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LS1a Fall 2014 Lab 1: Acidity and pKa

Goals of the lab:
To determine the pKa values of ionizable groups using titration.
To identify an unknown molecule by measuring its pKa values.
To relate pH, pKa, and Ka to one another.
To draw chemical structures showing the protonation state that would predominately exist at
different pH values given the pKa values of a molecules ionizable groups.

Required safety regulations and lab etiquette
Wear a lab coat, gloves, and safety glasses at all times.
Wearing appropriate clothes that protect the leg, foot, and ankle (e.g., jeans, socks, and sneakers).
No eating or drinking in lab.
Cleaning up bench space after completion of the lab.
[Points can be deducted from your post-lab assignment for not wearing the appropriate clothes, not
wearing the required personal protective equipment, or leaving a mess.]

Introduction
As you heard in lecture last week, acid/base chemistry is a fundamental feature of all living systems.
Many molecules found within cells are capable of donating or accepting one or more hydrogen ions
(which we also describe as protons), and this can strongly influence the ways in which these molecules
interact or react with other molecules. It is important to characterize the properties of molecules acidic
groups in order to predict how molecules will react and interact with one another. To that end,
acid/base titrations, like the one we will perform today, are a simple means of determining the chemical
properties of an acid.

In this lab, you will perform a titration assay using a solution that contains a molecule (an unknown
analyte) with several acidic groups. Your goals are to estimate the concentration of the acid and to
identify and characterize the molecule by estimating the pKa values of each of its acidic groups. In order
to do this, you will generate a titration curve by monitoring the pH of a solution of the unknown analyte
using a pH sensor as you slowly add defined volumes of dilute base.

The following paragraphs provide general background information on acidity and pKa, the analysis of
titration curves, and the proper use of burettes.

Background information
An acid can be defined as a molecule that donates a hydrogen atom to a water molecule when dissolved
in water. Acid dissociation reactions are a particular type of chemical reaction in which the reactant (the
acid molecule) donates a proton to water (H2O) and the products are hydronium (H3O
+
) and a
conjugate base.



A conjugate base is simply the part of the acid that continues to exist after the proton has been
donated. We therefore can also think of it as the deprotonated form of the acid.
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The example of an acid dissociation reaction that we learned about in class was carbonic acid:



Acids can be defined as molecules that give up (donate) hydrogen ions (hydrogen ions are often
referred to as protons). The released hydrogen ion binds to a water molecule to produce hydronium.
The acid is converted into its conjugate base after donating its hydrogen ion.

Acids come in all shapes and sizes, but regardless of what they look like, in the reactions that we care
about in this class, acids are molecules that donate a hydrogen ion. We therefore use a shorthand
notation and refer to acids as H-A, which indicates that the acid is bound to a hydrogen ion. In the
example above, the red H is the hydrogen ion and the rest of the blue molecule is the A, so we can
think of it as H-A. When the acid (H-A) loses its hydrogen ion (H
+
), the remaining molecule is the
conjugate base (A
-
).

Since a hydrogen ion is actually just a proton, we call H-A the protonated form of an acid, and A
-
the
deprotonated form of the acid.

While some acids can only donate one proton, many acids can release more than one proton. Maleic
acid is an example of a diprotic acid that can release two protons, and it does so in two discrete steps.
Each deprotonation is described by its own Ka:



Note that the HA shown above is a neutral molecule that gives rise to a negatively charged conjugate
base, but keep in mind that this is not true for all acids.

The strength of an acid (i.e., its inherent ability to donate protons) is described by the value of its acid
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dissociation constant, Ka.

K
a
=
[H
3
O
+
][A

]
[H A]


Strong acids dissociate much more readily into their conjugate bases and protons than weak acids and
therefore have larger Ka values. By convention we use a log scale to describe the strength of an acid
since Ka values can vary over many orders of magnitude from extremely small to extremely large. The
acidity of a molecule is therefore often described by pKa rather than Ka. Ka and pKa are related by the
equation:
pKa = log(Ka)

Therefore, strong acids will have smaller pKa values (and larger Ka values) than weak acids. Very strong
acids are practically 100% dissociated at equilibrium. Because the dissociation reaction for very strong
acids proceeds almost entirely to completion, it is typically not represented as an equilibrium but rather
as a one-way, irreversible reaction. For instance:

HCl(aq) + H2O(l) H3O
+
(aq) + Cl
-
(aq)

In this reaction one proton is released (and one H3O
+
ion is generated) for every molecule of HCl that is
present in an aqueous solution of hydrochloric acid.

Similar to strong acids, strong bases such as sodium hydroxide (NaOH) dissociate completely when
dissolved into water.

NaOH(aq) + H2O(l) Na
+
(aq) + OH
-
(aq)

Every molecule of NaOH that dissolves gives rise to one sodium ion (Na
+
) and one hydroxide ion (OH

).
We will take advantage of this property in todays lab in order to help us calculate the concentration of
an unknown molecule present in solution.

Titrations
Titrations can be used to analytically determine the amount of a given acid (or base) that is present in a
specified volume of solution. Note that this is typically not the same as determining the concentration of
H3O
+
(or H
+
) in solution. For strong acids dissolved in water, the concentrations of acid and H3O
+
are
indeed equal due to complete dissociation. However, this is not the case when dealing with weak acids
that have small Ka values (like the type we will be studying today). In these cases, only a small fraction of
the total acid has dissociated and the H3O
+
concentration is less than the total acid concentration at
equilibrium.

The total concentration of an acid (or a base) in solution can, however, be found by carrying out a
titration. Titrations are an analytical procedure used to measure the volume of one solution that must
be added to a different solution such that equivalent amounts of reactive molecules have been mixed
together. In an acid-base titration, the reactive molecule in one solution is the acid, while the reactive
molecule in the other solution is the base. In this lab we will be adding a base of known concentration to
an unknown acid to both identify the acid and determine its concentration.

Titrations are a type of volumetric analysis, owing to the fact that one is actually monitoring the volume
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of solution of one reactant that must be added to another reactant. This is accomplished by using a
special type of laboratory glassware known as a burette, which is a long, cylindrical glass tube that is
graduated along its length (see Figure 1). Controlled volumes of fluid of known concentration can be
accurately dispensed by operating a valve at one end of the tube. The solution that is used to fill the
burette is known as the titrant, and it is slowly dispensed into a beaker that contains a specified amount
of the other solution to be assayed (the analyte). The volume of titrant that was added to the analyte in
order to reach a desired outcome can then be recorded.

Fig. 1: Typical components used in an acid-base titration



Burette filled with titrant




Valve used to dispense titrant



Beaker or flask containing analyte




In this weeks lab you will be using a pH sensor to monitor the pH of the solution as titrant is added to
the analyte.

For an acid-base titration in which the concentration of the acid is to be determined, a base solution of
known concentration is used as the titrant while the acid is added to the beaker. For example, consider
the titration of an acid with a strong base (e.g., NaOH). When NaOH is added to the HCl solution,
hydroxide ions react with hydronium ions to form water.

OH
-
(aq) + H3O
+
(aq) 2 H2O(l)

The Keq for this reaction is far greater than 1. When all of the hydronium ions have reacted with an equal
number of hydroxide ions to form water, the pH of the solution will change from being acidic (pH < 7) to
being neutral (pH = 7).

Titration Curves
The progress of a titration can be followed by measuring the pH of the solution in the beaker using a pH
meter as increasing amounts of titrant are added. In the example of titrating a strong acid such as HCl
with a strong base like NaOH, the pH of the HCl solution would be expected to increase until the
endpoint (pH = 7) is reached and the solution is neutralized. If more NaOH were then added to the
solution, we would expect the pH to increase further (pH > 7). The results of such an experiment can be
shown on a graph that plots the volume of NaOH added to the beaker (on the x-axis) against the pH of
the solution (on the y-axis). This type of graph is known as a titration curve. An example of such a curve
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for the above reaction is shown in Figure 2. Notice how the pH of the solution rises slowly at first as
hydroxide ions are added to the flask containing HCl; this is because of the non-linearity of the pH scale.
Once a certain amount of NaOH has been added, the pH rises rapidly and then finally levels off again
gradually at higher pH values. On the graph in Figure 2, the equivalence point depicts the point at which
all of the H3O
+
ions have reacted with an equivalent quantity of hydroxide (OH

) ions. In the example

graph shown below, the equivalence point is reached when x mL of NaOH have been added to the HCl
solution.





Fig. 2: A hypothetical titration curve showing
the titration of HCl with NaOH.







Titrations of weak acids
Titrations can also be performed using a weak acid and a strong base. Weak acids (by definition) do not
dissociate extensively into H
+
ions and their conjugate bases when placed into solution on their own.
Nonetheless, when an aqueous solution of a weak monoprotic acid (such as acetic acid, which is found
in vinegar) is titrated with a strong base such as NaOH, the continuous addition of hydroxide ions (OH

)
will cause all of the ionizable hydrogen atoms to eventually dissociate from the acid. (Can you think of
why this is the case?) The equivalence point is reached once all of the released hydrogen ions (in the
form of H3O
+
) have reacted with the added hydroxide ions to form water.

Titration curves can be very useful for examining some of the inherent properties of weak acids as well
as any molecules that contain one or more ionizable (or titratable) groups. In particular, titration
curves for weak acids allow us to easily approximate the pKa values that are associated with the
different ionizable groups within a molecule. For instance, consider the titration of maleic acid, C4H4O4,
with NaOH. Maleic acid is a weak diprotic acid capable of donating two hydrogen ions. Each molecule of
C4H4O4 loses its two protons one at a time, and each dissociation step has its own, separate acid
dissociation constant (Ka). The corresponding pKa values (at 25 C) for each dissociation reaction, as
shown on page 2.

The pKa values show that the first proton that is donated (step 1) is the one that is most readily lost.
Nonetheless, if an aqueous solution of maleic acid were fully titrated with a strong base such as
NaOH, all of the acidic hydrogens would dissociate and react with hydroxide ions to form water.
Because the dissociation of the two ionizable hydrogens occurs in a stepwise manner, each
dissociation step has its own equivalence point on a titration curve.

This is shown diagrammatically in Figure 3 for a hypothetical titration of maleic acid with NaOH. At the
first equivalence point, all of the C4H4O4 will have been converted to C4H3O4
-
(maleate
1-
), while at the
second equivalence point, all of the C4H3O4
-
will have been converted to C4H2O4
2-
(maleate
2-
). At the
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point where exactly half of the C4H4O4 has been converted to C4H3O4
-
, there will be equal concentrations
of this conjugate acid/base pair present in solution. In other words, the ratio of [C4H3O4]/[C4H3O4
-
] will
be equal to 1. Based on the Henderson-Hasselbalch equation (covered in lecture), the pH value at which
this occurs is equal to the pKa of the first titratable group, and it can be easily approximated by eye from
the titration curve. The same reasoning applies to the estimation of the second pKa.
pK
a
= pH +log (
[HA]
[A

]
)

When [HA] = [A
-
], log (
[]
[

]
) = log (1) = 0, such that pKa = pH



Fig. 3: A hypothetical titration curve showing the
titration of maleic acid with NaOH. At the points
where there are equal concentrations of the
corresponding conjugate acid/base pairs, the pH of
the solution is equal to the pK
a
of the respective
titratable group. In this example, the acid has two
titratable groups with pK
a
values near 2 and 6.




Regardless of the acid being analyzed, titrations allow us to monitor the volume of titrant (e.g.,
NaOH) that must be added to reach the corresponding equivalence point(s). In order to calculate the
number of acid molecules that are present in our initial analyte sample, and thus the concentration
of the solution being titrated, we need to determine the actual quantity of base that is contained
within a specified volume. The following section serves as a brief review of how concentration is
measured. If you are already familiar with this material, you can skip ahead.

Units of concentration
In the above text, we have been referring to individual molecules of HCl and NaOH. In reality, it is
impossible to accurately measure out individual molecules in a solution that contains such a large
number of small particles. Likewise, it is impossible to weigh out single atoms and molecules that
weigh such tiny amounts. Because atoms are so small, a much larger unit must be used to measure
the quantities of atoms and molecules in a way that is feasible in the laboratory. A standard unit that
is used to measure the quantities of atoms and molecules is the mole (abbreviated mol). One mole of
a particular atom or molecule consists of Avogadros number of particles, namely 6.022 x 10
23
particles.
Thus, one mole of protons corresponds to exactly 6.022 x 10
23
protons, while one mole of hydroxide
(OH
-
) ions corresponds to exactly 6.022 x 10
23
individual OH
-
ions.

It turns out that there is a simple way to convert the number of grams of any pure substance to its
corresponding number of moles (and vice versa). A mole of a substance corresponds to its atomic mass
in terms of grams. In this case, a mole of protons will consist of 6.022 x 10
23
protons and weighs 1 gram
(because a proton equals 1 atomic mass unit). Similarly, a mole of hydroxide ions consists of 6.022 x 10
23

hydroxide ions and weighs 17 grams (because oxygen has an atomic mass of 16 and hydrogen has an
atomic mass of 1).
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When molecules are dissolved in a solution, their concentrations are often expressed as the number of
moles of the molecule per liter of solution. This is known as the solutes molarity, or molar
concentration. The molarity of a solute can be calculated by dividing the number of moles of the
substance by the volume of the solution, expressed in liters.

Molarity (M) = moles of solute
liters of solvent

Thus, a 1 M (pronounced one-molar) solution contains exactly one mole of solute per one liter of
solution. Likewise, a 0.5 M solution contains exactly 0.5 moles of solute per one liter of solution.

The examples below are meant to illustrate simple ways to calculate these parameters. Make sure
you feel comfortable performing these calculations so that you can complete the exercises at the
end.





Reading Burettes
You will be using burettes to record the volume of titrant added to your solution. Please review the basic
information provided below. Take care to not damage the burettes and not to harm yourself. If not
handled with care, they will break easily.

Burettes are filled with a titrant solution and the valve used to dispense this solution is found at the
Example 1: Calculating the molarity of a solution

Question: A solution was prepared by dissolving 0.075 moles of sodium chloride (NaCl) into
water. The final volume of the solution was 300 mL. What is the molarity of the solution?

Solution: To calculate the molarity, you simply need to divide the number of moles of
solute by the final volume of the solution, expressed in liters.

Molarity = 0.075 mol NaCl
0.3 L solution

= 0.25M NaCl

Example 2: Determining the number of moles in a given volume

Question: How many moles of Na
+
are present in 45 mL of a 0.6 M solution of NaCl?

Solution: In this case, you can use the molar concentration of NaCl and adjust for the new
volume of solution.

In 45 mL: 0.045 L solution x 0.6 mol NaCl = 0.027 mol Na
+
.
1.0 L solution
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bottom. It is important to know how to read burettes correctly. Unlike a graduated cylinder or beaker, a
burette has the zero mark at the top rather than at the bottom, and the values of the numbers
increase as one reads down the burette. This is helpful when adding up the incremental amounts of
solution that are being dispensed. One of the trickier parts about using a burette is to accurately read
the level of the liquid inside.

The top of the solution inside a burette appears as a meniscus when viewed from the side
(see images below). Burettes are read by aligning the bottom of the meniscus (not the top!) with the
appropriate scale bar. The examples below help to illustrate this feature. It can sometimes help to
visualize the meniscus more clearly by holding one of your fingers (or a piece of white paper) behind the
burette just where the meniscus is located.



6.90 mL 46.35 mL

Lab Protocol: Titration of an unknown analyte using NaOH
In this lab, you will be given the task of constructing a titration curve to determine the chemical
properties of an unknown molecule (or analyte). The analyte of interest has several titratable groups,
each with its own pKa value. In the sample you will use in lab, all of the titratable groups were
protonated prior to dissolving the sample in water.

You only need to generate one titration curve for your sample. Below are general instructions on
how to use the pH sensors in the lab in conjunction with software that allows you to record the
progress of your titration. Work with your lab partners to set up the titration and divide up the work
to make it more efficient. Your TF can help you to answer questions about the procedure. Make sure
that you record the following:

The concentration of NaOH used for the titration:

The initial volume of sample you are titrating:

Using the pH sensors

You will be using a software program called Logger Pro to record and plot the pH readings of your
sensor. This will allow you to generate a titration curve to be used for answering questions for your lab
assignment. The pH sensors that you will be using today have already been calibrated prior to starting
the lab, and it will not be necessary to re-calibrate them. These sensors are very delicate, and you
should always be careful when handling them. Importantly, dont leave them out of solution for a
Bottom of meniscus
(at 6.90 mL)
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prolonged period of time. Please ask your TF if you have any questions.

General Protocol Guidelines
1. Fill your burette with NaOH (available in the lab) above the 0 mL line and then dispense NaOH into a
waste beaker until the meniscus reaches the 0 mL line. There are containers at the bench labeled
Used Chemicals that you can use to collect used or excess chemicals. Be sure to write down the
concentration of the NaOH solution that youll be using.

2. Obtain a 50 mL plastic beaker and add your sample solution. The beakers may already be at your
bench. If they are, you should rinse the beakers about 3 times with distilled water to make sure that
there is no remaining acid or base in the beaker. It is not necessary to completely dry the beaker, as
water wont affect the titration in any manner. We recommend that you add 15 mL of the unknown
analyte solution. Whatever you decide, just make sure you make note of how much volume you add
to your beaker.

3. To start the experiment, open the file called LS1A Titration Lab in the Downloads folder. This will
open the Logger Pro program.
a. Log in to the Student profile using the password g3n0m3.
b. If Logger Pro starts up automatically, close the "Logger Pro" window that first appears.
c. Hit "Don't save" at the prompt.
d. Click on the "Documents" folder at the bottom of the computer screen and select Life Science
Labs.
e. Double click on the program called LS1A Titration Lab.cmbl to open Logger Pro with all of the
appropriate configurations set.
f. [If the file is not there, you can download it from the Laboratory page of the course website.]

4. Lift the pH sensor out of the storage solution in which it is submerged and set the storage vial aside
until you are finished. Before you submerge the pH sensor in your sample, rinse the tip of the pH
sensor with the distilled water to wash away any residual solution. Do so by using a squirt bottle and
hold the sensor over the sink.

5. Submerge the tip of the pH meter probe into your sample and make sure you get a stable pH
reading. You can secure the pH sensor to the fastening rod using the supplied clamp if you wish. The
tip of the pH meter should be fully submerged. Then position the burette such that you can dispense
NaOH into the beaker while the pH sensor remains submerged.

6. You will see the pH of your sample solution displayed in the bottom left corner of the screen.
Once you have everything set up, click on the green start button in the toolbar menu. The
button will now change and appear as a red stop button.

Next, click on the keep button that is located next to the red stop button. The image below
shows where these buttons are located.

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7. After you click the keep button, a new window will appear as shown below:



To enter the current pH reading into the table on the screen, type in the total volume of NaOH that
has been added to the beaker. To enter the initial pH reading, type in 0 (the number zero) and
click OK. This will enter the initial pH of your sample into the table.

8. Begin the titration by adding NaOH in 0.5 mL increments to the beaker, briefly swirling the contents,
and then waiting for the pH reading to stabilize. It is not crucial for you to add exactly 0.5 mL, but
try to be as close as possible. Once you have a stable reading, enter the total amount of NaOH
added to the beaker up to that point in order to track the pH as a function of NaOH added to the
beaker.

9. Whenever you observe noticeable increases in slope of the titration curve, add NaOH in smaller
increments to increase the precision of your curve. See how well you can do!

Saving your titration curve
10. Once you are satisfied that you have enough information to characterize all of the titratable groups
(which you can tell when the titration curve settles around pH 12-14), stop the titration by clicking
on the red stop button in the toolbar menu. Use the Grab program to capture and save an image
of your titration curve on the desktop, but do not save the Logger Pro file. Please email the picture
to yourself once it has been saved (it is part of your post-lab assignment due next week!) Your TF
can show you how to use Grab to capture an image. Before next week you will need to print off a
copy of the titration curve that you send yourself and staple it to your post-lab assignment.

11. After you are finished, rinse the tip of the pH sensor with distilled water, blot the end of the sensor
with a kimwipe, and place it back into the storage solution.

WASTE DISPOSAL and CLEAN UP

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Discard all of the solution inside your beaker into the 5 gallon waste drum in the fume hood at the
back. The 5 gallon waste drum should be labeled with LS 1a and should contain only the basic
waste product of this titration exercise.
Rinse out the beaker with tap water three times, discard the rinse into the sink, and then rinse the
beakers twice with distilled water and leave them at the bench.
Make sure your bench is clean and organized for the next group. Check with your TF before you
leave.