Lab Report #1 - Stating the Chemical Compositions of Cells Through the Uses of Indicators and PDF

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Stating the Chemical Compositions of Cells Through the Uses of Indicators and Solutions
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Diana Bocina, Devon Rotolo Bio 150 23 September 2016

Stating the Chemical Compositions of Cells Through the Uses of Indicators and Solutions

Introduction: All organisms consist of a basic unit of life called an atom. Biomolecules are usually associated by and with organisms. In this lab, I studied the three of the four major units of life, which were lipids, proteins, and carbohydrates. To form these large macromolecules, dehydration synthesis occurs to join smaller molecules to create a larger molecule. And by doing this, water is taken out. To reverse this process, adding water to separate a large molecule to a smaller molecule is known by hydrolysis. With proteins, smaller subunits are called amino acids and with carbohydrates and lipids, their subunits are called monosaccharides and monomers. Peptide bonds hold amino acids together to form proteins by carbon to hydrogen covalent bonds and as well as carbs and lipids. Nonpolar bonds combine carbon and hydrogen bonds together in lipids. Based on my observations of the proteins, carbohydrates and lipid substances that were used in this lab, the protein pepsin would test positive of being chemically composed of amino acids rather than the other substances like distilled water, albumin, and starch through the Biuret reagent. If the protein pepsin turns to the color of purple it indicates amino acids are present hence claiming that it's positive. For the carbohydrates, the potato juice and starch suspension will chemically react to the Iodine (IKI) solution to show starch is present out of the other substances that will test negative such as the onion juice and glucose. The potato juice and starch suspension will turn a dark blue and black color indicating starch is present. Thirdly, to test the sugars in this lab, I predicted from my observations that the glucose and onion juice will show the chemical compositions of sugars present by using the Benedict's reagent. And so,

these substances will turn red or orange and positive unlike the other liquids like the potato juice, water and starch suspension. Lastly, I predicted that the emulsifier in tube two in the lipid experiment would work and be able to see the fat disperse throughout the water showing emulsification.

Materials and Methods: To test the presence of proteins, be sure to have Biuret reagent to test specifically for peptide bonds of amino acids. 1. Label 4 clean test tubes 1-4. Set in test tube rack. 2. Use a designated transfer pipet and add 1mL of the experimental solutions that will be used. (Distilled water, Albumin, Pepsin, and Starch) 3. Add 5 drops of the Biuret reagent to the tubes labeled 1-4, swirl contents so they can mix effectively. 4. As soon as you add the 5 drops, reactions occurs almost instantly. Record your data. 5. After your results are written down, keep in mind if any of the substances turned purple, peptide bonds or proteins were present, and if so, put a + mark next to the substance to indicate the positive chemical reaction, put a - mark if the substances maintained blue or their original color. To test for the presence of starch, make sure to a have Iodine solution (IKI). 1. Label 5 clean test tubes 1-5. Set in test tube rack. 2. Use a designated transfer pipet and add 1mL of experimental solutions that will be used. (Water, Starch Suspension, Onion juice, Potato juice, and Glucose solution) 3. Add 5 drops of the IKI solution to the tubes labeled 1-5 at the same time. 4. After your results and final color changes have been made, write down your data. Keep in mind, if any substances turn black/blue, or even an orange color with black specs at the bottom, it indicates that there was a positive reaction to starch present. Put a + mark

next to your recorded data. If any substance shows up yellow, put a - mark which indicates there was no starch present. For the Experimental Procedure: Microscopic Study For the Potato: 1. With a scalpel, use precaution, and slice a very thin piece of a potato. 2. To set the potato on a microscope slide, lay the piece of potato down first, add 1 drop of water on top, and carefully lay the coverslip on top to finish your slide. 3. Place the slide under the microscope and get it into focus. 4. Be able to find major components of the cell like, the cell wall (Large, geometric components) and the starch grains (Numerous clear and oval-shaped objects). 5. Now without removing the coverslip, add 2 drops of the iodine solution onto the microscope slide so the iodine is in contact with the cover slip. 6. Grab a small piece of paper towel and gently place it on the opposite side of the microscope slide to absorb the iodine solution through the potato. 7. Microscopically examine the potato close to where the iodine solution was placed. 8. Record and describe what you see. For the Onion: 1. Peel a layer of onion from the bulb and you’ll find a thin transparent layer of onion skin. 2. Place the onion skin on a new microscope slide. 3. Add a large drop of iodine solution. 4. Record what you see happen to the onion skin under the microscope. To test for the presence of sugars, make sure to have Benedict’s reagent to test specifically monosaccharides and disaccharides. 1. Prepare ahead of time boiling water to 100 degrees and label 5 clean test tubes 1-5. 2. Use a designated graduated transfer pipet, add 1 mL of the experimental solutions. ( Water, Glucose solution, Starch suspension, Onion juice, and Potato juice).

3. Add 5 drops of the Benedict’s solution to each of the test tubes at the same time. 4. Place all of the test tubes in the boiling water bath at the same time. 5. Wait approximately 15 minutes and you can see the color changes. 6. Remove all tubes from the hot bath and record your observations. 7. After your results are written down, keep in mind that the red color change or even orange color change indicates sugars are present and mark your data with a + mark. If the substances colors are green or blue, there was very low to no sugar present and you can mark your data with a - mark to indicate a negative reaction. To test the presence of fats, make sure to have a piece of loose leaf paper or brown paper. 1. Place a small drop of water on a square piece of brown paper. Watch what happens to the water. 2. Place a small drop of vegetable oil on a square piece of brown paper. Watch what happens to the oil. 3. Wait at least 15 minutes to record your observations. And let the paper dry. 4. Keep in mind, oils and fats do not evaporate. Evaluate which substance penetrates the paper and which is subject to evaporation. To test the emulsification of fats, make sure to have an emulsifier and the solutions needed. (Oil, water and milk). Label 3 clean test tubes 1-3 and use graduated transfer pipet to add solutions. Tube 1: 1. Add 3 mL of water and 1 mL of vegetable oil. Shake. 2. Observe for the initial dispersal of water. 3. Let the test tube sit for 5 minutes. 4. Label a microscope slide as #1 5. Use a dropped and place a drop from the sample onto the slide, add a coverslip and examine under low power on a light compound microscope. 6. Record your data/observations.

Tube 2: 1. Add 2 mL of water, 3 mL of vegetable oil, and 1 mL of emulsifier. (Either bile salts or Tween). Shake. 2. Describe the distribution of oil in tube 2 as compared to in tube 1. 3. Let the tube set for 5 minutes. 4. Label a microscope slide ad #2 5. Use a different dropper to remove some of the sample that is just below the layer of oil. Place drop on slide and carefully add a coverslip and examine under low power on a light compound microscope 6. Record your data/observations. Tube 3: 1. Add 1 mL of milk and 2 mL of water. Shake well. 2. Use a different dropper to remove a sample of the solution. Place drop on slide and carefully add a coverslip and examine under low power on a light compound microscope. 3. Record your data/observations. Results: Figure 1: Biuret Test for Proteins Tube

Contents

Final Color

Conclusion (+ or -)

1

Distilled Water

Blue

-

2

Albumin

Blue

-

3

Pepsin

Purple

+

4

Starch

Blue

-

The results of the protein test experiment with the Biuret reagent showed that three of the four substances chosen came out to be positive. The Pepsin came out positive to the chemical

reaction of the Biuret reagent due to either peptide bonds and amino acids present in the substance. Because this substance turned purple in color than the rest of the substances, it clearly states that only ¼ of the chosen substances contain amino acids and that’s due to the fact of that Pepsin is a large protein molecule which is shown above in Figure 1.

Figure 2: Iodine (IKI) Solution to test for Starches. Tube

Contents

Final Color

Conclusion (+ or -)

1

Water

Yellow

-

2

Starch Suspension

Black/Blue

+

3

Onion Juice

Light Yellow

-

4

Potato Juice

Black/Blue

+

5

Glucose Solution

Orange

-

The results of the Iodine solution test showed that only two out of the five substances were reported as positive which states that starch was present. The positive reaction that was given off of the potato juice and starch suspension shows that the final color was a black/blue color which indicates the polysaccharide, starch is molecule present in those substances and that is clearly stated about in Figure 2.

Figure 3: Benedict’s Solution to Tests for Sugars Tube

Contents

Color (After Heating)

Conclusions

1

Water

Blue

-

2

Glucose Solution

Red

+

3

Starch Suspension

Blue

-

4

Onion Juice

Orange

+

5

Potato Juice

Green/Yellow

+

As for the results of sugars present in specific substances that are stated above in Figure 3, a red, orange and yellow color appearance states that sugars are present in the substances like the glucose suspension, onion juice and potato juice. The glucose and water contents in this specific testing for sugar molecules are the positive and negative controls to base the color after heating information off of and these are the trends that were present in this specific experiment.

Figure 4: Brown Paper Test for Fats Sample

Observations

Conclusions

Water Spot

water dispersed

Damp towel

Oil Spot

Oil never dispersed

Left an oily spot

As the results of Figure 4, it clearly states that the water does not contain oil since the water did disperse on the brown paper during this test and after 15 minutes of leaving the water droplet on the paper, the paper didn’t remain saturated as the oil spot left. Therefore, showing the results of that oil does not evaporate, it left an oily stain on the paper towel after the 15 minutes.

Figure 5: Emulsification Test for Fats

Tube

Contents

Observations

Conclusions

1

Oil & Water

Globules were made

Water = polar Oil = nonpolar

2

Oil, Water & Emulsifier

Many globules were made in sections

Emulsifier broke down oil

3

Water & Milk

More globules present in water

Water = polar = nonpolar

Milk

From the results of the emulsification test, it clearly states in Figure 5 that the contents of oil, water and the emulsifier broke down the fat molecules present in the oil. The tube was shown with sections of three to show the emulsified fat, and water were separated out by density. And while under the microscope, a naked eye can not see the fats emulsion which had occurred with the oil, water and emulsifier contents in tube 2. Figure 6: Testing Unknown Substances for Presence of Fats, Sugars, Proteins and Starches

Sample Name

Protein (Biuret)

Starch (Iodine)

Sugar (Benedict’s)

Fat (Brown or loose-leaf)

Gatorade

None (-)

Light Brown (-)

Orange/Brown (+)

Evaporated (-)

Parmalat Milk

Cloudy Purple (+)

Cream Color (-)

Cream Color (-)

Evaporated (-)

Quinoa Whole Grain

None (-)

Black Speckled Grains (+)

Green (-)

Not evaporated(+)

Life Cereal

None (-)

Black (+)

Pale Yellow (-)

Not Evaporated (+)

Unknown A (Corn)

None (-)

Brown Speckled Grain (+)

Orange (+)

Not Evaporated (+)

Unknown B (Milkbone)

Cloudy Purple (+)

Black (+)

Yellow/Orange (+)

Not Evaporated (+)

As the results show in Figure 6, some of these foods test positive for all or more than one organic compound. To show positive for protein, a substance needs to turn a purple color, to show positive in starch, a black color is needed, to show positive for sugar after a hot boiling bath, and orange/ red is needed, and lastly for fat molecules to be present, they specific substance should not evaporate. As we see from the figure, sometimes with our normal everyday life foods, we figured out that their are surprisingly more organic molecules present

that can be used for our bodies in a good or bad way.

Discussion and Conclusion: As for the Biuret reagent experiment on the different contents that were used, distilled water, albumin, pepsin, and starch, the only substance that became positive during the test was pepsin. Pepsin is a large protein molecule which is composed of amino acids and to join those amino acids are peptide bonds. These amino acids and peptide bonds present made a positive chemical reaction to the Biuret reagent making the liquid substance turn to the color of purple indicating that protein is present. I have accepted my hypothesis to this experiment since I did observe that Pepsin is a protein due to its suffix, “in”, indicating it is a name of a protein. Since this reaction is often immediate, pepsin tested positive for proteins, even though it is the positive control in this test, and so did the unknown B in Figure 6 and the parmalat milk. Both foods and substances tested positive rather quickly after the Biuret reagent was added to these three substances. Therefore, my conclusion to the protein Biuret test accepted my hypothesis before the experiment and shows peptide bonds, proteins and even the subunits of proteins, amino acids were clearly present. As for the Iodine (IKI) solution experiment to test the presence of starch in substances, the contents used during this test, water, starch suspension, onion juice, potato juice, and glucose solution, only the starch suspension and potato juice tested positive. The starch suspension is the positive control for this experiment and the water is the negative control. As for the water, it was only dyed the color of the solution and not black/blue indicating there is no starch present. On the other hand, the potato and starch solution chemically reacted positively to the iodine solution stating that there is starch present. Also, the foods and unknowns from Figure 6, contained starch as well. The milkbone (Unknown B), life cereal, and the quinoa whole grain tested positive for starch with the iodine solution added. I accepted my hypothesis at the

end of this experiment because both my substances that I observed to be contained of starch are present by turning black in color. It was again accepted when I looked under the microscope of a thin slice of potato, and the starch granules began to fill up with the iodine indicating starch was present. To add on, some of the weaknesses that occurred during this experiment like the glucose solution, it has turned orange and I was rather confused on the fact if glucose actually did or did not contain starch. I found out that the black specs at the bottom of the tube did indicate that glucose does contain small amount of starch. Therefore, my conclusion to this procedure was positive towards the contents like the positive control, the starch suspension, the potato juice and glucose solution. As for the Benedict’s test for sugars present in either, water, glucose solution, starch suspension, onion juice and potato juice, only three came out positive for sugars. The glucose solution which is the positive control, turned a bright red color which indicated a high amount of sugar present. The onion juice turned a dark orange color which indicated not such a significant amount of sugars are present but some. And lastly the potato juice turned a yellow/green color which indicated a low amount of sugar. These recordings were stated after a hot, boiling bath of 100 degree water. The onion juice compared to the potato juice, the onion juice contains more sugar, due to its color change and the potato juice contains a lot more starch than sugar which again shows why the color change was yellow, not an orange. As for the other substances like water and the starch suspension, these both turned blue which is the same color as the indicator, showing no sugar is present. The water in this experiment is the negative control which shows if any substance is the color blue, it automatically shows there is no sugar present, just like in the starch solution which is not composed of sugar at all, only starch molecules. For the anonymous food and substances in Figure 6, the life cereal, the corn (Unknown A), and the milkbone (Unknown B) all tested positive for the presence of sugar. Again, an orangey color was expressed as a few sugars present which was shown by the corn, and the cereal and milkbone showed a yellow color which indicates a low amount of sugars

present. On the other hand, I have accepted my hypothesis to an extent. I did not include that the potato juice would be composed of sugar molecules rather than the onion juice and glucose solution. To add on, one source of error to this specific experiment might have been the temperature of the hot bath. In class, the two hot baths were used were recorded at different temperatures which can cause a source of error and even the amount of time the test tubes were left inside. Maybe if my group and I left the test tubes in a little bit longer, the potato juice might have turned a more vivid yellow rather than being a green/yellow color. Therefore, the conclusion to this experiment was that the glucose solution, onion juice, potato juice and lastly the life cereal, and both unknowns were composed of sugars due to their chemical color change. And lastly, as for the fat paper test, water and oil were tested to see whether or not those substances contains fats and evaporated. After the 15 minutes of waiting after one drop of each substance, it came to the conclusion that the oil does not evaporate due to the oil spot staying in one place on the brown paper and as the water spot dispersed, the paper became damp/almost dry showing water does evaporate off of paper or even a loose leaf paper. As for the unknown substances and foods on figure 6, the quinoa whole grain, life cereal, corn (Unknown A) and milkbone (Unknown B) were tested and was shown that these substances do contain fats/oils because they did not evaporate. On the other hand, the gatorade and parmalat milk did evaporate which shows it does not have or a ...