Bio103 Laboratory Students Guide John Jay College, C.U.N.

Lab #2: pH and Buffers

I. Introduction the ions of water a. The cell is the basic unit of life, the smallest structural level at which life can exist. Water forms the bulk of nearly all living systems. For instance, humans are approximately 70% water by mass. Both cells and intercellular spaces are mainly composed of water. This should not surprise us given the properties of water (see Lab I). These properties, especially waters solubility, play a vital role in facilitating the numerous chemical reactions required to maintain the cells viability. b. Although the covalent bonds holding the water molecule together are strong, in a solution of water, at any one time, a very small portion of water molecules will spontaneously break down into OH- and H+ ions. At the same time, other H+ and OH- ions will spontaneously re-associate and form H2O. c. Thus, there is equilibrium, according to the following equation: H2O OH- + H+ d. In a neutral solution of distilled water, this equilibrium is heavily favored to the left and most molecules in a water solution exist as regular H2O, with less than 0.000001% of the molecules in the dissociated form at any one time. But remember, just one drop of water will contain more than 1020 molecules of water, so there will still be about 1013 molecules of OH- and H+ ions in that single drop. e. (Actually the H+ ion typically combines with a molecule of H2O to form H3O+ (hydronium ion), but this is generally ignored and we will simplify the discussion by referring solely to the H+ (hydrogen ion). ) f. Even though the amounts of these two ions are, relative to H2O, very small, they are extremely reactive and give water many of its special properties, specifically those that are related to pH.

II.

Measuring the concentration of the ions of water a. In a neutral solution of pure distilled water, the amount of these two ions, H+ (hydrogen ion) and OH(hydroxide ion), will be equal, at a concentration of 0.0000001 moles per liter of water. i. The term moles per liter has its own unit, molar (abbreviated M) and this is the metric unit for concentration of molecules in a liquid solution. ii. 0.0000001M is usually written in scientific notation: 1 x 10-7M iii. The term concentration can also be abbreviated simply by placing brackets around something. Thus, the term [H+], means, concentration of H+. b. The concentrations of OH- and H+ in a solution of water are always directly related to each other. Only in perfectly neutral conditions (pH 7, explained below) will the concentrations be the same. More often, there is more of one ion or the other. When there is more H+ than OH-, this is said to be an acidic solution. When there is more OH- than H+, this is a basic solution. But the relationship between the two ions is fixed by the following equation:

[H+] x [OH-] = 10-14

c. This is a fixed relationship and when the concentration of one ion goes up, the other goes down. Compounds that, when added to an aqueous solution, cause an increase in the [H+], would, at the same time, also cause a decrease in the [OH-]. Such a compound is called an acid. The opposite kind of compound, a base, would cause an increase in [OH-] and thus a decrease in [H+].

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Bio103 Laboratory Students Guide John Jay College, C.U.N.Y

d. Because the concentrations of H+ and OH- are typically very small, it is often convenient to convert these values to a logarithmic scale called the pH scale. These conversions are performed using the pH equation, which only considers the concentration of H+: +

pH = -log([H ])

e. This converts a number like 0.0000001M (1 x 10-7M) to simply, 7. To quickly make these conversions in your head, you simply count the decimal places to the right of the right of the decimal. Or, if the value is already expressed in scientific notation, you simply take the exponent and remove the negative sign. HQ-2. Convert the following to pH values: If [H+] = 0.0001M; pH = f. __ If [H+] = 1.0 x 10-9M; pH = ___ If pH = 2; [H+] = _

If we know the pH or the [H+], we also know the [OH-], since they are fixed in a direct relationship. Your instructor will give you more info about the pH equation and conversions, and you will do some practice together. The chart below helps you visualize the relationships between pH, [H+] and [OH-]:

pH
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
III.

[H+]
1 0.1 0.01 0.001 0.0001 0.00001 0.000001 0.0000001 0.00000001 0.000000001 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10 1.0 x 10
-10 -11 -12 -13 -14

[OH-]
1.0 x 10-14 1.0 x 10
-13 -12 -11 -10

pOH
14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

Example
Battery Acid Stomach Acid Lemon Juice Vinegar Orange Juice Coffee Milk Distilled Water Sea Water Baking Soda (bicarbonate) Laundry Detergent Milk of Magnesia Ammonia Bleach Liquid Pipe Clog Remover

1.0 x 10 1.0 x 10

1.0 x 10

0.000000001 0.00000001 0.0000001 0.000001 0.00001 0.0001 0.001 0.01 0.1 1

Strong vs. weak acids (and bases) a. Recall that acids increase the [H+] in solution and bases decrease the [H+] in solution. Acids and bases are considered relatively strong or weak depending on the degree to which they ionize in solution. For example: 1) Hydrochloric acid (HCl) ionizes completely and is an extremely strong acid: HCl H+ + Cl2) Acetic acid (vinegar) does not completely ionize and is a weak acid: CH3COOH H+ + CH3COOLab #2 Page 2

Bio103 Laboratory Students Guide John Jay College, C.U.N.Y

3) Water always dissociates and re-associates and is thus considered both a weak acid and a weak base: 2H2O H3O+ + OH- (simplified: H2O NaOH Na+ + OHH+ + OH-) 4) Sodium hydroxide (NaOH) ionizes completely and is an extremely strong base: 5) When sodium bicarbonate (baking soda) dissolves in water, the sodium and bicarbonate ions complete dissociate. However, the bicarbonate ion can then partially ionize by binding to a free H+ from the solution, forming H2CO3. Because this lowers the [H+] in the solution, bicarbonate is a weak base: NaHCO3 Na+ + HCO3- ; then: HCO3- + H+ (from the solution) H2CO3 b. It is important to note that the terms strong and weak refer to whether or not an acid or base ionizes completely or only partially, but this does not give any direct information about the pH of a solution containing these compounds. The pH of a solution of HCl, for example, depends on how concentrated the HCl is. A 1M solution of HCl will have a much lower pH than a 1mM solution of HCl. Further, a very concentrated solution of acetic acid could have a lower pH than a very diluted solution of hydrochloric acid (HCl), even though HCl is a strong acid and acetic acid is a weak one. c. In todays lab, we will be working with solutions of HCl, NaOH, and solutions with very low and very high pH, thus you need to handle them very carefully, wear gloves, and use safety glasses, etc. IV. pH Buffers a. A pH Buffer is a compound that protects an aqueous solution from large changes in pH. Buffers accomplish this by either donating or absorbing H+ ions, as needed, in order to minimize the impact of H+ (or OH-) being added to a solution. b. There are two general kinds of buffers. Weak acids, when dissolved in water, partially dissociate and release H+ ions and the conjugate base into the solution. (Thus they lower the pH of a solution when they are added.) However, once equilibrium is reached, these weak acids will then act as pH buffers because, if additional H+ is later added to the solution, some of those H+ ions will bind to the conjugate base, thus re-forming the weak acid, and minimizing the change in pH that will occur. However, if OH- ions are added to a solution, additional weak acid will dissociate, releasing more H+ ions, and minimizing the pH change.
carbonic acid hydrogen ion bicarbonate ion

H2CO3 H + HCO3weak acid hydrogen ion conjugate base

c. On the other hand, some weak bases absorb H+ ions when they are dissolved in an aqueous solution and form a conjugate acid. (Thus, they raise the pH as H+ ions disappear.) However, they can then act to buffer the solution from pH changes if OH- ions are later added, by releasing those H+ ions back into the solution and reforming the weak base. Similarly, if additional H+ ions are added, they can combine with the weak base, and once again, pH changes are minimized
ammonia hydrogen ion ammonium ion

NH3 + H
weak base

NH4+
conjugate acid

hydrogen ion

d. A solution that contains both a weak acid and a weak base will be well buffered against the addition of OH- or H+. For example, human blood is precisely buffered at pH 7.4 multiple weak acids and bases.

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Bio103 Laboratory Students Guide John Jay College, C.U.N.Y

LABORATORY PROCEDURES Lab #2

For all lab work students must wear safety goggles and latex gloves at all times and work in PAIRS, taking turns with measurements, and all data must be recorded neatly on a separate sheet of paper that will go into the lab notebook. I. Experiment A pH Calculations a. pH Practice Problems Your instructor will place on the board some practice problems for pH calculations. Neatly place your work in your lab notebook. Experiment B Establish reference standards using a pH-sensitive pigment a. Cabbage Extract a purple liquid will be provided for you. This was made fresh, just before your lab session started, by boiling fresh cabbage leaves in water. The purple pigment leaks out of the cabbage and into the water. This purple pigment, anthocyanin, changes color in different pH solutions. b. Known pH Standards prior to class, a range of clear pH-buffered solutions were prepared for you. These solutions are buffered to a precise known pH, as labeled on the beakers. The solutions are: pH 2.0, 4.0, 6.0, 7.0, 8.0, 10.0, and 12.0. A pH buffer is a solution that resists changes in pH when an acid or a base is added. Their properties will be investigated more directly later in todays lab. Note: the pH 2.0 and 12.0 solutions are corrosive/caustic and will burn your skin on contact handle them with extreme care. Wear gloves at all times. c. You will soon be adding 2mL of cabbage extract to each of the 8 test tubes. You will also shortly thereafter be adding more cabbage extract to a series of test tubes. However, up to four pairs of students are sharing one beaker of cabbage extract per table. Thus, each pair should take enough cabbage extract from the common stock to supply their entire experiment, and take it back to their seats in a small beaker. Although you should only need approximately 40-50mL of cabbage extract, you should take a bit more (70-80mL or so) than you need to allow for pipetting error, mistakes, spills, etc. Procedure 1. Using a crayon, label a 1L beaker, WASTE. This will be your liquid waste container. Dump or squirt all liquid waste in this beaker. DO NOT POUR ANY WASTE down the sink. When the beaker gets half full, ask the instructor or lab tech to empty it for you. 2. Set up a series of seven test tubes in a rack. Using the crayon, label each test tube as follows: 2; 4; 6; 7; 8; 10; and 12. 3. Using the 2mL transfer pipette, place 4mL of each of the known pH-buffered solutions to the appropriate pre-labeled test tube. (Avoid contamination by rinsing the pipette at least TWICE with water in between each ingredient. ALWAYS wash glassware, if there is any doubt!) 4. Using the plastic transfer pipette, add 2mL of cabbage extract from your working stock, into each test tube (containing the known pH solutions). Cover the test tubes with Parafilm and lightly mix. 5. The color of the extract will change based on the pH of the solution. Wait 5 minutes to obtain the final color of the pH 12 solution. Once you have established the range of pH-induced colors of the cabbage extract solution, you can use these colors as a reference standard for measuring the pH of

II.

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Bio103 Laboratory Students Guide John Jay College, C.U.N.Y

an unknown solution. Record the final color for each pH value in Table 2.1. Remember to keep these test tubes to use as a reference for the remainder of the lab period. III. Experiment C Independent Verification a. pH Paper Your instructor will explain how to use the pH paper. pH paper works the same way as the cabbage extract there are pH-sensitive dyes imbedded in the pH paper. b. Blind Measurement You have already tested the pH of the known pH solutions using the cabbage extract. Now you will test a selection of these (pH 4, 6, 7, 8, and 10) using pH paper to verify that they are the correct pH. This should be done blindly, so that one member of the lab pair goes to get the solution, but the other member does the reading, WITHOUT KNOWING what the solution is. Procedure 1. The pH papers should only be handled from the non-colored end and should be placed on a sheet of clean paper towel and not directly on the lab table. 2. Using a plastic transfer pipette, one member of the lab pair should drop two small drops of a known-pH solution and drop it onto a strip of pH paper, WITHOUT telling the other person which solution is on the paper. The solutions to test are: pH4.0, 6.0, 7.0, 8.0, and 10.0. Do these in RANDOM ORDER. 3. The other lab partner then reads the pH paper using the provided scale. 4. Switching roles, the lab pair should read all five the known pH solutions with pH paper to verify that they are the correct pH. IV. Experiment D Using pH to help determine the identity of unknowns a. In forensic casework, investigators will commonly encounter liquids and substance whose identity is not known. These unknowns, must be fully described and observed before any attempt at deducing their identity can be made. With liquids, one simple, but very important and informative measurement, is pH. b. For this experiment, you will be measuring the pH of several different unknown liquids, using the pH paper, and cabbage extract. Procedure 1. Identify the brown dropper bottles at your table that are labeled with single capital letters these are the unknown solutions. 2. Measure the pH of the unknown solutions, first using the cabbage extract procedure and the pH paper. (Follow the procedures as described above!) Each team at the table should plan their use of equipment and materials so as not to interfere with other teams. Remember to use fresh samples for each test, rinse glassware first, etc. 3. Record all procedures, results, and conclusions in a chart/table in your notebook. Leave a space in the chart for your guess regarding the identity of the unknown solution, and for its true identity. 4. The instructor will list the five possible liquids that each unknown solution could be. Using this list, make an hypothesis about the identity of each unknown. At the end, the instructor will reveal the true identity of which is which, this should also be recorded in your data chart.

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Bio103 Laboratory Students Guide John Jay College, C.U.N.Y

5. Clean all glassware at the end of each experiment. (Keep your cabbage reference standards.) V. Experiment E testing the action of pH buffers a. In this experiment, you will be given two solutions, one buffered and one not. Your job is to figure out which is which by observing the pH changes when you add acid and base. One is simply water, so we will call this unbuffered. Another contains 0.1M of a phosphate buffer. This buffer contains both of the following: 0.1M of the buffer Na2HPO4. (We will call this dibasic.) 0.1M of the buffer NaH2PO4. (We will call this monobasic.)

However, the solutions are labeled only I and II. Your task is to discover which is which. Procedure 2. Using a crayon, label two 50mL beakers with I and II. 3. Bring these two beakers to the front lab bench and pour 20mL of each of the unknown solutions into the beakers and bring them back to your bench. 4. Now, using the pH paper, measure the pH of each solution and record your results. 5. Using a clean pipette, add 1ml of 0.1M HCl to each beaker and gently stir. 6. Now, read the pH value again, using new pH paper 7. Dispose of the solutions in your liquid waste beaker and thoroughly rinse the beakers. 8. Repeat the above procedure, except at step #4, add 1mL of 0.1M NaOH , instead of 0.1M HCl VI. Final Wrap-up a. Your instructor will give you directions about the proper construction of your data chart and what the results of the acid and base attack should have been. PAY CLOSE ATTENTION, because next week, you will be required to turn in your results, procedures, and conclusions from all of the experiments from the first two weeks labs.

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Bio103 Laboratory Students Guide John Jay College, C.U.N.Y

VII.

Post-Laboratory Homework Questions Lab #2

1. Complete these conversions:

If [H+] = 0.01M; pH = If [H+] = 0.01M; [OH-] = If [OH-] = 0.001M; pH =

__ __ __

If [H+] = 1.0 x 10-12M; pH = If [OH-] = 1.0 x 10-11M; [H+] = If [OH-] = 1.0 x 10-12M; pH =

___ ____ ____

If pH = 1; [H+] = If pH = 9; [OH-] = If pOH = 10; [H+] =

___ ___ ___

2. Is the [H+] of a pH 7.6 solution higher or lower than the [H+] of a pH 9.4 solution? Explain.

3. If solution A has 10,000 times as many H+ ions as solution B, what is the difference in pH units between the two solutions? Explain.

4. Make a statement relating hydrogen ion concentration [H+] to the acidity and basicity of solutions.

5. You have made the following two solutions:

X) hydrogen ion concentration = 1 x 10-5M Y) pH = 4

State which statements below are true and which are false: a. Solution X contains 1 x 10-9M hydroxide ions. b. Solution X contains more H+ ions than Solution Y. c. Solution Y contains 1 x 104M hydrogen ions. d. Solution X is basic. e. Solution Y is more acidic than solution X. f. Solution X has ten times more OH- ions than solution Y. 6. Write the equation for the neutralization of NaOH by HCl.

7. State two reasons why pH maintenance (buffering) is important to biological systems.

8. Normal pH in biological systems is not usually pH 7 as we might think. Find two examples of non-pH 7 systems in humans. Explain why this is the case for one of these examples.

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