I have 3 lab simulations that need to be done. have the paperwork to go with them.

Solution

s& Dilutions, Acids &

Bases – Breaking Bad Badge

(Adapted from Biology Laboratory Manual

1

0th Edition, by

Darrell Vodopich & Hayden-McNeil Lab Simulations)

Solutions and Dilutions

Chemicals in living systems are in solution. A solution consists of a solute dissolved in a solvent. For example, salt water is a solution in which salt (the solute) is dissolved in water (the solvent). The concentration of solute in a solution can be expressed as either

a percentage of the total solution as weight/volume OR as a measurement of the Molarity of the solution. We will work on figuring out solution concentrations using both of these methods as we will be using several different solutions over the course of the semester.

Percentage (weight/volume)

For the percentage method, the percentage of solute is the number of grams of solute per

10

0 mL of solution (weight/volume). It does not matter what chemical or liquid the solute or solvent are – this is strictly a percentage. For example, a

3

% solution of sucrose is prepared by dissolving 30g of sucrose in 1L (1000 mL) of water (30 is 3% of 1000). If we wanted the same concentration of sucrose in final volume of only 100 mL, we would dissolve on 3g of sucrose in 100 mL of water (0.0

3

x 100 mL = 3g; 3g is 3% of 100). If we wanted 3% sucrose solution in a final volume of

5

00 mL; we would multiple 500 mL by 0.03 (3%), which equals

15

. In order to make 500 mL of a 3% solution, we would dissolve 15g of sucrose in 500 mL water. Now – let’s practice:

1) How many grams of sugar would you add to 100 mL of water to make a

2

5% solution?

25g

2) How many grams of calcium chloride would you add to 100 mL of water to make a 25% solution? __

25g

3) How many grams of calcium chloride would you add to 500 mL of water to make a 25% solution? _

12

5g

4

) What percentage solution would you have if you mixed 5g of sugar in 500 mL of water?

4. 1% solution as 5g is just 1% of 500ml (0.01×500 = 5)

1

5) If you only have 20g of sugar, what percent solution would you make if you dissolved it in 20 mL of water? 50%

What percent solution would you have if you dissolved the 20g of sugar in 200 mL of water? 10%

. What percent solution would you have if you dissolved it in 2L of water? 1%

Molarity

Molarity is the most common measure of concentration. It does matter what solute you use in this method of measuring concentration. The weight of

1

mole (M) of a chemical equals that chemical’s molecular weight in grams. A chemical’s molecular weight is the sum of the atomic weights of its component elements. For example, the molecular

weight of water is 1

8

g (2H = 2×1=2; O=1

6

; 16+2=18). A mole of water weighs 18g. A solution with a molarity of 1.0M would be the molecular weight of the solute dissolved in 1L (1000 mL) of solvent. For example, a liter of solution containing 58.5g of NaCl (MW=58.5g) is a 1M solution of NaCl. Like in the percentage method, if your final volume is less than 1L, you would reduce or increase the number of grams of solute proportionally. For example, if you only wanted 500 mL of a 1M NaCl solution, you would dissolve half of the amount of solute that you would in 1L (58.5g/2=2

9

.25g), since 500 mL is one half of the volume… Let’s practice!

1. How many grams of NaCl (MW=58.5g mole-1) would you dissolve in water to make a 2M NaCl solution in 1L final volume?

11

7

g

2. What is the molecular weight of Calcium chloride (CaCl2)?

110.98g/mol

. How many grams of CaCl

2

would you dissolve in water to make a 1.5M CaCl2 solution in 1000 mL final volume?

166.47g

In order to save space and time in the lab, scientist often make very concentrated solutions called stock solutions that they can later dilute with water to the molarity they need. This process is called dilution. The formula to determine how much of the stock solution is required for the desired molarity is:

Vi x Mi = Vf x Mf

Where Vi is the initial volume, Vf is the desired final volume, Mi is the initial molarity, and Mf is the desired final molarity.

For example, if you want to make a 1M solution of NaCl in 500 mL and your stock solution is 2M, then:

2

Vi x 2M = 500 mL x 1M

Vi = 500 mL x 1M / 2M = 250 mL

SO you would take 250 mL of the 2M NaCl and add 250 mL of water to get 1M NaCl solution in 500 mL.

Let’s practice determining concentrations and dilutions in the exercises below:

1. How many mL of a 6M NaCl stock solution should you add to make a 0.5M NaCl solution in a final volume of 250 mL?

20.83mL

2. If you only have 150 mL of 2M NaCl stock solution, do you have enough solution to make 200 mL of a 1M NaCl solution?

Yeah the solution of 150mL is more than enough because only 100mL is required.

3. How many mL of water would you need to make 250 mL of a 0.1M HCl solution? You have a 1M HCl stock solution already made.

225mL of water is added.

Acids and Bases

There are millions of chemical substances in the world. Some of them have acidic properties, others, basic properties. Acids are substances which release hydrogen ions (H+) when they are mixed with water. Bases are substances which release hydroxide ions (OH-) when they are mixed with water. (This freeing of ions is called dissociation in both cases). Free hydroxide ions react with the hydrogen ions producing water molecules: H+ + OH- = H2O. In this way, bases diminish the concentration of hydrogen ions. A solution rich in hydrogen ions is acidic, a solution poor in hydrogen ions is basic, or alkaline. Some acids dissociate only in part and they are called weak acids; others dissociate completely, freeing large amounts of hydrogen ions. These are called strong acids. In the same way, the bases can be stronger or weaker. Diluted acids and bases are less concentrated and less aggressive in their actions. The acidic or basic degree of substances is measured in

pH

units. The scale used spans from 0-

14

. Substances with

pH

lower than 7 are considered acids, those with pH equal to 7 are considered neutral, and those with pH higher than 7 are considered bases. Substances with low pH are very acidic, while those with high pH are highly basic. Concentrated acidic and basic substances are very corrosive and dangerous.

pH is the measure of the concentration of hydrogen ions in a solution. As this concentration can extend over several orders of magnitude, it is convenient to express it by means of logarithms of base ten. As this concentration is always less than one, its

3

logarithm always has the minus sign. To avoid having to always write the minus sign, it has been agreed to write this value with the positive sign. (This is the same as using the logarithm of the reciprocal of the hydrogen ion concentration). So, the pH is the logarithm of the concentration of hydrogen ions, with the sign changed: pH = -log [H+]. Thus, when pH has low values, the concentration of hydrogen ions is high.

There are substances which have the property of change their color when they come in contact with an acidic or basic environment. These substances are called pH indicators. Usually, they are used as dissolved substances, for example phenolphthalein and

bromothymol

blue

. Often, to measure the pH special papers which have been soaked with indicators are used. These papers change color when they are immersed in acidic or basic liquids. This is the case of the well-known litmus paper.

Procedure 1: Determine the pH of Various Substances 1. Find 10 samples around your house to test. They need to be in liquid format in order to be tested. Some examples of what you can test are coca-cola, mouthwash, soap (make sure to dilute with water first!), milk, water, lemon juice, etc. Determine the pH using the commercial pH indicator paper provided to you in your at-home kit. Record your results in the table provided.

a. pH papers – Immerse an end of the paper in the liquid you wish to examine and remove it immediately. The pH of the liquid is determined by comparing the color of the paper to the scale of colors printed on its packet. Record the pH reading in the table below.

Samples pH by pH paper

2

3

5

6

0	battery acid
	2	lemon juice, vinegar
	3	orange juice,
vinegar
soda
			4	tomato juice
				5	black coffee
bananas
				6	urine,
milk

1. Which substance was the most acidic? Which substance was the most basic?

4

The substance with the least pH is most acidic and the one with highest pH is the most basic.

2. Were there any solutions that had a neutral pH? If so, which ones were they?

The substance having the PH of 7 is considered neutral as it’s neither acidic nor basic. E.g. in the sample,

Water

has the neutral pH.

3. Were you surprised by any of your results? Why or why not?

Yes I was surprised to see that milk is acidic in nature

Log-in to the Hayden-McNeil lab simulation website (http://courses.haydenmcneil.com) and click on the “Acids, Bases, and pH Buffers” simulation. Read through the background material provided and then click on the gray arrow at the bottom of the page. Open up the simulation by clicking on the green button. The simulation directions are available on the website and below. Use the simulation for Procedures 2-4.

Procedure 2: Introduction to pH Indicators

1. Take six small test tubes from the Containers shelf and place them onto the workbench.

2. Label the test tubes 1 – 6 by clicking on the tube and typing in the name and add a reagent to each test tube according to the table below. All reagents can be found on the Materials shelf.

3. Take a pH meter from the Instruments shelf and place it in the first test tube. To properly attach the meter, remember to place your cursor over the test tube when dropping the pH meter onto it. Repeat this procedure for test tubes 2 – 6. Record the color and pH of all six solutions to reference later.

4. Take a 50 mL beaker from the Containers shelf and place it onto the workbench. 5. Add

5 mL

of

bromothymol blue

to the beaker.

6. Take a dropper from the container shelf and place it on the workbench.

7. Place the dropper into the beaker of bromothymol blue. You should observe the dropper filling with the liquid.

8. Using the dropper, add 2 drops of bromothymol blue to each test tube. Record the color and pH of all six solutions again after adding this material.

5

2

3

4

5

6

Test Tube Number	Solution	pH before addition of bromothymol blue	Color before addition of bromothymol blue	pH after addition of bromothymol blue	Color before addition of bromothymol blue
	1	10 mL water
10 mL acetone
10 mL 5 M citric acid
10 mL 5% vinegar
10 mL 4 M ammonia
10 mL diluted bleach

9. Clear your workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink.

Why is bromothymol blue considered a pH indicator?

A pH indicator is a chemical compound existing in neutral dyes and having the ability to detect change in pH via colour changes. Here, Bromothymol Blue is a weak acidic indicator showing colour change over a range of pH of 6 to 7.6.

How accurate of an indicator is the bromothymol blue?

Bromothymol Blue is considered a to have a good accuracy as it shows very distinct colours at even slight changes in pH.

Does it change to a different color for every pH examined?

No, Bromothymol can indicate the change in pH only in its specific range. Despite its accuracy, it’s unable to detect the pH range outside 6 – 7.6

Does the addition of indicator change the pH of the solutions at all? Explain why or why not.

As it’s a weak acid, addition of a small amount have Bromothymol have no significant change in pH pf the solution.

6

Based on your results with bomothymol blue, what color would a solution that was pH

13

.5 be?

A this pH the indicator would show Blue colour as it’s the Basic pH. Procedure 3: Phosphate Buffer System

Part 1: Set-up

1. Take four small test tubes from the Containers shelf and place them onto one side of the workbench.

2. Label the test tubes 1 – 4 and fill the test tubes with solutions from the Materials shelf, according to the table below.

Note: The combination of the two phosphate solutions in the fourth test tube is the standard phosphate buffer.

Read the labels of the solutions on the shelf carefully, so you do not confuse the two phosphate solutions. Sodium hydrogen phosphate (

Na2HPO4

) and sodium dihydrogen phosphate (

NaH2PO4

) are not the same thing.

1

5 mL

5 mL

Test Tube Set-up
Test Tube Number		Water	0.1 M Sodium Dihydrogen Phosphate	0.1 M Sodium Hydrogen Phosphate
		5 mL
2
3
4	2.5 mL	2.5 mL

Part 2: Response to Hydrochloric Acid

1. Take a pH meter from the Instruments shelf and place it into test tube 1. Repeat for test tubes 2 – 4.

2. Record the contents of each solution along with its pH in the results table below. 3. Take a 50 mL beaker from the Containers shelf and place it onto the workbench. 4. Add 10 mL of 0.5 M hydrochloric acid (HCl) to the beaker.

5. Take a dropper from the Containers shelf and place it onto the workbench.

6. Place the dropper into the beaker of HCl. You should observe the dropper filling with hydrochloric acid.

7. Move the dropper onto test tube 1 and add two drops.

8. Repeat step 7 with the other three test tubes.

7

9. Record the new pH of each test tube in the results table below. Indicate whether it was a positive or negative change.

10.Clear your workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink.

Part 3: Response to Sodium Hydroxide

1. Set up your workbench with four test tubes, as in Part 1.

2. Repeat the procedure outlined in Part 2, steps 1 – 10, using 0.5 M sodium hydroxide (NaOH) in your beaker instead of hydrochloric acid.

pH

Water

Results from Procedure 3: Phosphate Buffer System
HCl Results	NaOH Results
Test Tube Contents			pH	pH after HCl	Change in pH	pH after NaOH	Change inpH
NaH2PO4
Na2HPO4
NaH2PO4 and Na2HPO4

Questions:

1. Which solution was the least sensitive to the addition of acid or base?

The combined solution of NaH2PO4 and Na2HPO4 proves to be the best buffer due to the presence of both a weak acid and base. So, it’ll be least sensitive to the addition of acid or base.

2. Which of the four solutions tested is the best buffer against changes in pH caused by the addition of an acid or a base?

The combined buffer having both NaH2PO4 and Na2HPO4 proves to be the best buffer against the addition of both acids and bases.

8

Procedure 4: Buffering Capacity of a Phosphate Buffer

Part 1: Addition of Acid

1. Take a small test tube from the Containers shelf and place it onto the workbench. 2. Add 2.5 mL of 0.1 M sodium hydrogen phosphate (Na2HPO4) and 2.5 mL of 0.1 M sodium dihydrogen phosphate (NaH2PO4) to the test tube.

3. Take a pH meter from the Instruments shelf and place it in the test tube. Record the pH of the phosphate buffer solution in the results table below.

4. Take a 50 mL beaker from the Containers shelf and place it onto the workbench. 5. Add 20 mL of 0.5 M hydrochloric acid (HCl) to the beaker.

6. Take a dropper from the Containers shelf and place it onto the workbench.

7. Place the dropper into the beaker of hydrochloric acid. You should observe the dropper filling with hydrochloric acid.

8. Add 1 drop of hydrochloric acid to the test tube.

9. Record the pH every time you add another drop of HCl in the table below.

10.Repeat steps 8 and 9 until the pH falls below 3 or until you have dispensed a total of 15 drops, whichever is reached first. Make sure that you record the pH after each drop is added.

11.Clear your workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink.

Part 2: Addition of Base

1. Repeat the procedure outlined in Part 1, steps 1 – 10, using 0.5 M sodium hydroxide (NaOH) in your beaker. Add the base until the pH rises above 12 or until you have dispensed a total of 15 drops, whichever is reached first. Record results in the table below.

2. Clear your workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink.

9

pH

1

1

2

2

3

3

4

4

5

5

6

6

7

7

8

9

9

10

11

12

13

14

15

Results from Procedure 4: Buffering Capacity of a Phosphate Buffer
Drops Acid	pH	Drops Base
	8
	10
	11
	12
	13
	14
	15

Questions:

1. The strong acid and strong base used in this lab are the same concentration. Thus, the magnitude of the pH change caused by addition of a single drop will be the same on either side of the starting pH. Addition of acid lessens the pH and addition of a bases raises the pH.

Construct a graph of the pH versus the number of drops of acid or base added to the phosphate buffer. To show the relative magnitudes, use a negative value for each drop of acid and a positive value for each drop of base. For example, use -7 to write 7 drops of acid and 5 to show 5 drops of base. Copy the graph you construct and paste into this document to turn in.

10

2. Suppose you had a buffer containing 0.5 moles of sodium dihydrogen phosphate and 0.5 moles of sodium hydrogen phosphate. How many moles of hydrochloric acid would this phosphate buffer be able to accept before the pH of the solution began to change drastically?

As the pKa value for the ionization reaction of NaH2PO4 and Na2HPO4 is 7.21. A pH change of 1 (unity) is considered a drastic change. So according to Henderson’s Hesselbach’s equation,

pH= pKa + log[A-]/[HA]

Considering a change in pH pf 1,

pH= pKa-1= 7.21-1

pH= 6.21

So,

6.21=7.21+log[A-]/[HA]

-1=log[A-]/[HA]

0.1=[A-]/[HA]

From this ratio we can change moment to moment change in pH by addition of acid.

As the buffer pair is of 0.5M each, we can say

0.1=(0.5-x)/(0.5+x)

0.05+0.1x= 0.5-x

1.1x=0.45

x=0.41 moles

So the number of moles pH HCl before the drastic change in pH is 0.41moles 11

3. Which chemical provides the conjugate base in the buffer containing NaH2PO4 and Na2HPO4?

A strong acid gives the conjugate base. For this buffer pair, Carbonic Acid (H2CO3) gives the conjugate base.

4. Explain why phosphate buffer needs both NaH2PO4 and Na2HPO4in order to resist changes in pH.

An excellent pH buffer has the ability to resist the change in pH in either direction i.e. by the addition of an acid or a base. By the addition of acid, there’s a weak base to neutralize it and vice versa. In this case, NaH2PO4 is the weak acid and Na2HPO4 is the weak base. So this buffer uses both of them for it’s working.

5. Predict what might happen if you made up phosphate buffer with only half as much NaH2PO4 compared to Na2HPO4.

As NaH2PO4 is a weak acid, in this condition, the buffer will have a reduced ability to resist the change in pH by the addition of a base. So, the addition of half the original quantity of base will cause a drastic change in pH.

6. Your lab mate attempts to use bromothymol blue to differentiate between two solutions – one that should be pH 7.3, and another that should be pH 6.7. What is your advice to your lab mate? Do you agree with his decision?

Yes I agree with the decision. As the normal range of pH for Bromothymol Blue is 6 – 7.6. So it’s an excellent decision to use this indicator for the solutions having pH within its range.

I would advise him to be precarious about having a contact of Bromothymol blue to his eyes, clothing or skin. Moreover, strictly avoid inhalation or ingestion of the solution.

12

2

>

Macromolecules: Carbs,

Proteins

, and

Lipids

–
Good Eats Badge

(Adapted from Biology Laboratory Manual

1

0th Edition, by Darrell Vodopich and Randy Moore)

Most organic compounds in living organisms are carbohydrates, proteins, lipids, or nucleic acids. Each of these macromolecules is a polymer made of smaller subunits, or monomers. These monomers are linked together in a reaction known as dehydration synthesis, which is an energy-requiring process in which a molecule of water is removed and the two subunits are bonded covalently. We can break the bonds between the subunits to release energy and separate the monomers gain by adding a water molecule in a process known as hydrolysis.

Scientists have devised several biochemical tests to identify the major types of organic compounds in living organisms. Each of these tests involves two or more treatments: (1) an unknown solution to be identified, and (2) controls to provide standards for comparison. As its name implies, an unknown solution may or may not contain the substance that the investigator is trying to detect. Only a carefully conducted experiment will reveal its contents. In contrast, controls are known solutions. We use controls to validate that our procedure is detecting what we expect it to detect and nothing more. During the experiment, we compare the unknown solutions’ response to the experimental procedure with the control’s response to that same procedure.

In our scientific method lab, we introduced the concept of a control group: a group that is subjected to the same conditions as the experimental group EXCEPT for the independent variable. This was to show that any differences observed were due to the independent variable alone and not to other factors within the procedure. But what if there were no differences detected between the groups? Could you be sure that it was due to the independent variable and not that there was a problem with the experimental procedure? A way to be sure is to introduce a new kind of control group: the positive control. A positive control contains the variable for which you are testing and should react in a predictable way in the procedure. For example, if you are testing a solution to see if it contains protein, an appropriate positive control is a solution that is known to contain protein and there should result in a positive reaction in your test. A positive reaction indicates that your test is working as expected and shows you what a positive result should look like. You can then compare the unknown and other samples with this known positive to check for the presence of the molecule.

Even with a positive control we still need to show that any positive reaction is due only to the variable that we are testing. Therefore, we still need the negative control. The negative control does not contain the variable for which you are searching. It contains only the solvent (often distilled water with no solute) and should not react in the test. A negative control shows you what a negative result looks like.

In this lab, we are going to use chemical tests to detect macromolecules in known and unknown solutions. We will employ both negative and positive controls to be sure that our tests are working properly and that we can trust our results. You will be working in groups of

4

students (your table) to be “food detectives” and solve the mystery of your unknown solution.

Experimental Procedures

Log-in to the Hayden-McNeil lab simulation website (

http://courses.haydenmcneil.com

) and click on the “Biological Molecules” simulation. Read through the background material provided and then click on the gray arrow at the bottom of the page. Open up the simulation by clicking on the green button. The simulation directions are available on the website and below. Use the simulation for Procedures 1-5.

Carbohydrates:

Carbohydrates are made up of monosaccharides. Many monosaccharides such as glucose and fructose are reducing sugars, meaning that they possess chemical groups that can reduce weak oxidizing agents like the copper in Benedict’s reagent. Benedict’s test identifies reducing sugars based on their ability to reduce the cupric ions (blue) to cuprous oxide (green to reddish orange) at high pH. A green solution indicates a small amount of reducing sugars, and reddish orange indicates an abundance of reducing sugars. Non-reducing sugars like disaccharides (sucrose) are not able produce a color change.

Starch

is a form of carbohydrates produced by plants. It is a highly coiled polymer of individual glucose units. Iodine interacts with only coiled polymer molecules of glucose (like starch) and becomes bluish black. Iodine does not interact with uncoiled sugars such as disaccharides (sucrose) and monosaccharides (glucose) and therefore remains yellowish brown in the presence of these sugars.

In summary – what color changes are we looking for with each test?

Color Changes	Positive Result	Negative Result
Benedict’s Test
Iodine/Starch Test

Procedure 1: Test for

Reducing Sugars

1. Take a constant temperature bath from the Instruments shelf and place it onto the workbench.

2. Set the constant temperature bath to 100 °C.

3

. Take a test tube from the Containers shelf and place it onto the workbench.

4. Take the 5% glucose solution from the Materials shelf and add 6 mL to the test tube. Record the color of the solution in the table below.

5. Take Benedict’s solution from the Materials shelf and add 6 mL to the test tube. Record any color change in the table below.

6. Move the test tube into the constant temperature bath. Wait a few moments to see if the color changes. The color change usually begins at temperatures greater than 60 °C, but will finish by the time the solution reaches 100 °C. Take a thermometer from the Materials shelf and attach it to the test tube to monitor its temperature. Record any color changes.

7. Empty the test tube in the waste bin, then place the empty test tube in the sink.

8. Repeat steps 3 – 8 two more times, replacing the 5% glucose solution in step 4 with:

· 5% sucrose

· water

9. Clear your station by dragging all instruments back to the Instruments shelf and by emptying all containers in the waste bin and then placing the empty containers in the sink.

			Solution				Initial Color	Color with Benedict’s Solution	Color after Heating
5% Glucose
5% Sucrose
			Water

Which solution(s) did not contain reducing sugars?

What was the purpose of the water?

Procedure 2: Test for Starch

1. Take two test tubes and a 250 mL beaker from the Containers shelf and place them onto the workbench.

2. Add 90 mL of water and 10 mL of Lugol’s iodine from the Materials shelf to the 250 mL beaker. Rename the beaker “diluted Lugol’s iodine” by double-clicking on it.

3. Take the 2% starch solution from the Materials shelf and add 6 mL to one test tube.

4. Take the water from the Materials shelf and add 6 mL to the other test tube.

5. Record the color of the solutions in the test tubes in the table below.

6. Add 6 mL of the diluted Lugol’s iodine solution in the 250 mL beaker to each test tube.

7. Observe what happens in each test tube. Record your observations.

8. Empty the two test tubes in the waste bin, then place the empty test tubes in the sink. Keep the beaker of diluted Lugol’s iodine on the workbench to use in Experiment 5.

Solution

Initial Color

Water

Starch Test Results
Color with Iodine Solution
	Starch

Which solution(s) reacted with the iodine solution?

Which solution is considered to be the negative control?

Based on your knowledge of what potatoes are made of, develop a hypothesis for testing whether they contain starch or not. What is your prediction for the outcome of this experiment?

Procedure 3: Test for Proteins

Proteins are polymers made up of amino acids. A peptide bond forms between the amino acid and the carboxyl group of an adjacent amino acid and is identified by a Biuret test. Specifically, peptide bonds in proteins complex with Cu2+ in Biuret reagent and produce a violet color. A Cu2+ must complex with four to six peptide bonds to produce a color; therefore individual amino acids to not react positively. Proteins have many peptide bonds and produce a positive reaction.

Biuret Test for Proteins

1. Take two test tubes from the Containers shelf and place them onto the workbench.

 

2. Take the 35% egg albumin from the Materials shelf and add 6 mL to the first test tube. 

3. Take the water from the Materials shelf and add 6 mL to the second test tube.

4. Record the colors of both solutions in the table below.

5. Take the biuret solution from the Materials shelf and add 6 mL to each test tube.

6. Observe any color changes in the two test tubes. Record your observations.

7. Empty the test tubes in the waste bin, then place the empty test tubes in the sink.

Solution

Initial Color

Water

	Biuret Test Results
Color with Biuret Solution
35% Egg Albumin

Which solution(s) contained protein?

Which solution is the negative control?

Procedure 4: Test for Lipids

Lipids include a variety of molecules that dissolve in non-polar solvents such as ether, acetone, methanol, or ethanol, but not as well in polar solvents such as water. Triglycerides are abundant lipids made of glycerol and three fatty acids. Tests for lipids are based on a lipid’s ability to selectively absorb pigments in fat-soluble dyes such as Sudan III.

Sudan III Test for Lipids

1. Take two test tubes from the Containers shelf and place them onto the workbench.

2. Take the corn oil from the Materials shelf and add 6 mL to one test tube. 

3. Take the water from the Materials shelf and add 6 mL to the other test tube.

4. Record the initial colors of the liquids in the test tubes in the table below.

5. Take the Sudan III solution from the Materials shelf and add 6 mL to each test tube. 

6. Observe any potential color changes in the two test tubes. Record your observations.

7. Empty the test tubes in the waste bin, then place the empty test tubes in the sink.

Biuret Test Results

Solution

Initial Color

Water

Color with Sudan III Solution

Corn Oil

Which solution(s) contained lipids?

Which solution was the negative control?

Procedure 5: Test the Contents of

Food

s

For each food sample, do the following:

1. Take four test tubes from the Containers shelf and place them onto the workbench. Based on steps 2 and 3, label each test tube with the material and test being performed. For example, label the test tube used to test onion juice for the presence of reducing sugars as “

Onion Juice

Benedict’s Solution”.

2. Add the reagents to the test tubes as directed in the table below. Be sure to use the diluted Lugol’s iodine you made in Experiment 2.

Test Tube	Reagent
1	6 mL Benedict’s solution
2	6 mL diluted Lugol’s iodine solution
3	6 mL Sudan III solution
4	6 mL biuret solution

3. Take the potato juice from the Materials shelf and add 6 mL to each of the four test tubes. Observe and record all color changes in the table below. Remember that the test tube with the Benedict’s solution requires heating in a 100 °C constant temperature bath for the color change in the presence of reducing sugars to occur. Recall that you can use a thermometer to monitor the heating.

4. Empty the test tubes in the waste bin, then place the empty test tubes in the sink.

5. Repeat steps 1 – 4 for the onion juice, whole milk, and skim milk. Be sure to record all of your observations.

6. After you record the color changes in the 4 foods. Record whether each macromolecule was present or absent from each of the four foods by recording a plus (+) sign when the macromolecule is present. Record a negative ( – ) sign if the macromolecule is absent from the food group.

7. Clear the bench of all materials, containers, and instruments, then return to your course page to complete any assignments for this lab.

Starch

Color

+/-

Color

+/-

Color

+/-

Results of Food Test
Food	Reducing Sugars	Lipids	Proteins
			Color				+/-
Potato Juice
Onion Juice
Whole Milk
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Diffusion and Osmosis –
Osmosis Jones Badge

(Adapted from Biology Laboratory Manual

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th Edition, by Darrell Vodopich and Randy Moore)

All molecules display random thermal motion or kinetic energy: this is why a dissolved molecule tends to move around in a solution. Kinetic energy cause molecules to diffuse outward from high concentration to lower concentrations. The process of molecules moving from an area of higher concentration to an area of lower concentration is called diffusion. This random movement is constant, but the net movement of molecules from areas of high concentration to areas of low concentration continues until the distribution of molecules is even throughout the solution. This point is called equilibrium. At equilibrium, the random motion continues, but there is no net change in the concentration of solute through the solution. In some cases, the areas with differing concentrations are separated by a membrane that will only let water and not solutes pass through it. In this case, the movement of water down its concentration gradient is called osmosis.

Heat cause an increase in the random motion of molecules that can passively move in biological systems. This motion was originally described by Robert Browning and so it is called Brownian motion (or movement). Although we cannot directly see molecules move, we can see small particles move after they collide with moving molecules. In this lab exercise, we are going to observe diffusion and what causes diffusion rates to change, diffusion through a semi-permeable membrane, and osmosis.

Procedure 1: **ONLINE SIMULATION**

Login to the Hayden-McNeil Lab Simulation (

https://courses.hayden-mcneil.com/local/ecologin/

). Click on the blue notebook next to the “Expanded Osmosis and Diffusion” module. Read through the introduction information and click the forward arrow at the bottom of the page. Click on the green button to launch the simulation. Complete the following exercises in this module.

Part 1: Membrane Size Selectivity

1. Take a diffusion bag and a 2

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mL beaker from the Containers shelf and place them onto the workbench.

2. Fill the bag with

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5 mL of water and 5 mL of Lugol’s iodine solution from the Materials shelf. 

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. Fill the beaker with 150 mL of water and 50 mL of 2% starch solution. Record the color of the liquid in the beaker and of the liquid in the diffusion bag to reference later.

4. Move the diffusion bag into the beaker.

5. Watch the beaker and bag for signs of color change. After 30 seconds, move the diffusion bag out of the beaker and onto the workbench. Answer questions below.

6. Empty the beaker and diffusion bag into the waste bin, then place the empty containers in the sink.

Questions:

1) Initially, Lugol’s iodine was placed inside the diffusion bag, and starch was placed outside the bag. Which of these materials was able to pass through the diffusion membrane? How do you know?

2) Which material did NOT diffuse across the membrane? Why not?

Part 2: Dependence on the Rate of Diffusion on the Concentration Gradient

7. Take four diffusion bags from the Containers shelf and place them onto the workbench. Double-click the diffusion bags and change the labels to 1, 2, 3, and 4.

8. Make up the following four solutions in the four dialysis bags with items from the Materials shelf.

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Dialysis Bag	10% Acetic Acid (mL)	Water (mL)	Final Concentration of Acetic Acid (%)
	1	50	0
	2		25
	3	12.5	37.5
	4	5	45

9. Calculate the final concentration of acetic acid (%) in the diffusion bags. Record these values to reference later.

a. The first diffusion bag has 10% acetic acid.

b. The second bag contains a total of 50% (50 mL of 100 mL total) of 10% acetic acid. Therefore, the second bag contains a final concentration of 5% acetic acid (50% multiplied by 10%).

10. Take four 250 mL beakers from the Containers shelf and place them onto the workbench. Double-click the beakers to label them 1 – 4. Fill each beaker with 200 mL water. 

11. Take four pH meters from the Instruments shelf and place one into each beaker. Record the pH of each beaker under the “

pH at 0 min

utes” row in the table below.

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2

3

4

Diffusion Bag

Acetic Acid Concentration

pH at 0 min

pH at 1 min

12. Take diffusion bag 1 and place it into beaker 1. Start a timer or note the time on the lab clock. This reaction should run for 1 minute.

13. After 1 minute, remove the diffusion bag from the beaker and record the pH of the liquid in the beaker in table above.

14. Repeat steps 6 – 7 for the remaining diffusion bag and beaker pairs.

15. Clear the workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink. Answer the question on the next page.

Question:

Explain how the rate of diffusion changes according to the concentration gradient.

Part 3: Evidence for Osmosis

1. Take a 250 mL beaker from the Containers shelf and place it onto the workbench.

2. Add 200 mL of 35% egg albumin solution from the Materials shelf to the beaker. Record the contents of this beaker below.

Observations:

3. Take a balance from the Instruments shelf and place it onto the workbench. Place the beaker on the balance. Click the Zero button to set the starting mass to zero. Move the beaker from the balance to the workbench.

4. Take a diffusion bag from the Containers shelf and place it onto the workbench.

5. Add 50 mL of water from the Materials shelf to the diffusion bag. Record the contents of the diffusion bag below.

Observations:

6. Move the diffusion bag into the beaker containing the egg albumin solution. Start a timer or note the time on the lab clock and wait 1 minute.

7. While you are waiting for the 1 minute to elapse, take two test tubes from the Containers shelf and place them onto the workbench.

8. Add 5 mL of biuret solution from the Materials shelf to each test tube. Record the color of the liquid below.

Observations:

9. When the 1 minute has elapsed, move the diffusion bag out of the beaker and onto the workbench.

10. Place the beaker on the balance and record its mass. Write down whether the beaker has gained or lost mass, no matter how slight.

Observations:

11. Perform a biuret test for the presence of protein in both the beaker and diffusion bag. This test will determine if the dissolved albumin moved out of the beaker and into the diffusion bag or whether only water moved out of the diffusion bag and into the beaker.

  

 

a. Take two droppers from the Containers shelf and place them onto the workbench.

b. Place one dropper into the solution in the beaker. You should observe the dropper filling with liquid.

c. Place this dropper onto the first test tube containing biuret solution. Select the Pour All option. Record any color changes in the test tube. If there is protein present in the solution from the dropper, the biuret solution will turn purple. Record your observations below.

Observations:

d. Place the second dropper into the diffusion bag. You should observe this dropper filling with liquid.

e. Place the dropper in the second test tube filled with biuret solution. Dispense the entire contents of the dropper. Look for any color changes in the test tube. Record your observations.

Observations:

12. Clear the workbench by dragging instruments back to the Instruments shelf and by emptying containers in the waste bin and then placing the empty containers in the sink. Answer question below.

Questions:

1) What was the amount of mass change for the solution in the beaker at the end of the experiment? Report 0 for no change, and use + or – signs to indicate a mass gain or mass loss respectively.

2) Explain what caused an imbalance in osmotic pressure on either side of the diffusion bag membrane.

Part 4: Concentration Gradients and Osmosis

1. Take four diffusion bags from the Containers shelf and place them onto the workbench.

2. Double-click the diffusion bags to label them 1, 2, 3, and 4. Fill the diffusion bags according to the chart below with items from the Materials shelf.

Diffusion Bag

20% Fructooligosaccharides Solution (mL)

Water (mL)

Final % of Fructooligosaccharides

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50

0

2

37.5

12.5

3

25

25

4

12.5

37.5

3. Calculate the final percent (%) fructooligosaccharides in each diffusion bag. Record these calculations in the table.

a. The first diffusion bag has 20% fructooligosaccharides.

b. The second bag contains a total of 75% (75 mL of 100 mL total) of 20% fructooligosaccharides. Therefore, the second bag contains a final concentration of 15% fructooligosaccharides (75% multiplied by 20%).

4. Take four 250 mL beakers from the Containers shelf and place them onto the workbench.

5. Fill each of the four beakers with 100 mL 20% fructooligosaccharides solution and 100 mL of water from the Materials shelf. Record the final concentration of fructooligosaccharides in each of these beakers. _____________________.

6. Take a balance from the Instruments shelf and place it onto the workbench.

7. Weigh each of the full beakers and record these initial masses in the table below.

8. Place one diffusion bag next to each beaker. You will run four osmosis trials of 1 minute each by doing the following:

a. Place one diffusion bag into its corresponding beaker.

b. Immediately start a timer or note the time on the lab clock.

c. When the minute has elapsed, remove the diffusion bag from the beaker and place the beaker on the balance. Record the new mass of the beaker in the table below. Record whether it increased in mass, decreased in mass, or did not change in mass in the table below.. 

d. Repeat steps a – c for each diffusion bag and beaker combination.

Diffusion Bag

1

2

3

4

Beaker Initial Weight

Beaker Final Weight

Change in Beaker Mass

9. Double-check that you have recorded all of the necessary information. Clear the bench of all materials, containers, and instruments. Answer the questions below.

Questions:

1) For each of the four bag/beaker systems explain which substance moved in order to cause change in mass and what caused the substance to move.

2) Suppose you are given a diffusion bag that is permeable to both water and a protein. You fill the diffusion bag such that it contains 99% water and 1% protein. You place the diffusion bag into a beaker filled with 98% water and 2% protein. Predict what will happen next.

3) Define Osmosis.

4) Suppose you are given a container that has a membrane permeable only to metabolic wastes. The container is filled with 5M metabolic waste in an aqueous solution. You wish to reduce the amount of waste dissolved in the container. You see a 5L beaker of pure water sitting across the room on a shelf. What is your plan?

Part 5: Osmosis in Living Cells

1. Take two empty slides from the Containers shelf and place them on one side of the workbench. 

2. Add Elodea from the Materials shelf to each slide. One Elodea leaf and a drop of aquarium water will automatically load onto the slide for you.  

3. Take a microscope from the Instruments shelf and place it onto the workbench.

  

4. Examine one slide with the microscope. Increase the magnification, if necessary. Identify the cell walls, cell membrane, cytoplasm, and chloroplasts. How close is the cell membrane and cytoplasm to the cell wall? Take a screenshot of your image and upload below.

5. Move the slide from the microscope back to the workbench.

6. Take a test tube and a dropper from the Containers shelf and place them on the workbench.

7. Add 10 mL of hypertonic solution from the Materials shelf to the test tube.

8. Use the dropper to transfer one drop of the hypertonic solution to the slide. 

9. Return the slide to the microscope. Record any changes you observe. Are your observations what you expected? Record your answer and an explanation for your answer to reference later. Save a screenshot of the Elodea in the hypertonic solution in order to upload below.

10. Remove the slide with hypertonic solution from the microscope and empty its contents into the waste container. Note: Elodea is an invasive species in many areas and thus should not be released directly to the environment. Place the empty slide in the sink.  

11. Empty the dropper and test tube in the waste bin, then place them in the sink.

12. Take a test tube and a dropper from the Containers shelf and place them on the workbench.

13. Add 10 mL of the hypotonic solution from the Materials shelf to the test tube. Use the dropper to transfer one drop of the hypotonic solution to the second slide. 

14. Examine the slide with the microscope. Record your observations. Again, be sure to observe the cell membrane, cell wall, cytoplasm, and chloroplasts. Save a screenshot of the Elodea in the hypotonic solution in order to upload below.

15. Clear the bench of all materials, containers, and instruments, then return to your course page to complete any assignment for this lab.

Questions:

1) Upload your Elodea picture from step 4 here.

2) Upload your Elodea picture from step 9 here.

3) Upload your Elodea picture from step 14 here.

4) How did the Elodea cells change when aquarium water was replaced with hypertonic solution? What caused those changes?

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