Polarity, pH, and Buffers

Before we Begin

● You will be utilizing chemicals in the following laboratory procedures, please be sure you
○ Have a clear space to conduct the experiments such as a kitchen table or if it is nice outside a flat area of driveway or picnic table.
○ Wear gloves and eye protection.
○ Read instructions thoroughly and completely prior to beginning.
○ For chromatography use forceps, tweezers or a small pair of kitchen tongs to handle the paper with gloved hands. It is important the paper does not touch skin as the skin has oils on it and can be absorbed by the paper and impact the experiment.
● There are several activities in this laboratory, please be sure to complete all activities. Questions and ‘next steps’ will be indicated in bold in the text.

Overview

In the world of molecular bonding there are several types of bonds molecules use to attach to one another: covalent bonds, ionic bonds, hydrogen bonds and van der waals interactions. The first part of this laboratory will focus on covalent bonds, specifically polar and nonpolar covalent bonds. Following our polarity lab exercise, part two will discuss the power of hydrogen ions and test the effectiveness of two buffers and tap water.

Part I: Polarity

Learning Objectives

● Define polar and nonpolar bonds
● Differentiate between polar and nonpolar bonds
● Identify polar and nonpolar pigments using paper chromatography

Background

Chromatography is a technique that is used to separate molecules from each other on the basis of their solubility in particular solvents. A common solvent for chromatography is acetone, which is less polar than water. When the chromatography paper is spotted with pigments and set in acetone, the solvent wicks up the paper and the more nonpolar pigments move with it. The more nonpolar a pigment, the more soluble it is in the nonpolar solvent, and the faster and farther it proceeds up the chromatography paper. The chromatography paper is made of cellulose and is a polar substance. The more polar pigments will be attracted to the polar chromatography paper and will migrate up the paper slowly (See figure 1). Paper chromatography utilizes differences in polarity between the solvent and the paper to separate mixtures of polar and nonpolar molecules.

Figure 1. Schematic of chromatography experiment. (1) acetone, (2) chromatography paper, (3) sample containing polar and nonpolar pigments (4) samples that contain ‘more polar’ or ‘more nonpolar’ pigments as evidenced by their migration up the paper. More nonpolar pigments migrate farther up the paper than polar pigments. (5) final line of migration when experiment is finished. (6) Initial line where all extracts were dotted prior to placing in acetone. Figure credit:
https://en.wikibooks.org/wiki/ Structural_Biochemistry/Chromatography/Paper#

Why do we care?

Molecules range greatly in polarity. Polarity will determine how molecules interact; for example, water is polar and therefore will dissolve polar molecules. Polarity is important for two main reasons; first, polar molecules ‘orient’ with respect to other molecules. Due to this, polarity plays a role in the three dimensional structure of molecules, like fatty acids. Fatty acids have a polar end and a nonpolar end; when added to water the molecules flip so the nonpolar ends are away from the water, on the inside, and this gives the basic structure of membranes (ie. phospholipid molecules), important for all living organisms. Secondly, polarity is important for understanding the geometry and chemical characteristics of large molecules, like proteins. The geometry or structure of a protein is, in part, due to it’s polar groups. Polar groups stabilize our proteins and allow them to interact with other proteins, necessary for metabolism and other functions that keep us alive. Have a look at Figure 2; the diversity of polar and nonpolar substances that you may interact with while going about your day.

Figure 2. Polarity of different solvents and their most common contemporary or historical uses.

This lab

For this lab we will be using leaves which contain both polar and nonpolar pigments. A pigment is a substance that absorbs light. The principal pigment in plants is chlorophyll a which is nonpolar; however has polar groups (contain oxygen). A measure of how nonpolar versus polar a pigment is can be determined by counting the number of oxygen groups the pigment’s chemical structure has.Plants with leaves that exhibit bright color changes such as oak or maple leaves contain anthocyanins which are polar molecules. Other nonpolar pigments include chlorophyll b, carotenes, and xanthophylls, play a secondary role in photosynthesis and are found in many plants.

Below are examples of pigments from plants. Predict which ones are polar or nonpolar based on their structure. Which would you expect to travel higher on the chromatography paper?

1. Circle your answer below for each pigment in the figure below. Predict pigment polarity keeping in mind:
(i) To determine polarity of the pigments below, count the number of oxygens in each molecule. The more oxygens, the more polar.
a. Which pigment is most polar?
b. Which pigment is most nonpolar?

2. Given the four pigments above hypothesize where they will be on the chromatography paper if you ran an experiment with these specific pigments. Label the chromatography paper below with chlorophyll a, chlorophyll b, xanthophyll and beta-carotene.

Keep in mind that:

1) Polar molecules are attracted to polar molecules, and nonpolar molecules are attracted to nonpolar molecules.
2) The solvent, acetone, is relatively nonpolar.
3) The chromatography paper is polar.
4) The most nonpolar pigment will be attracted to the nonpolar solvent and travel up the chromatography paper the fastest.

Procedure 1: Polarity

1. Head outside and grab 2 leaves preferably different colors or different shades of green at the very least. The more different the colors the more interesting your chromatography results may be. Oak and maple leaves work well for this experiment.
2. Decide which leaf will be leaf #1 and #2
3. Put on gloves and eye protection
4. Grab 2 ziplock bags, one for each leaf you collected. Label one for each leaf, #1 and #2.
5. Cut up each leaf and put it in its own zip lock bag. Add 2 tablespoons of nail polish remover and seal bag, removing as much air as possible, by zipping it and then using scotch tape to further tape the zipped end to help prevent liquid from seeping out while you mash the leaves.
6. Time to crush those leaves! Using a rolling pin or the bottom of a glass, take turns mashing your bagged leaves for 2 minutes alternating a ‘rolling’ motion with a ‘pounding’ motion.
Checkpoint: You should now have 2 separate, labeled, zip lock bags with mashed up leaf in them.

7. Place out 2 pieces of aluminum foil, one for each leaf you crushed (#1 and #2). Label the foil sheets #1 and #2 and shape so they are ‘curved’ by bending up the sides and corners so the fluid can’t escape when you dump it into the foil ‘holder’.
8. Cut some cheesecloth and place it over the foil ‘holder’ you just created.
Checkpoint: You should now have 2 separate, labeled foil holders with some cheesecloth draped over each one.

9. Move all contents of mashed leaf #1 to the bottom of it’s zip lock bag and cut open the top of the ziplock with scissors.
10. Pour the contents onto the cheesecloth which should be draped over your foil holder, being careful to pour into the center of the cheesecloth to capture as much leaf debris as possible as the liquid filters into the foil holder.
11. Fold over the cheesecloth and give it a squeeze to get the liquid all out.
12. Discard cheesecloth and the trapped leaf debris.
13. Move all contents of mashed leaf #2 to the bottom of it’s zip lock bag and cut open the top of the ziplock with scissors.
14. Pour the contents onto the cheesecloth which should be draped over your foil holder, being careful to pour into the center of the cheesecloth to capture as much leaf debris as possible as the liquid filters into the foil holder.
15. Fold over the cheesecloth and give it a squeeze to get the liquid all out.
16. Discard cheesecloth and the trapped leaf debris.
Checkpoint: You should now have 2 labeled foil ‘holders’. Each one should have some liquid in it from your mashed leaf. Put those off to the side for a moment.

17. Put on fresh gloves and have your forceps ready.
18. Pull out the chromatography paper, handled by its edges with gloved hands or with forceps/tweezers. It is very important that you do not get skin oil on the paper, it will mess up the experiment!
19. Using a pencil and a ruler, draw a straight line 1 inch from the bottom of the paper. This is simply a guide to help place your leaf extracts.
20. Divide your paper into 2 equal sections by drawing a line down the middle with your pencil. Label one side #1 and the other #2.
21. Check your set up with the figure below.

22. Take your first capillary tube (included in your kit) and draw up some liquid from foil holder #1 (leaf #1) by sticking the end of the capillary tube into the fluid. You will see it start to draw up automatically. Once the liquid is drawn up, use your finger to seal the opposite end. This prevents the liquid from running out the capillary tube.
23. Releasing your sealing finger, streak the capillary tube across the bottom line on the leaf #1 section of your chromatography paper, leaving some white space on the edges, see figure. Allow to dry.

24. Repeat steps 22 and 23, 12-15 times with drying in between. Make sure to allow the extract to dry before each subsequent application.
25. Now take your second capillary tube and repeat the process for leaf #2 (Steps 22-24). Your final chromatography paper should look like the below figure.

26. Make sure all lines are dry.
Checkpoint: You should now have a cylinder of chromatography paper you put leaf extract lines onto.

27. Pour nail polish remover (acetone) into the beaker/chamber provided to you in the kit until it is about ½ inch high.
28. Gently place your paper in the chamber/beaker, line side down (at the bottom sitting in nail polish remover). You can tape it to the side of the chamber if you are worried about it falling over and to help keep it upright. It is important the paper bottom is level, otherwise solvent will run up one side faster than the other.
29. Cover with the lid or a piece of foil.
30. Wait for the pigment to migrate up the paper until they are 1-2 inches from the top. This may take 3-10 minutes.
31. Pull the cylinder out and allow it to FULLY dry on a piece of clean foil.
32. Remove the staples and place flat on a clean piece of foil.
33. On the DRY chromatography paper, take a pencil and write/label where the nonpolar pigments and polar pigments are on the paper.
Final Step: Snap a picture of your finished chromatography paper. You will be uploading this to the D2L dropbox.

You are not finished with this week’s lab, please continue to the next page for the pH and Buffers lab.

Part II: Understanding buffers and pH

Learning Objectives

● Explain the pH scale
● Define and acid and a base
● Provide examples of acids and bases
● Relate pH to how we determine if something is basic or acidic
● Describe a buffer
● Discuss the role of buffers in living organisms
● Complete an experiment to investigate the buffering capacity of solutions.

Background

pH

The pH scale is a measure of the concentration of hydrogen ions (H+) in a solution. When some substances are dissolved in water, they can separate (ionize) into charged fragments, including hydrogen ions (H+) and hydroxyl ions (OH-). An acid is a substance that has a high concentration of H+. A base has a low concentration of H+. The purpose of this lab is to introduce you to the pH scale and the function of buffers.

An acid is a substance that increases the H+ concentration of a solution. An example of an acid is hydrochloric acid (HCl). When hydrochloric acid is added to water, hydrogen ions separate from the chloride ions: HCl –> H+ and Cl-.

A base is a solution that decreases the H+ concentration of a solution. An example of a base is sodium hydroxide (NaOH). When sodium hydroxide is added to water, hydroxide ions (OH-) separate from the sodium ions (Na+), and the hydroxide ions (OH-) then combine with hydrogen ions (H+) to form water (thus lowering the total H+ concentration of the solution): NaOH –> Na+ and OH-. Bases result in lowering the H+ concentration in a solution.

The pH scale ranges from 0 to 14. A pH value less than 7 signifies an acidic solution, a pH value greater than 7 signifies a basic solution. A pH of 7 is a neutral solution (see figure on next page).

The pH scale is logarithmic, which means there is a ten-fold difference between pH units. For example, a solution with a pH of 4 is 10 times more acidic than a solution with a pH of 5. Please note, H+ concentrations increase as pH decreases.

Credit: OpenStax college, commons.wikimedia.org

Buffering Capacity: A buffer’s ability to resist changes in pH

Organisms must be able to maintain stable pH values for our normal physiological processes to occur. Very acidic solutions and strong bases can denature and damage proteins which drive many of the pathways in our body. For example the pH of our blood is approximately 7.4, and our body has mechanisms to help maintain this pH, including a high “buffering capacity”.

A buffer is a solution that resists changes in pH when an acid or a base is added. A buffer works by accepting H+ ions from a solution when they are in excess, and donating H+ ions when needed.

A thought question, before we move on:

Aspirin is an acidic compound (pH 3). Many people who take large amounts of aspirin also take a stomach medicine such as Maalox. Explain why.

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In this activity you will run an experiment to test the effectiveness of 3 solutions as buffers; tap water, 1X McIlvaine buffer and 0.1X McIlvaine buffer. Use the instructions that follow to walk yourself through the experiment and logging of results. You will then use these results to write a Results Section for this experiment.

Central Question: Is buffering capacity affected by changing buffer concentration?

Write a hypothesis and reasoning for the central question above

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Write a prediction:

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What is the dependent variable? Explain.

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What is the independent variable? Explain.

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Procedure 2: Buffering capacity

1. Solution 1: Tap Water
a. Using the graduated cylinder in your kit, measure out 5 mls of water from your faucet (tap water) into the provided beaker (in your kit).
b. Measure the starting pH of the water by using 1 pH strip, submerging it into the water for 10 seconds and removing the strip.
c. Read the color result and record the pH in the Results Table below.
d. Add 1 drop of lemon juice to the water, stir with the glass stir rod, wait 10 seconds and then measure the pH as in step b and record in your table below.
e. Repeat step d, adding 1 drop at a time, stirring, measuring pH, recording pH in the table until you have reached 10 drops.
f. Record the final pH of the water in your table.
g. Thoroughly wash and dry the beaker.
2. Solution 2: 0.1X McIIvaine Buffer
a. Using the graduated cylinder in your kit, measure out 5 mls of 0.1X McIIvaine buffer provided in your kit and pour in the beaker.
b. Measure the starting pH of the buffer by using 1 pH strip, submerging it into the water for 10 seconds and removing the strip.
c. Read the color result and record the pH in the Results Table below.
d. Add 1 drop of lemon juice to the buffer, stir with the glass stir rod, wait 10 seconds and then measure the pH as in step b and record in your table below.
e. Repeat step d, adding 1 drop at a time, stirring, measuring pH, recording pH in the table until you have reached 10 drops.
f. Record the final pH of the buffer in your table.
g. Thoroughly wash and dry the beaker.
3. Solution 3: 1X McIIvaine Buffer
a. Using the graduated cylinder in your kit, measure out 5 mls of 1X McIIvaine buffer provided in your kit and pour in the beaker.
b. Measure the starting pH of the buffer by using 1 pH strip, submerging it into the water for 10 seconds and removing the strip.
c. Read the color result and record the pH in the Results Table below.
d. Add 1 drop of lemon juice to the buffer, stir with the glass stir rod, wait 10 seconds and then measure the pH as in step b and record in your table below.
e. Repeat step d, adding 1 drop at a time, stirring, measuring pH, recording pH in the table until you have reached 10 drops.
f. Record the final pH of the buffer in your table.
g. Thoroughly wash and dry the beaker.
Results table is on the next page.

Results Table.
Drops of Lemon Juice Tap Water 0.1X McIIvaine’s buffer 1X McIIvaine’s buffer
0 7
8
8

1
4
8
8
2 4

7 8

3
4 4

8
4
3
4
8
5
3
3 8

6 3
3
8

7
2
2
8
8
2
2
8
9 2

2 7

10
2
2 7

Which solution had the best buffering capacity? Explain (how do you know?).

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Go on to the next page…
Write a Results Section for this experiment. Be sure to refer to numerical trends or patterns evident in the data. Construct your own version of the table above and generate an appropriate figure (graph) of the data. Be sure to include all appropriate details with your table and figure.

Writing Assignment: Results Section Grading Rubric (for reference).

_____/0.25 pt Section header present

_____/3.0 pts Paragraph reporting the data
– Accurate data with appropriate units
– Comparison of treatments (solutions)
– Straightforward presentation
– Trends presented
– Reference to table
– Reference to figure (graph)
– If needed, data problems noted that were caused by human error during the experiment.

_____/3.0 pts Data table
– Professional looking
– Data accurate
– Table number present and correct
– Table title present, descriptive, and appropriate
– Row and column labels appropriate
– Appropriate units

_____/3.0 pts Figure (graph)
– Professional looking
– Graph type correct & fits data
– Data accurate
– Easy to interpret
– Figure number present and correct
– Figure title present, descriptive, and appropriate
– Axis labels present and appropriate
– Appropriate units

_____/0.75 pt Format
– Passage written in paragraph form
– Uses past tense
– Sentence structure, grammar, punctuation, and spelling all correct
– No quotes
– Line spacing: double
– Table formatted to be complete on one page – does not break across pages
– Figure formatted to be complete on one page – does not break across pages
– Printed single-sided (instructor preference), no more than two pages