm the best. PS I is also called  P700, because it absorbs a wavelength of 700 nam the best.In PS II light enters it and stimulates the electrons. Water also enters it and excites the electrons by splitting water, H+ remains in PS II and O2 leaves the photosystem. The electrons bounce around till they reach the chlorophyll a molecule. The electrons are then sent to the primary electron acceptor and makes them 680+. 
          After the electrons are sent to an electron transport chain. Electron transport chains help lower the energy in electrons. It also helps drive chemiosmosis  to make ATP.  Some of the electrons that are in the transport chain get sent to the chlorophyll a molecules of PS I. PS I electrons come from light and the electrons from PS II. PS I  is similar to PS II, in having the chlorophyll a molecules pushing electrons to the primary electrons acceptor. The difference is in the end. Instead of going to an electron transport chain to make ATP,  P700+ goes through the electron transport chain to be reduced by the enzyme, NADP+ reeducase to make NADPH. Both of these process are meant to make ATP and NADPH for the Calvin cycle. 
The purpose of this experiment was to test how enzymes (in this experiment the enzyme was DPIP) would effect how much is transmitted. We wanted to see what would happen if we used boiled and unboiled chloroplast. We would see if the boiled maybe decreased the prcent transmitted.  Also we wanted to see the effect of having the curettes being placed in the light and dark. We could test if curettes kept in the dark would lower the percent transmitted.  In this experiment the independent variable was the DPIP and the dependent was the precent transmitted.
       First we set up the 5 different test tubes. The first test tube was a blank. It had 1 mL of phosphate buffer, 4 mL of distilled H2O, no DPIP, and 3 drops of unboiled chloroplast. The second test tube 1 mL of phosphate buffer, 3 mL distilled H2O, 1 mL of DPIP, and 3 drops of unboiled chloroplast. The third test tube had 1 mL of phosphate buffer, 3 mL of of distilled H2O, 1 mL of DPIP, and 3 drops of unboiled chloroplast. The fourth test tube had 1 mL of phosphate buffer, 1 mL of DPIP, and 3 drops of boiled chloroplast.  The fifth test tube was 1 mL of phosphate buffer, 3 mL of distilled H2O, 1 mL of DPIP, and no chloroplast. When the test tube was ready to be measured in the spectrophotometer the contents in the test tube would be put in to a cuvette. For each test tube the chloroplast was not added until it was put into a cuvette. After we got all the test tubes ready, we filled up a big beaker of water and behind the beaker of water was a huge floodlight. The first test tube was a blank that was used to calibrate the spectrophotometer. After it was used to calibrate it was kept in the light. Because we ran out of time we measured all the test tubes at the same time. We would put test tube 2 in the spectrophotometer then get it's reading. Then we would take the test tube out and put it in the light for 5 minutes. But the 2nd test tube is covered in foil to prevent the light from getting in. Right after that we would put in test tube 3, get a reading on the spectrophotometer and then put it in the light for 5 minutes. This same thing happened to test tube 4 and 5. After every 5 minutes we would take the test tube out of the light and measured in in the spectrophotometer we would go up to 15 minutes. We would take measure if the test tubes in the order of 2,3,4,5.
We repeated this experiment again. This time the experiment was slightly changed. The content that was put in each test tube was the same except that for every 1 drop of chloroplast we would add 3 drops of water  to try to dilute the chloroplast. This took place in test tubes 1-4. It did not happen in test tube 5 because it had no chloroplast in it. Everything else in the experiment was the same like the process of measuring with the spectrophotometer. 

                                                    Zero minute mark 

                                                      Five minute Mark 

                                                Ten minute Mark 
                                                    Fifteen minute mark 
                   Test tubes with Solutions 
             Cuvettes Sitting in the Light 

Our group had to perform the experiment twice because of inaccurate data. The first time we ran the experiment, the percent transmittance jumped well past 100% for several cuvettes at the 0 minute mark, which is clearly inaccurate. After having the chloroplast added to the rest of the solution for less than a minute, there shouldn't be that strong of the ratio of intensity of the light that has passed through the sample to the intensity of the light when it entered the sample. This led us to believe that our data was not correct. We believe that the solutions were using up the chloroplast too fast, causing the unreliable data. However, after performing the experiment a second time, we received more accurate data, especially since we deluded the chloroplast with one drop of water per one drop of chloroplast. In cuvette one there was no DPIP added. The data stayed at zero the whole time. Photosynthesis was not able to take place then because there was nothing to act as an electron acceptor, similar to how DPIP would have done. In cuvette two, unboiled chloroplast was added and the cuvette was wrapped in tinfoil, leaving it in the dark. Photosynthesis was not able to occur because the light reactions of photosynthesis need light to take place, but since the cuvette was wrapped in tinfoil, there was no light to provide light energy for the reaction. No electrons were made, and the color didn't change. The transmittance at each 5 minute mark stayed within a three percent range of one another for this particular cuvette for these reasons. In cuvette three, unboiled chloroplast was added, and it was placed in front of the light. The cuvette was placed in front of the light so this provided light energy for the light reaction to take place. This is why the transmittance of this cuvette went up by a total of about 9% throughout the experiment, the most by far of any cuvette in the experiment. In cuvette four, boiled chloroplast was added, and the cuvette had been placed in front of the light. It was hard for photosynthesis to occur in this cuvette because since the chloroplast was boiled, this denatures it. Photosynthesis is not able to take place with unliving chloroplast. This is why the percent transmittance for this cuvette went down. No reaction was able to take place at all. In cuvette five, there was no chloroplast added and the cuvette was not placed in front of the light, however, it was not wrapped in tin foil in the dark either. The percent transmittance stayed the same for this cuvette since there was an absence of chloroplast and light to get photosynthesis going. 
           Since we ran the experiment twice there were two conclusions. As shown in the methods and discussion, The first time we ran the experiment the percent transmittance was over 100 for the cuvettes, which was not what was supposed to occur. We came to the conclusion that we needed to delude the solution in order for the usage of chloroplasts to slow down. In the second experiment we changed the procedure by adding an extra drop of water with every drop of chloroplast. After this slight change the data showed that the percent transmittance was no longer sky rocketing but a gradual increase. The adding of water to delude the chloroplast definitely made an impact on our results and made significantly more accurate. Yay go chloroplasts!


Paper chromatography is used to separate and identify pigments and other molecules in a solution that contain many complex molecules. The paper creates stronger and weaker bonds with certain solutes (molecules). How strong the bond is depends on the solubility of the solute in the solvent. The further a pigment (or molecule) travels up the paper, the more soluble that solute is in the specified solvent and forms weaker hydrogen bonds with the paper itself. 
Carotene forms no hydrogen bonds with cellulose; therefore, it is very soluble in the solvent. Xanthophyll forms hydrogen bonds with cellulose which slows down its progress up the the chromatography paper because it is less soluble.  Chlorophyll contains oxygen and nitrogen which causes it to form stronger bonds with the paper and move up more slowly.
The distance the pigment moved divided by the distance the solvent moved is called the Rf constant. It illustrates the relationship between the two factors.

This lab tries to show how many different pigments are present in a plant through paper chromatography. This allows all the pigments to separate due to their solubility and bonding.  When we calculate the Rf constant we can more easily compare which pigments are more soluble and form certain type of bonds because the distance the solvent moved is already calculated in as a comparison. 

Many materials were used in this experiment, such as filter paper, solvent, cylinder, a coin, and a spinach leaf. As instructed, we cut a 1.5 cm point on the tip of the filter paper and crushed a spinach leaf with a penny in order to extract pigments of the leaf cells. By doing so, a green line of spinach particles appeared near the end of the paper. We then placed the paper into a graduated cylinder, and let it sit until the pigments traveled almost to the top. We removed the filter paper right away and marked the furthest migration with a pencil, then all the others. The Rf was thus calculated from the distance of the pigment of which it traveled.


       Measurement of Migration of Pigments 

      Measuring the Migration of Pigments 

        Spinach Leaf Pigments in Solvent 

In the lab we found that xanthophyll moved the furthest up the paper. Also, it had an Rf constant of one. This shows that this pigment was the most soluble and most polar because it was able to easily travel up the paper with the water.  Carotene was next. It had an Rf factor of .547. Then, chlorophyll A with an Rf of .444. Finally, chlorophyll B with an Rf of .282. We can observe that the closer the Rf factor is to one the more soluble and polar it is because the distance the pigment traveled is closet to that of the distance traveled by the solvent. The separation of pigments in chromatography allows us to examine the different colored pigments present in plants. We can from this predict which light and wavelengths of light will be most absorbed by this plant because the plant will reflect the colors present in the leaf and not absorb as much of them, This means that green, yellow, and orange light will be reflected in some way or form in the spinach leaf. The other from least absorbance to greater absorbance is in the respective order.

         By performing this lab and analyzing its results, our lab group found that the many pigments found in chloroplast take part in the process of absorption of energy from the sun. The filter paper and its color spectrum showed each pigment's solubility from which we measured each pigment's migration


1 comment:

  1. Graph of your data? I see the data tables but no graphs. It is hard to follow which run is for which cuvette. Good Information and very informative.