Plant Pigments & Photosynthesis

                   Plant Pigments & Photosynthesis


In this lab, our lab group separated plant pigments via chromatography and measured the rate of photosynthesis in chloroplasts. We also measured the percent transmittance within chloroplast and DPID solutions. Much went on and this is by far one of our biggest labs. IT all starts with the spinach! Enjoy!


               Part A: Plant Pigment Chromatography

     Before we could start experimenting, we had to make chromatography paper. We did this by cutting out a slip of paper around 6 in long, and then cut the tips on the bottom to make it pointy, like in the picture below. 

        After this, we drew a line around 1.5 cm from the bottom. This will be our pigment origin, which is where we will rub the spinach cells onto. 

        This next part was the fun one. We took a spinach leaf and started crushing it with a coin. We then put the spinach guts onto the pigment origin line we made earlier.

        Once our chromatography paper was ready to go, we hooked it to a cork then placed it into a flask filled with 1 ml of the solvent.We made sure that when the chromatography paper was in the cylinder, the solvent was not touching the pigment. When he solvent was about 1 cm from the top of the paper, we removed the paper from the flask and marked the solvent front.  In this trial, we found 5 bands. We then measured the distance from the pigment origin to each band. 

Analysis: After looking at the results, we concluded that there are many factors that went into how the chromatography paper was separating the pigments. It mainly depended on how soluble each pigment was with the solution that we used as the solvent. That is why there are different bands and some of are different color. They could only go as far as their solubility with the solvent. Ones that were less soluble were farther away from the solvent front. Also, if we had used a different solution as the solvent, the results may have been much different. For the different solvent could be more or less soluble with the different pigments found in the spinach.

                        Part B: Photosynthesis/The Light Reaction

This chart shows what needed to be in the specific solution.

           In this section we filled 5 different cuvettes according to the chart above.  3 out of the 5 cuvettes called for light, so during the duration of the experiment they were placed in front of the incubator. . By placing the cuvettes in the Lab Pro contraption after 5, 10, and 15 minutes we were able to find the % transmittance in each different cuvette. What seemed to of happened is that for the cuvettes that had living chloroplast in them, the longer we let them absorb light the lower the % transmittance got. What seems to be going on here is the living chloroplast  use this light to preform photosynthesis, which gives off ATP which makes the solution more absorbent. The other cuvettes, were supposed to be out control. One was covered in tin foil when left in front of the incubator, and the other had boiled (dead) chloroplasts in the solution.

Analysis: We looked at our results and concluded many observations. The boiled chloroplast solution did not have that much of a change in percent transmittance because there was no reaction going on, thus keeping the solution staying the same. This also follows for the cuvette solutions that were covered in tin foil. They did not have much of a change in percent transmittance because they were not given light to react.

BUT WAIT THERE IS MORE!

So, we decided to take on the Extra Credit challenge, not entirely sure what question(s) we were suppose to answer but here is our explanation on why the spinach glows red under the light and how we got it to glow.

So, our materials consisted of:

Test tube, leaf of spinach, alcohol, light, scissors, beaker and filter paper

First, we cut the leaf of spinach in little pieces and placed it in the beaker. After, we poured a bit of alcohol into the beaker and let it sit for a couple of minutes. Next, we filtered out the pieces of spinach into a clean test tube. At this point, we were only left with the liquid consisting of alcohol and chlorophyll. Lastly, we shined the light at the liquid and saw that it was glowing red!

The point of this was to extract the chlorophyll, next is why it glows red.

Spinach soaking in chlorophyll

So, chlorophyll absorb light and that is how they derive their food source. Chlorophyll is able to absorb different colors of light as well. 

Since the light that is shining gives off a blue light, the chlorophyll absorbs this and it excites the electrons A LOT! Naturally, chlorophyll is not able 

to stay at this high excited state and the electron falls very quickly, giving off the red "glow" as it moves into a new state of being less excited.

WOAH GREEN TO RED!

The solution of alcohol and chlorophyll

C-C-C-CONCLUSION

There is a lot to take in with this whole lab. We applied our knowledge about electrons, absorbency, transmittance, incubation, and chromatography to chlorophyll and chloroplasts. As a group, we were able to discuss and determine all that went on chemically and physically with our lab. Since this lab was so big, we decided to break up the analysis discussion questions at the end of each part of this lab. So in case you missed it, all of our wonderful work and deduction is above! 

Definitely more labs will be posted in the future. with CoolerThanAbsoluteZero's blog.

Cell Respiration!

Cellular Respiration!

In this lab, we compared the rate of cellular respiration between glass beads, mung beans, and peas. Before we tell you more about this extremely exciting experiment, you have to know some background info on cellular respiration. Cellular respiration takes place in a cell's mitochondria, and produces energy in the form of adenosine triphosphate, commonly known as ATP. Here's the equation:

          C6H12O6 + 6O2 -----> 6CO2 + 6H2O + ATP (energy)

This is a diagram of Cellular Respiration within the cell.

As you can see, cellular respiration requires sugar (glucose) and oxygen in order to take place, and then produces carbon dioxide (CO2), water, and ATP. In this lab, we were able to compare the rate of cellular respiration between glass beads, mung beans, and peas by using sensors that measured the amount of oxygen and carbon dioxide in the chamber where the specimens were placed. We compared the amount of CO2 being produced to the amount of O2 being used up in order to find the rate of respiration. Cooler Than Absolute Zero predicted that the mung beans would have the highest rate of respiration. Let's find out if we were right!

Note: That if the cell does not have oxygen to work with, it will not continue onto the Citric Acid Cycle. It breaks off in to two parts from glycolysis; Lactation or alcoholic fermentation.

Procedure: 

In this lab, we were measuring the respiration rate by focusing on CO2 and O2 production. First, we decided that it was a good idea to have a control group to check our other gathered data with. Our control group in this experiment were solid marbles. We placed these marbles in a clear container, closing it off with a lid. This lid contained two inputs for our CO2 and O2 respirators. We secured these into place and were ready to conduct the experiment. After a minute of waiting, we started calculating the percentage of CO2 and O2 in the container. The system we used was connected to a screen tablet that displayed a graph, and allowed us to see the amounts of these two gases. Shortly after waiting ten minutes, as written in the instructions, we began assessing the data collected. The graph showed that the amount of CO2 and O2 stayed at a constant rate. This data makes sense because the marbles are abiotic and not going through the process of cellular respiration. Next, we cleaned out the container and placed germinated peas to calculate their respiration rate. Following the instructions, we proceeded to do all the steps stated previously to receive our data for the peas. Again, after ten minutes of waiting; we finally got to assess the graph or our data. This time, the graph showed that CO2 progressively increased while,O2 progressively decreased. This data is accurate being the germinated peas are consuming O2, a clear indication that cellular respiration is taking place. Lastly, the group measured the respiration rate in mung beans. This graph showed an increase in CO2 and a decrease in O2; the exact same data in the germinated peas.

Not only did we test the mung beans and peas in normal temperature environments, we cooled the temperature to see if that had any affect on them. Basically, we did the same experiment stated above. However.. Before placing the mung beans and germinated peas into the container, we let each one of them soak in ice water for a minute. After, we simply dried them off and placed them into the container to measure the rate of cellular respiration. The graph for the mung beans showed that CO2 increased and O2 decreased, but at a slower rate. Then, the graph for the germinated peas displayed the same results.

Analysis questions...

1. The effect of germination on the rate of cellular respiration in peas, is that the rate of O2 will become higher. We know this because, when a pea is germinating it means that it is growing. When it keeps growing, the need for O2 consumption grows alongside. Resulting in the rate being higher, as stated previously. 

2. There are many pieces of evidence that cell respiration occurred in peas. One reason that the germinated peas still were undergoing cellular respiration because they were still alive. We know this because the O2 levels increased while the CO2 levels decreased.

3. Germinated peas undergo cellular respiration because they are still producing sugar for themselves

Conclusion:

In this lab, we really started to see how each chapter we learned about starts building on each other. Without knowing vital information from chapters on sugars and proteins, we would not be able to analyze thoroughly on how cellular respiration and photosynthesis works. We see how temperature affects proteins and denaturizes them so these processes are unable to function, and we see how certain molecules behave. Overall, we learned how by changing specific factors, we can change the rate of cellular respiration.

Group Discussion:

We got to observe and accurately measure cell respiration in the lab by using high tech, high quality equipment to measure levels of Oxygen and CO2 within a closed chamber of germinating peas and beans. With dry beans we saw no drastic changing in Oxygen or Carbon Dioxide. When using peas that were germinating, we saw that oxygen was slowly dropping and carbon dioxide levels was rising. This made sense because plants like peas release carbon dioxide during cell respiration. When we soaked the peas in really cold water, the rate at which carbon dioxide was being release drastically slowed down. Is proves that plants have a ideal point of temperature to grow in and hotter/colder climates could slow down their rate of respiration.

Catalase Titration Lab

Catalase Titration Lab

In this lab, we were using enzymes (yeast) to break down hydrogen peroxide. But after a set time (10 seconds, 30, 60, 180, 360), we injected sulfuric acid to stop the catabolic process. We then titrated a 5 ml sample of the solution with potassium permanganate to see how much hydrogen peroxide was left to consume the potassium permanganate in the 5 ml solution. This experiment consisted of three different parts. 

Part B: In this section of the experiment, we determined the amount of hydrogen peroxide (H2O2) initially present in a 1.5% solution without adding a catalase, or enzyme, to the reaction mixture. The amount we find will be our base line, or constant, for the upcoming parts of the experiment. 

                 We started this experiment by measuring out 10 ml of the 1.5% hydrogen peroxide into a plastic cup. (Insert photo) Then, we added a mL of water. For this section, we added water instead of an enzyme solution in order to find constant data that we could compare to the data of the solutions that contained enzymes. After this we added 10 mL of sulfuric acid and mixed the solution. This stopped the reaction by denaturing the enzymes. Next we removed a small sample of the final solution and placed it into the titration cup. As might have guessed, we then titrated the solution. This means that we added small amounts of potassium permanganate until the solution permanently changed to a light pink or brown color. We compared the initial amount of potassium permanganate in the burette to the final amount in order to find out how much potassium permanganate was needed to discover how much hydrogen peroxide was used up. 

Part C: In this portion of the lab, we wanted to calculate the rate at which H2O2 turns into H2O and O2 (or decomposes) in an uncatalyzed reaction. An uncatalyzed reaction simply means that an enzyme was not present. Additionally, this results in the activation energy of the reaction to be higher. After the solution was left to sit uncovered for twenty-four hours, it was then tested to gather this information.

      Natalie made a great video for the paragraph below

To prep for the experiment, we first placed about 15mL of H2O2 in a beaker and let it sit over night. After, we added 1mL of H2O (also known as, water) because we were calculating data for an uncatalyzed reaction. Next, we added 10 mL of H2SO4 (also known as, Sulfuric Acid) to the solution. If the solution we were testing had an enzyme, the acid would denature the enzyme since it is a protein. The enzyme would denature because the acid would lower the pH of the solution and enzymes have a set pH level in which they can function normally. Then, we mixed the contents of the beaker and drew a 5 mL sample of the solution. We took this 5mL sample and added KMnO4 (also known as, the titrate in this experiment) drop by drop, to measure the amount of  H2O2 still remaining in the solution. During this step, we also swirled our sample to insure that the titrate distributed evenly. It only took one drop of KMnO4 to give us the data that we needed for this experiment. Below is the data collected from this portion of the experiment.

                

Part D: In this part of the lab, we wanted to figure out how much H2O2 was eaten up depending on how long we left the H202 to react with a catalase. In this case we used yeast! We noticed a lot from the results when looking at the different amount of times we left the H2O2 with the yeast.       

When we prepared this we had five different beakers that each had a time label. "10 seconds, 30, 90, 180, 360". Each time label represented how long we would let the H2O2 react with the yeast before we applied 10 ml of sulfuric acid in order to stop the process. We added 10 ml of H2O2 and then 1 ml of yeast but the second that we added the yeast, we immediately started timing accord to the label on the beaker. After waiting, we add the acid and then extracted a 5 ml sample from the beaker. Proceeded to place it under the burette and calculated how much potassium permanganate it took to turn the sample a slight pink showing that there wasn't anymore H2O2 to react and "erase potassium permanganate.

 We noticed from our results (after graphing the data) that the longer we left H2O2 to sit with the yeast, the less amount of potassium permanganate was needed to turn the sample a slight pink. Showing that the catalase (yeast) was reacting with the hydrogen peroxide and reacting to make H2O and oxygen.

OUR DISCUSSION

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So in order to determine the course of an enzymatic reaction, we had to measure out how much substrate was disappearing over certain time intervals. So we recorded our data for the amount of hydrogen peroxide used in every test, and made a graph to better depict this information. As you can see below, (insert graph here) the rate of the amount of H2O2 in a certain amount of time is the highest between 180 and 360 seconds. This means that the difference of the amount of hydrogen peroxide used up is greatest between 180 and 360 seconds, most likely because that is the biggest time interval. On the other hand, the smallest rate is between 0 and 10 seconds. This is because it is the shortest gap of time, so hydrogen peroxide does not have nearly the same amount of time to become used up as it does between the 180 and 360 second gap.

CONCLUSION

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Overall, our group experienced a hands-on lab that taught us more about enzyme activity. The rate of the reaction had to do with the substrate concentration. As the concentration went higher, the reaction rate increased as well. At one point, the rate stays the same since the enzymes are always loaded with what they are catalyzing. In order to collect the right data on the reaction rate, we used an acid to denature the enzyme, and then titrate to see how much substrate was left over. In order to better this part, we could have been more precise in our titration. Other than that very tiny error, this lab went very smoothly!

This was our enzyme catalysis lab, hope you enjoyed reading!

Osmosis and Diffusion Lab

Cooler Than Absolute Zero's Osmosis and Diffusion Lab

No sucrose, dialysis bags, or potato cores were harmed in the experimenting of this biology lab.

1.A: Small solute molecules and water molecules can move freely through a selectively permeable membrane but larger molecules will pass through more slowly or not at all. The size of the minute pores of the dialysis tubing determines which substances can pass through the membrane.

The purpose of this section was to measure the amount of diffusion of small molecules through dialysis tubing, which is an example of a selectively permeable membrane.

   

Dialysis definition: The movement of a solute through a selectively permeable membrane.

METHODS -->We placed a dialysis bag of glucose and starch into a small cup of iodine. After waiting 30 minutes, iodine entered the bag turning its color black and glucose exited the bag into the cup.

A dialysis bag glucose and starch submerged in an iodine liquid. The bag turned black.

Our lively discussion: We noticed that glucose was exiting the bag whilst iodine was entering the bag. This is an example of diffusion. The bag was a hypotonic solution due to the fact that iodine was entering the bag. We knew that the glucose was exiting the bag because the bag was turning a blackish color and iodine detects starch-like substance. After testing the liquid with a glucose test strip,  the cup results were positive. This must mean that the molecules of iodine are small enough to pass through the membrane of the dialysis bag.

1.B: 

       In this section of the experiment, our lab group used these thin plastic tubes called dialysis tubing to find the relationship between solute concentration and the movement of water through selectively permeable by a process called osmosis. Osmosis is the tendency of  water to pass through a semipermeable membrane into a solution where the solvent concentration is higher.

A dialysis bag of sucrose being submerged in a beaker or cup of water.

      So in the experiment, we made six bags with the dialysis tubing. In each bag, we added a different solute. For example, Bag #1 had distilled water, Bag #2 had 0.2 M Sucrose, Bag #3 had 0.4 M Sucrose, and so on. After that, we put each of the bags in cups filled with water, completely submerging the bag. Then we waited thirty minutes, and let osmosis take place. Next, we massed each of the bags in order to get our data. Here is a picture of our data chart!
 (insert picture of 1B data chart)

The percent change of the class's average compared to our own data.

Before the experiment, the dialysis bags were in a hypertonic solution. This means that there was less water inside the bag than outside. After the procedure, the environment of the bags turned into a hypotonic solution since the water rushed inside the bag. We gathered this conclusion after discovering that the final mass was larger than the initial mass, meaning that the water entered the dialysis bag.

1.C

      This awesome experiment was all about potatoes! And water potential. :( What is water potential? Well, I'm glad you asked. Water potential is the tendency of water to leave one place in favor of another place. There are two factors that affect water potential. One factor is the addition of solute. This lowers water potential. The other is pressure potential. To raise the water potential, you would need to increase the pressure. In biology, water potential is used to predict the movement of water into or out of plant cells.

In the lower half of this collage, we noticed that as the molarity went up from each beaker, the cores began to float. But the lower the molarity, the core would sink.

Day One: Before we could begin our experiment, we had to obtain potato cylinders. In order to make this, we punctured the potato with a cork borer (a plastic straw) 24 times. After, we made sure that the cylinders did not contain skins by cutting the remaining skin off with a razor blade. What fun! We divided the potato cylinders into groups of four, and then massed each group. During this step, we massed the potato cylinders incorrectly. This made our data invalid, so in order to fix our mistake, we found the average of three other groups' data. Next, we placed each group of cylinders into a cup of sucrose solution. Each cup differed in the molarity of the sucrose. Then, our lovely, god-like biology teacher wrapped the beakers with plastic in order to prevent evaporation. Finally, after a long day's worth of working with potatoes, we left the beakers to sit overnight. 

Our class's average of percent change in mass with the potato cores in beakers of sucrose.

    Side note: During this experiment, we made a quick observation. We saw that some of the potato cores floated while others sank. This is because in some cases the potato cylinders had more water than the molarity of sucrose, causing them to sink. On the other hand, those with less water than the solute floated.

Day Two: We started off our morning by removing the potato cores from the beakers, and then determined their total mass. Then, we filled out the rest of our data table by calculating the mass difference and percent change in mass. This is the moment when we realized our data was severely miscalculated. (put in good chart and bad chart)

    We noticed after analyzing our data that the mass of the solute in some of the beakers decreased. This is because water is moving from a higher water potential to a lower water potential, which means the water is leaving the potato and entering the solute. This makes the mass of the potato cylinders decrease. We discovered that the higher the molarity of sucrose is, the more the mass of the potato decreases. 

We were given a set of data for the same experiment expect the cores were Zucchini. The results were very similar.

1E.

Plasmolysis is the process in which cells try to create equilibrium when placed in a hypertonic solution. Due to the difference in molarity on the inside and outside if the cell, water rushes out in an effort to create a balance. When the onions were placed in the 15% NaCl solution, the onion shriveled because of this effort to make equilibrium. Compared to the onion that was prepared with just water on the slide, this onion was smaller. This is due to the difference in the level of water in each onion. The cells in the water stayed at equilibrium, or at most took in water, because the molarity of NaCl was greater or the same inside the cell. The opposite was true for the 15% NaCl mixture, thus causing water to rush out of the cell.

Here is a photo of a plasmolyzed plant cell. As you can see, it is lacking water, due to the hypertonic environment. 

                                                

Milk Lab

 What's this "milk" lab all about?

Well we are glad you asked, the purpose of the milk lab was to determine the percentage of protein in Prairie Farms non-fat milk. Once this percentage was determined, it was compared to the percentage found on the Prairie Farms milk carton.  The lab dealt with the denaturing of proteins due to the dramatic change in the pH level of milk.

Day 1.. The Cooler than Absolute Zero crew takes on the "super sexy" milk lab.

First, as all.. Super responsible, A.P students; we read through the procedure at home before the lab.

Next, we began to go through the lab procedures which were written on our worksheet; along with writing in our collected data in the table provided. 

In these pictures, we are massing out the milk and adding in acetic acid to denature the milk, resulting in curds forming.

Fun fact #1: Milk curdles when acid is introduced into the milk which causes the milk to drop in pH levels and that puts the milk into a state of denaturalization. Thus causing proteins to lump together.

After, we let the milk sit for 5 minutes.. (Tick.. Tock.. Tick.. Tock) & then filtered it through.

(Of course, we did this part twice because our filter paper broke the first time.. And just our luck!)

ALERT! ALERT! ALERT! RANDOM TANGENT ON THE WAY! ALERT! ALERT!

 During this 5 minute wait... Our super awesome, A.P Biology teacher was giving a speech; we decided to photograph this for memory. (We are still waiting on that autograph, Filipek!)

Next, after.. That nerdy distraction; we added Biuret Reagent to distilled water and our failed-filtered milk... (In normal people words, it basically tests the protein presents in a substance).

When a protein is present, it turns a purplish, blue color! As seen above :) (In this case, protein is present in the milk!)

Fun Fact #2: Yogurt is made by fermenting bacteria in milk, allowing the bacteria to churn out lactic acid which helps give yogurt its creamy texture. If you heated fresh milk and left it at 100 degrees Fahrenheit for a few hours, it would turn into yogurt.


Day 2..... Continuation of the "super sexy" milk lab.

After the grueling first day.. We simply massed the filtered paper and calculated all of our data! 

Fun Fact #3: Casein is a phosphoprotein commonly found in mammalian milk. It plays a major role in cheese, is a binder for the end of safety matches, and is responsible for giving us so many important Nutrients.

                                                           Data

Da

Here is the data we collected during our milk lab. As you can see, we had to find the mass of the filter paper, both the filter paper and dry protein, and the dry protein alone. As you can see in the picture above, our group found two sets of numbers. This is because we had a mishap and tore our first filter paper, which destroyed the hole filtering process. To clear an confusion, our data chart has a mass of our first filter paper which is labeled under the column "reg", and the mass for our second filter are under the "coffee" column.  To make our data a bit more accurate, we decide to add up both masses and put it under the column titled, "total". With our results, we recorded less protein in the milk than what we said to be on the carton. Our percent error was -70.31% due to a couple different factors. The first time we tried filtering the milk, we punched a hole through the filter, letting most of both; the liquid milk and solid protein through. We left this filter aside to dry and record the mass of protein. What we did, was take all of what went through the first filter and put it through a second one. This time we used a coffee filter (look at us, stepping up our game. Come at us, bro.. #sorrynotsorry). Using test tube tongs, we squeezed what was in the filter as a way to quicken how much liquid was filtered at one time. This was successful for a decent amount of time until the filter paper gave out once more, and broke. (Just our luck..) This time, less of the milk spilled out of the paper. We did the same thing that we did the first filter, and massed it. Altogether, we massed .74grams of protein, a significant difference in the amount shown on the carton. This was due to the filter paper breaking, and therefore; our results are not valid. 

Conclusion

So, at the end of all this.. We, as a team, concluded that the carton containing milk had more proteins than the milk we made. Our team's milk contained only 4.75% of protein, which is equal to .74grams. The Priaire Farms milk contained 16% of protein which is a huge difference compared to our results. We do have a wicked, totally awesome.. super nerdy explanation for this! We accidentally poked a huge hole in the filter paper. (Ivan's fault; yes I am throwing you under the bus.. JK, we love you tie-guy) This definitely created some, tiny error in our experiment. This incident is most likely going to cause our collected data to be inaccurate, but hey; we tried our best!

Works cited

<"Casein." Wikipedia. Wikimedia Foundation, 22 Sept. 2014. Web. 22 Sept. 2014.
http://en.wikipedia.org/wiki/Casein>

<"A Quick Guide to Yogurt Preparation and Variations." About. N.p., n.d. Web. 22 Sept. 2014."
http://homecooking.about.com/od/cookingfaqs/f/faqyogurt.htm>

<"Why Does Milk Curdle -- and When Is It a Good Thing?" About. N.p., n.d. Web. 22 Sept. 2014.

foodreference.about.com/od/dairy/a/why-does-milk-curdle.htm>