Molecular structures, water, and pH

Biology I Lab

Part A: Exploring three-dimensional shapes of biological molecules using molecular modeling kits Hydrogen, carbon, nitrogen, and oxygen, are the four most abundant elements found in living organisms. Atoms of all of these elements form covalent bonds by sharing a pair of electrons with another atom in order to fill their outer energy shells with atoms. The energy shell closest to each atomic nucleus holds two electrons. The second energy shell holds eight electrons arranged in pairs. The third energy shell can technically hold more than eight electrons, but an element is stable with eight in its 3rd energy shell, so we biologists think of a 3rd shell with eight electrons as being full. Since the atomic number of an element is equal to the number of protons and electrons in a neutral atom of the element, we can figure out how many covalent bonds an atom can make by figuring out how many electrons are in the outer shell and how many more are needed to fill that energy shell. For example, oxygen has two electrons in the first shell and the remaining six electrons in the 2nd shell. It needs two more electrons to have eight electrons in its outer (2nd) energy shell, so oxygen makes two covalent bonds. How many covalent bonds will each nitrogen atom make? _____________________ In this lab exercise students will build small molecules that are important for living organisms using molecular modeling kits and then write the molecular formulas, structural formulas, and electron dot structures for each molecule. A molecular formula lists the numbers and types of each atom in a molecule. The structural formula shows how the atoms are arranged using short lines to represent each covalent bond. An electron dot structure shows each electron pair in the outer-most energy shell as a pair of dots. Electron dot structures differ from electron distribution diagrams (as in Hillis Fig 2.1) because the latter also show electrons in lower energy shells The dots are distributed in four pairs, which are as far apart from each other as possible, because the negative charges of electrons repel each other. Unpaired electrons are represented as a single dot. There are examples of each way of representing chemical molecules in the table below. Name Molecular Formula NH
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Structural Formula
H N H H

Electron Dot Structure

Ammonia

Water

H2O
H H C H H

Methane Elemental Oxygen

CH4

O2

O=O

Building three-dimensional models will allow biology students to see the actual shapes of these molecules, which is not obvious when they are represented in structural formulas or Lewis dot structures. In addition to becoming familiar with different ways of representing molecules and their actual 3D shapes, students will become used to the color schemes used to represent atoms of different elements: black (or dark gray) for carbon, red for 1

Molecular structures, water, and pH

Biology I Lab

oxygen, blue for nitrogen, and white (or light gray) for hydrogen. This will make it easier to understand figures in the text and online assignments. Students will work in learning teams. Please be careful to keep the pieces of your molecular modeling kit together and find any that fall on the floor immediately so that they are not lost. First become familiar with the molecular model kit. Carbon atoms are black, oxygen atoms are red, nitrogen atoms are blue, and hydrogen atoms are white (and smaller, because they have one fewer energy shells with electrons than the other atoms). The shorter gray connectors represent single covalent bonds. The flexible, longer, darker-gray connectors are used to make double covalent bonds. The very short white connectors also represent single covalent bonds; they allow you to generate space-filling models, whereas the gray bonds are of an exaggerated length that let you see molecular architecture better in some cases. You will need to use a beige tool to remove the shorter white connectors from the atoms. Procedure: 1. Build each of the molecules listed in the table on page 1 of this handout. For speed, each member of the team can build one molecule. Keep these molecules intact until you finish answering the questions on this page. What is the shape of the 3D structure formed by methane? ______________________________________ Describe the shape of the 3D structure formed by water:

Do the 3D shapes of these molecules change if you rotate atoms on either end of single covalent bonds?

Which molecules have polar covalent bonds? How are polar covalent bonds different from nonpolar covalent bonds?

Draw the structural formula for ammonia. Use the symbols + and to show the partial charges across any polar covalent bonds on your structural formula of ammonia.

Molecular structures, water, and pH

Biology I Lab

2. Build a molecule with the molecular formula CH3CH2OH. This is ethanol (ethyl alcohol, the alcohol in beer and other alcoholic beverages). Remember to think about how many covalent bonds carbon, hydrogen, and oxygen wants to make to fill its outer energy shell. Rotate atoms on either side of single covalent bonds? There is free rotation around single covalent bonds, so this molecule does not really have a single 3D shape, like H2O or methane. (The ethanol dogs legs can be up or down.) Write the structural formula and Lewis dot structure for ethanol: Structural formula Lewis dot structure

Use the symbols + and to show the partial charges across any polar covalent bonds on your structural formula of ethanol. 3. Build a molecule with two carbon atoms and four hydrogen atoms. Remember that each carbon atom makes four covalent bonds and each hydrogen atom makes one covalent bond. Describe this molecule (it is called ethene). What is its 3D shape?

Draw the structural formula and Lewis dot structure of ethene. Structural formula Lewis dot structure

Is there free rotation around a double covalent bond? (In fact, the rotation, or lack of rotation, around covalent bonds in these kits is realistic.)

Molecular structures, water, and pH 4. Follow the steps below to build an amino acid, a small biological molecule found in proteins. a. Put four single covalent bonds on a single carbon atom. b. Attach a single hydrogen atom to one of the covalent bonds on the carbon atom.

Biology I Lab

c. Use the shorter white pegs to attach two hydrogen atoms to a blue nitrogen atom (to make NH2) and attach the nitrogen by one single covalent bond to the carbon atom. d. Attach one oxygen atom to a different carbon atom by a double covalent bond. Now attached a second oxygen atom to the same carbon and use a small white peg and hydrogen to fill the oxygens outer energy shell. (This will make COOH.) Fill this carbons outer energy shell by connecting it to one of the single covalent bonds on the first carbon atom. e. Use the shorter white pegs to attach three hydrogen atoms to one carbon atom to make CH3. Attach this to the fourth single covalent bond on the central carbon so that all carbon atoms are making four covalent bonds. f. Congratulations: you have assembled an amino acid named alanine.

Draw the structural formula for alanine. Try it on your own first. If you need help, look at Table 3.2 on page 40 in your text for structural formulas of amino acids.

Find a carbon atom in the alanine molecule that has four qualitatively different things attached to it. This is called the central carbon. Draw an arrow pointing to the central carbon in your structural formula. Circle the amino functional group in alanine. Draw a rectangle around the carboxyl functional group. Are there any other functional groups in alanine? If so, which one(s)? 5. Make a second alanine molecule identical to the first. You might need to replace the gray single covalent bond connectors with shorter white connectors to attach hydrogen atoms to O, N, or C in order to have enough single bonds. Hold them next to each other to show that they are identical. Now swap two of the things attached to the central carbon in your second alanine to make a molecule that is a mirror image of the first alanine. We call molecule like this, mirror images of each other, enantiomers or stereoisomers. They do not have identical 3D shapes because you cannot superimpose one on the other. They differ in the way that the four different things are attached to the central carbon. Which of your body parts are mirror images of each other? How do you know that these mirror image body parts are not the same shape?

Molecular structures, water, and pH

Biology I Lab

6. Take apart your alanine molecules. Now build a structure for CH3CH2CH2CH2CH2CH2COOH, a fatty acid. Use gray single covalent bond pegs for all carbon-carbon bonds, but you can use shorter white pegs for carbonhydrogen bonds. Rotate atoms around single covalent bonds until your fatty acid is as straight as possible. Describe the 3D shape of this molecule.

Which do you think will dissolve more easily in water, the amino acid or the fatty acid? Explain.

7. Now take apart your fatty acid and build the molecular shape of CH3CH2CH=CHCH2CH2COOH, a different fatty acid. The = symbol indicates where to place a double covalent bond between two carbons. Fatty acids that contain one or more carbon-carbon double bonds are called unsaturated fatty acids because one could change the double bond to a single bond and add more hydrogen atoms, so the fatty acid is not saturated with hydrogen atoms. Again, rotate atoms around single covalent bonds until your fatty acid is as straight as possible.

Does your fatty acid look more like this?

This is a cis fatty acid.

Or does it look more like this?

This is a trans fatty acid.

Can you swap things attached to one of the carbons in the double covalent bond to switch between these two shapes? Two molecules like these fatty acids that have the same molecular formula, but different structures are called isomers. The different 3D shapes of fatty acid isomers make them interact differently with other molecules in cells, which is why trans fatty acids are not so great for you, but you need certain cis fatty acids in your diet to be healthy. 5

Molecular structures, water, and pH

Biology I Lab

Part B: The cohesiveness and adhesiveness of water. SAFETY: Work with methanol over a piece of absorbent bench paper (the plastic side should be down). Wear gloves and safety glasses while working with the chemicals. Polar water molecules stick to each other (cohesion) and to other polar molecules (adhesion), such as sugars. You can demonstrate how cohesive water is by carefully dropping water onto the surface of a penny and observing how many drops you add to the dome of water gets before it spills off of the penny. In contrast, methanol, which only has one hydroxyl group, does not form such a high dome. Demonstrate this to yourself. Use dry pennies. Place the pennies on a dry paper towel and have your lab partner observe the paper towel around each penny to see when it wets as the liquid spills off the over-loaded penny. Secure the lid on the methanol bottle when you arent using it, because methanol evaporates quickly. Why do you think methanol evaporate more quickly than water?

You can demonstrate adhesion by seeing how far water and methanol move up a glass capillary tube that is touched to the surface of the liquid. Touch the end of a glass capillary tube to each liquid on a penny until the liquid stops moving up the tube. Measure the heights of the liquid columns in the capillary tubes using a metric ruler. How high is the level of water in the capillary tube in mm? ___________ How high is the level of methanol in the capillary tube in mm? ___________ Which liquid adheres better to the glass, methanol or water? Do you think glass is polar or non-polar? Why?

Clean up: Empty your methanol into the beaker in the hood labeled Methanol waste. Empty water into the sink. Touch your capillary tubes against the bench paper or a paper towel to wick the liquid out and leave them on a paper towel next to the sink to dry. Turn a small beaker (make sure it is dry and clean) upside down and place one drop of water on the bottom of it. Use a metric ruler to measure the diameter of the water drop in mm. Now dry the bottom of the beaker completely with a paper towel. When it is dry, wax the bottom of the beaker with parafin paraffin. Once you have wiped off the excess lumps of paraffin, place a drop of water on the bottom of this treated beaker. Measure the diameter of this water drop in mm. Diameter of water drop on untreated beaker: __________ Diameter of water drop on waxed beaker: _________ 6

Molecular structures, water, and pH

Biology I Lab

Explain any differences in the sizes of the water drops on treated and untreated beakers, using the words cohesion and adhesion in your explanation.

Part C: Exploring pH and buffers Even in pure liquid water, some H2O molecules come apart (dissociate) because a covalent bond between one hydrogen atom and the oxygen atom absorbs enough energy from the surrounding environment to be broken. Oxygen atoms are more electronegative than hydrogen atoms (see Table 2.2 in your text), that is, oxygen atoms hold electrons much more tightly than hydrogen atoms, so the oxygen takes both electrons in the covalent bond, leaving the hydrogen atom without any electrons. Because the hydrogen atom still has its proton, it now has a positive charge; it has become a hydrogen ion. The OH (hydroxide) ion has a negative charge because it has the extra electron that originally belonged to the hydrogen ion. This reaction is reversible, because the hydrogen and hydroxide ions can join to form a neutral water molecule again. H2O

H+ + OH

The pH is a measure of the concentration of hydrogen (H+) ions in an aqueous (water-based) solution. Even though some water molecules dissociate, most water molecules in an aqueous solution to not dissociate, so the concentration of hydrogen ions is very small. For example, in pure water, the concentration of hydrogen (H+) ions is 1 x 107 M (M means molar, which is a unit that represents a specific number of molecules dissolved in the solution). Written in decimal notation, this is a concentration of hydrogen ions equal to 0.0000001 M. Writing such tiny numbers is tedious, so the pH scale abbreviates the number of place-holding zeroes by the formula: pH = log[H+] In pure water, pH = log[1 x 107] = 7

Thus, as the hydrogen ion concentration gets larger (say, 1 x 103 or 0.001), the pH value gets smaller: pH = 3. Solutions with a hydrogen ion concentration greater than that of pure water are acidic and substances that increase the hydrogen ion concentration when they are added to water are called acids. As the hydrogen ion concentration gets smaller (1 x 1011 or 0.00000000001), the pH value gets bigger: pH = 11. Solutions with a hydrogen ion concentration less than that of pure water are called basic or alkaline and substances that decrease the hydrogen ion concentration when they are added to water are called bases. Solutions with a pH = 7, the pH of pure water, are called neutral.

Molecular structures, water, and pH

Biology I Lab

This figure shows the pH scale with the most acid value (pH = 0) at the top and the most basic value (pH = 14) at the bottom. Hydrogen ion concentration is given on the left and pH value on the right. Altering pH of body fluids is destructive to living organisms, which is why excess acids and bases are toxic to the environment.

Procedure: SAFETY!! WEAR GLOVES to protect your skin from the acids and bases! 1. Obtain a vial of pH paper. The paper will change color when dipped in an aqueous solution, with the final color determined by the pH of the solution. The color key is shown on the outside of the vial. 2. Use forceps (tweezers) to hold one end of a pH paper, dip part of it into the pH 7 solution, and compare the color of the damp pH paper to the color key on the vial. If the color looks intermediate between two pH values on the vial, you can estimate an intermediate pH (such as pH 6.5 if it is somewhat more yellow than pH = 7, but not as light as pH = 6). Does is match the color for pH 7? Record the measured pH below. 3. Using a fresh piece of pH paper for each pH standard, record values as estimated with pH paper for each pH standard, 4, 7, and 10.

Measured pH of pH 4.0 standard: _____________________ Measured pH of pH 7.0 standard: _____________________ Measured pH of pH 10.0 standard: _____________________

How close are the measured pH values to the pH values named for the standards?

What is the H+ ion concentration in each pH standard? (Use this formula: pH = log[H+] ) H+ ion concentration of pH 4.0 standard: _____________________ H+ ion concentration of pH 7.0 standard: _____________________ H+ ion concentration of pH 10.0 standard: _____________________ 8

Molecular structures, water, and pH

Biology I Lab

4. Several aqueous solutions that you are familiar with are present in the lab. Test and record the pH for four of these solutions, recording the name of the solution and its pH in the table. Remember to use fresh pH paper for each solution. Use the pH equation to calculate the hydrogen ion concentration of each solution in the table. Name of solution Measured pH Hydrogen ion concentration

Remember, pH = log[H+], for example, in pure water, pH = log[1 x 107] = 7

Which solutions are acidic?

Which solutions are basic?

Did the pH of any of these solutions surprise you? If so, which ones?

5. A buffer is a mixture of a weak acid and a weak base that together resist changes in pH when an acid or base is added to the buffer. Bicarbonate buffer and phosphate buffer both are present in blood and other body fluids to resist changes in pH that can harm our cells. Obtain three test tubes and a test tube rack. Label the test tubes with the terms water, bicarbonate buffer, and phosphate buffer. Use transfer pipettes to transfer 10 ml of dH2O (pure water), bicarbonate buffer, and phosphate buffer into the appropriate beakers. Measure the pH of the buffers and dH2O and enter in the No HCl added column of the table. (Remember to use a fresh piece of pH paper for each solution.) Add two drops of 0.50 M HCl (an acid) to each test tube and mix well by holding the test tubes near the top and shaking the bottom back and forth. Then test the pH and record it in the table on the next page. Repeat with a three more drops of 0.50 M HCl (5 drops total), measuring the pH after mixing. Repeat with 5 more drops of 0.50 M HCl (10 drops total). Repeat with 5 more drops of 0.50 M HCl (15 drops total). 9

Molecular structures, water, and pH

Biology I Lab

Solution

0.50 M HCl added

No HCl added

2 drop

pH after mixing 5 drops 10 drops

15 drops

Deionized H2O Bicarbonate buffer

Phosphate buffer

Which solution resisted changes in pH the best? Which resisted changes in pH the worst?

What is the effect of a buffer on the pH of an aqueous solution?

6. Look at the properties of the carboxyl and amino functional groups in Figure 2.7 (page 23) of your text. Which is basic? Which is acidic?

Obtain two test tubes and label one CH3COOH and the other CH3NH2. Place 2 ml of each solution in the appropriate test tube. Use the pH paper to measure the pH of a 1% CH3COOH solution and a 1% CH3NH2 solution. How do these measured pH values compare to those predicted in table 2.7? pH of 1% CH3COOH ___________________ pH of 1% CH3NH2 ____________________

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Molecular structures, water, and pH

Biology I Lab

Use your understanding of the numbers of covalent bonds made by C, H, and O, and your knowledge that this molecule has a carboxyl functional group to draw the structural formula of CH3COOH. Show how it dissociates in water and explain why it has the pH that it does.

Use your understanding of the numbers of covalent bonds made by C, H, and N, and your knowledge that this molecule has an amino functional group to draw the structural formula of CH3NH2. Show how it accepts a proton in water and explain why it has the pH that it does.

7. Clean up: Pour the contents of the test tubes into the acid and base waste. Submerge your test tubes into the white basin containing water and detergent near the sink and push the bottoms down to fill them with water. You may throw pH papers, paper towels, and empty transfer pipettes away in the trash. Return any reagents and test tube racks to the cabinet or bench where you got them.

Extra credit - Part D: Viewing representations of biological molecules using modeling software (Optional complete this section for 3 points extra credit after you have finished the rest of the lab.) Open source software has been developed that models the three dimensional structures of molecules, even very large and complex molecules like proteins and DNA. Some open source molecular modeling software can be downloaded for free and installed on your hard drive, after which you can use it to view molecular structure files, but the Jmol applet allows viewers to visualize molecular model files accessed online without downloading the viewer. Jmol requires that you have Java on your computer. In this exercise, you will use Jmol to view the amino acid alanine, a saturated and unsaturated fatty acid, and ribose, molecules that you built in Part A. Procedure: 1. Go to http://www.biotopics.co.uk/JmolApplet/jcontentstable.html, a web page with a table of biological images that you can view with Jmol. Just below the top sentence on this page you will see the gray letters JMOL rotating if you already have Java on your machine. If you do not see these rotating letters, you will need to download Java at the link in the top sentence (this is a free download).

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Molecular structures, water, and pH

Biology I Lab

2. When you can see the rotating letters JMOL, scroll down the page to the red part of the table, which has links for structures of amino acids. Find the link for alanine and click on it to open a new window with the alanine structure. You will probably need to wait for a few seconds for Java and the structure to load. 3. If you put your cursor on the molecule and click and drag, you can rotate the molecule in any direction to view it from different angles. To test your control, move the alanine molecule so that the blue nitrogen atom is closest to the top of the Jmol window. If you right click on the molecule it pulls up a menu. Put your cursor on Spin and click on On to start the alanine structure rotating. 4. Notice the COOH (carboxyl) group with two red oxygen atoms and the NH2 (amino) group with its blue nitrogen atom. If you click on the show amino link to the right, the NH2 (amino) group will be highlighted. If you click on the show acid link, the COOH (carboxyl) group will be highlighted. All amino acids have an amino group and a carboxyl group. 5. Go back to the Jmol contents window and find the red amino acid portion of the table again. Now click the link that says 20 amino acids to pull up 20 small Jmol structures, one of each of the 20 different amino acids found in proteins. It will take longer for this page to load, as there are 20 molecular structures opening instead of just one. Find alanine, right click on it, and turn on the spin. Now pick a few more amino acids, right click on them, and start them spinning. To compare them more easily, click and drag to put the blue nitrogen atom toward the top of the window for each one. Watch them for a while until you figure out how they are different. How are the 20 amino acid structures the same? How are they different?

6. Go back to the Jmol contents page and scroll down to the green lipids part of the table. Click on the link saturated and unsaturated fatty acids to open a Jmol viewer showing two fatty acids with the same the same number of carbon atoms. Move them around or start them spinning as you examine them. Read the text between the two figures. These two fatty acids have many more carbon atoms than the ones you modeled, because you did not have that many carbon atoms in your modeling kits. Fatty acids in cells usually have between 14 and 34 carbons. How are the structures of stearic acid and oleic acid similar? How are they different?

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Molecular structures, water, and pH

Biology I Lab

7. Scroll up to the yellow carbohydrates section of the table and click on the ribose link to open the Jmol viewer. Start the ribose structure spinning. Leave this window open while you go back to the carbohydrates section of the table and click on the deoxyribose link. Deoxyribose is a different sugar molecule, but it is not much different than ribose. Examine the two sugars carefully. How are ribose and deoxyribose different?

8. Now find a link for the sugar glucose in the contents table and open that structure. Compare glucose to ribose. How are ribose and glucose different?

9. Ribose and glucose are both monosaccharides. Maltose is a disaccharide. Click on the maltose link in the contents table to view maltose. How are disaccharides, such as maltose, different from monosaccharides, such as glucose and ribose?

10. Explore the molecular structures in the Jmol contents table as much as you wish. Notice that some of the biological molecules have a very large number of atoms! Molecules like ribose, alanine, and even stearic acid are small biological molecules, but molecules like DNA, collagen, and cellulose are macromolecules, extremely large biological molecules. Nevertheless, even macromolecules have specific three-dimensional structures.

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