Experiment 12 Overview Page |1

EXPERIMENT 12
pH in Blood

PRE-LAB REQUIREMENTS

PRE-LAB REQUIREMENTS FOR EXPERIMENT 12 ARE…...

1. Read the entire experiment 12: Overview.

2. Read the Online Introductory Material:

Read through the following techniques in the Experiment 12: Resources module:
Techniques:
• Measuring pH
• Setting up the Microlab program and Calibrating the drop dispenser

Virtual Lab:
Complete the Virtual Lab Experiment 12: Exploring Buffers
• Submit the Feedback Form, for Evaluation Question(s)
No late submissions will be accepted

3. Complete the Pre-lab portion of the Experimental Design Form:

As you design your procedure and complete the EDF (in pen):
• Use the Experimental Overview and Virtual Lab Tutorial to assist with designing your
procedure
• As you read through the Experimental Overview, choose glassware, equipment and
chemicals for each step from the Laboratory Stockroom

4. Complete the Pre-Lab Quiz (in Canvas)

• Experiment 12: Pre-lab Quiz
This quiz must be completed BEFORE the start of your Experiment 11 lab session

5. Learning Goals
• Read through the learning goals for Experiment 12 on the following page.

There is a lot of work for this experiment. Do no leave it until the last minute or you will be overwhelmed
Experiment 12 Overview Page |2

Experiment #12 Laboratory Learning Goals

A. Basic laboratory skills
A1. Adhere to instructions on laboratory safety, recognize hazardous situations, and act appropriately.
A2. Perform common laboratory procedures correctly (new and old). (Refer to A2.b & c in the General Lab LGs.)
A2.a: New laboratory procedures
• Perform pH probe and drop counter calibration procedures as directed
• Measure pH using a pH probe
• Prepare a buffer with necessary capacity for the problem
A3. Maintain an up-to-date laboratory record that is of sufficient detail that others could repeat any of your experiments if
necessary. (Refer to A3.a, b, & c in the General Laboratory LGs.)
A4. Apply appropriate methods of analysis to data and convert raw data into meaningful units.
• Convert mass to volume appropriately
• Illustrate a buffer region with a graph
• Calculate buffer capacity from experimental data
o Determine whether the acid or base buffering capacity of the blood model system is greater.
• Organize your data so that appropriate information can be found and interpreted quickly
• Determine how to restore the acid/base capacity of a buffer using your data
• Create an acute acidosis chart for a hospital based on the behaviour of your model buffer
A5. Perform logical troubleshooting of laboratory procedures.
• Set up the drop counter to minimize reading errors and monitor/adjust as needed.
B. Communication (Refer to B2 & B3 in the General Laboratory LGs.)
B3. Access (and properly cite) relevant information from the library and other resources.
• Perform literature research on the experiment’s topic (acidosis/alkalosis)
C. Responsibility (Refer to C2 in the General Laboratory LGs.)
C1. Effectively prepare in advance for laboratory work.
• Complete the Virtual Lab: Experiment 12 Exploring Buffers Tutorial
o Calculate the concentrations of acid and conjugate base required to create the needed buffer system
• Read the techniques: “Using the pH probe and drop counter”
• Complete online quiz and print the needed portion
• Prepare your experimental procedure based on lab manual information
D. Context
D1. Use the scientific method.
• Prepare an appropriate experimental design to examine the blood buffer model system based on lab manual
information to test the hypothesis
D2. Describe what your data does and does not say. Interpret the data
o Consider the differences and similarities between the model system and the real blood buffer system
▪ Consider the usefulness of models to examine problems indirectly
o Consider the limitations of the experiment (ex. atmosphere and the blood buffer)
o How can your graph of data be used? What information does the graph provide?
o Consider the limitations of the instruments (ex. pH probe precision)
E. Integration and application of knowledge/experience
E1. Apply critical thinking in the laboratory.
• Apply the principles of chemical equilibrium to explain how buffer solutions are able to keep the pH of a solution
within a particular pH range.
• Explain the changes in concentrations of specific ions in solution when a small amount of strong acid or base is added
to a buffer solution.
• Identify the buffer components responsible for neutralizing strong acid or strong base.
• Explain the differences between the model used to “treat the patient” and what would occur in an actual hospital
emergency room.
E2. Recognize whether results and conclusions "make sense". Example of question to consider:
• Did the pH change as expected when adding acid/base or when “saving the patient”?
Experiment 12 Overview Page |3

EXPERIMENT 12
pH in Blood

AIM
To examine the behaviour of buffers and to subsequently design an experimental model of the blood
buffer system.

OBJECTIVES:
❑ Design an experimental procedure to design a blood buffer system
❑ Determine the acid and base buffer capacities of the blood buffer system
❑ Calculate the amount of restoration solution needed to “save the patient”
INTRODUCTION
In order to function properly, the human body maintains a very narrow range of pH in blood, between
7.35 and 7.45. Maintaining the proper pH is critical for the chemical reactions that occur in the body. Any
condition in which the blood pH drops below 7.35 is known as acidosis. If the pH rises above 7.45, it is
known as alkalosis. If the pH drops below 6.8 or rises above 7.8, death may occur. Fortunately, we have
buffers in the blood to protect us against large changes in pH.

The Theory Behind the Experiment

Buffers
Buffer solutions are usually made of weak acid-base conjugate pairs, such as H2CO3 and HCO3-. One of
these buffer components will chemically remove H3O+ (strong acid) by turning it into weak acid, the other
will remove OH- (strong base) by turning it into weak base. In general, for a solution of weak acid, HA, and
its conjugate base, A-, the following equilibria must be satisfied simultaneously:
Ka
HA (aq) + H2O H3O+ (aq) + A- (aq) (1)
Kb
A- (aq) + H2O OH- (aq) + HA (aq) (2)
Kw
H2O + H2O H3O+ (aq) + OH- (aq) (3)

For reaction (1): For reaction (2):

+ −
[ H 3O ][ A ] [OH − ][ HA]
Ka = (4) Kb = (5)
[ HA] [ A− ]

If H3O+ ions are added, the equilibrium is disturbed, and reaction (1) will shift to the left. A-combines with
H3O+ and turns it into the weak acid HA. This causes the [H3O+] to decrease to close to the original amount,
and thus the pH stays fairly constant. If OH- ions are added, reaction (2) will shift to the left. HA combines
with OH- to give A-, a weak base. This will cause [OH-] to decrease to close to its original value, and the pH
will again remain fairly constant.
Experiment 12 Overview Page |4

Rearranging equation (4) allows us to express quantitatively the [H3O+], and thus the pH changes in the
buffer solutions:
[ HA]
[H3O+] = Ka
[ A− ]
If the concentrations of the weak acid (HA) and its conjugate base (A-) are equal in a buffer solution (a
“1:1” buffer), the term [HA]/[A-] = 1, and [H3O+] = Ka and the pH of the solution equals the pKa of the weak
acid. Other ratios of [HA] to [A-] give different pH values for the buffer.
When choosing an appropriate conjugate acid-base pair to form a buffer at a specific pH, the selected
conjugate acid's pKa should be within 1.0 pH units of the desired buffer pH.

Buffer Capacity
The buffer capacity is the amount of acid or base a buffer can accept until it can no longer maintain a
relatively constant pH. It is a value that quantifies the amount of H3O+ or OH- that a solution is capable of
neutralizing before the weak acid or conjugate base is used up and the pH of the solution begins to change
sharply (see figure below; tail ends of the pH curve).
Acid buffer capacity of a solution is defined as the number of moles of H3O+ per litre of buffer which are
required to lower the pH by 1 unit. Units are M.

Base buffer capacity of a solution is defined as the number of moles of OH- per litre of buffer which are
required to raise the pH by 1 unit. Units are M.
A buffer becomes increasingly resistant to changes in pH as the amounts of the conjugate acid-base pair
used in its preparation are increased. Buffer capacity, therefore, depends on the number of moles of HA
and A- present and not on the [HA] to [A-] ratio. The [HA] to [A-] ratio is, nevertheless, indicative of how a
buffer will respond towards the addition of H3O+ versus OH-. Accordingly, a “1:1” buffer has the capacity
to absorb both H3O+ and OH- with equal effectiveness. For such a buffer, the same amounts of weak acid
and conjugate base are available for neutralization of any added strong base or strong acid. The acid and
base buffer capacities of a “1:1” buffer are identical. For any buffer that does not have a 1:1 [HA]/[A-]
ratio, the acid and base buffer capacities will be different.
The figure to the right shows how the acid and base buffering capacity of a buffer solution can be
graphically determined, shown here using 25 mL of a pH = 5.00
acetate buffer solution. In this case, an experiment showed that
12.3 mL of 0.10M HCl was required to lower the pH by 1 unit,
whereas 7.65 mL of 0.10M NaOH was required to raise the pH by
1 unit.

(0.0123 L HCl)(0.10 M HCl) = 0.049 M acid buffer capacity

0.025 L buffer

(0.00765 L NaOH)(0.10 M NaOH) = 0.031 M base buffer capacity

0.025 L buffer volume acid added volume base added
Experiment 12 Overview Page |5

The pH Meter
The graph above was obtained using a pH meter. Using a pH meter in acid-base titrations provides much
versatility and is equally as accurate as using indicators in determining endpoints. Endpoints on a graph
of pH versus titrant added are easily identified since they occur where the slope is the steepest, i.e. when
small amounts of added titrant greatly change the pH. Where the slope is less steep, the solution is
buffered.
Preparing buffer solutions
A buffer solution contains appreciable amounts of weak acid-base conjugate pairs and can be prepared
from the pure weak acid (HA) by adding a strong base (NaOH) until enough conjugate base has been
produced and the desired pH (in the buffer region of the acid) is reached. Conversely, one could start
with the pure weak base (A-) and add the appropriate amount of strong acid (HCl) until the same point is
reached. In this lab, we will use the first method to prepare a buffer.
The buffer solution you will prepare must fulfill the requirements for the pH and the acid buffer capacity
(it must be able to resist the addition of a certain required amount of acid). Therefore, to prepare this
buffer, you must:
• Determine the ratio [HA]/[A-] to achieve the required pH.
• Determine the amounts (# moles) of A- and HA to achieve the required acid buffer capacity.
• Use a sufficient amount of weak acid (HA) for both the acid and the conjugate base (A-) components,
since all the conjugate base forms from the weak acid upon addition of NaOH.
The calculations required to prepare this buffer are explained in Virtual Lab 12: Exploring Buffers.
A buffer can also be prepared by mixing together a weak acid and its conjugate base in the correct ratio
for the desired buffer pH. This method is described in detail in your textbook.

The Carbonic Acid/Bicarbonate Buffer in Human Blood

The human body is designed to work in the specific pH range of 7.35 to 7.45. Because of this, the body
has many different mechanisms to prevent any drastic change in pH. One of these guards is the carbonic
acid/bicarbonate blood buffer.
Carbonic acid (H2CO3) is a weak acid that, when in solution, is in equilibrium with its conjugate base
bicarbonate anion (HCO3-) as shown below. This combination forms a buffer in the blood.
H2CO3 + H2O H3O+ + HCO3- Ka = 7.9 x 10-7
The pKa for carbonic acid is 6.1 and not close in value to the pH of blood. As shown in the calculations
below, under normal circumstances (ie, pH 7.4) there is approximately 20 times more bicarbonate present
in the blood than there is carbonic acid. As normal metabolism produces far more acidic than basic by-
products, the high conjugate-base concentration of blood is ideally suited to neutralize the acids
produced.

[ H 3O + ][ A − ] Ka [ A− ]
Ka = or =
[ HA] [ H 3O + ] [ HA]
pH = -log [H3O+] = 7.4 or [H3O+] = 4.0 x 10-8 M

[ A− ] Ka −7
= + = 7.9 x 10 −8 = 20
[ HA] [ H 3O ] 4.0 x 10
Experiment 12 Overview Page |6

Carbonic acid is not stable in aqueous solution and decomposes as shown below:
K2
H2CO3(aq) H2O + CO2 K = [CO2]/[H2CO3]
Accordingly, H2CO3 and CO2 always exist in blood in the same proportion. Alteration of blood CO2 levels,
therefore, has the same effect on the buffer as a similar alteration of H2CO3 levels. This second equilibrium
is significant, as it links together the blood buffer system with the respiratory system as shown in the
figure below. The respiratory system thus allows for constant hedging of blood pH by controlling blood
CO2 levels, thereby affecting H2CO3 concentrations.

respiratory system blood buffer system

2 H2O + CO2 H2CO3 + H2O H3O+ + HCO3-

exhaled from lungs produced via metabolism

Acidosis: Failure of the Human Blood Buffer

During strenuous exercise, there is danger of suffering from two different kinds of acidosis (lowering of
blood pH). One is respiratory acidosis while the other is metabolic acidosis.

Respiratory Acidosis: When one becomes active, body metabolism increases, and more food molecules
such as glucose are metabolized for energy
C6H12O6 + 6 O2 → 6 CO2 + 6 H2O (7)
The CO2 product shifts the H2CO3/HCO3- buffer towards bicarbonate, and hence towards H3O+, thereby
acidifying the blood. Breathing rates increase so that more CO2 is exhaled, restoring the buffer. Acidosis
results when the lungs fail to eliminate CO2 as fast as it is produced.
Metabolic Acidosis: If exercise continues, the oxygen supplied to the tissues by breathing may not be
sufficient to carry out reaction (7) all the way. Metabolism continues, but only part way, through the
breakdown of glucose into lactic acid:
C6H12O6 → 2 CH3C(H)(OH)COOH (lactic acid, C3H6O3)
Lactic acid is a weak acid and lowers blood pH by this non-respiratory process. This causes a familiar
aching and fatigue in the muscles. In extreme cases, intravenous injections of weak bases, like
bicarbonate, must be administered to restore the blood buffer back to a safe pH level.
The carbonic acid buffer is regularly analyzed in clinical practice. However, due to the decomposition of
H2CO3, as shown above, study of the carbonic acid buffer is impractical in the general chemistry laboratory
as it is too difficult to prevent the loss of carbon dioxide into the room’s atmosphere. Therefore, in this
lab we will model the behaviour of the blood buffer using the phosphate buffer that is formed between
the weak acid dihydrogen phosphate (H2PO4-) and its conjugate base hydrogen phosphate (HPO42-). This
buffer behaves similarly to the carbonic acid buffer but does not suffer from a decomposition limitation.
H2PO4- + H2O H3O+ + HPO42- Ka = 6.3 x 10-8
Experiment 12 Overview Page |7

The Experiment
LAB PROJECT SUMMARY
While you read through the procedure, consider the general concepts and think about the questions in
purple text, using your answers to help you develop your own experimental procedure and improve
your lab skills. The answers to these questions do not need to be included in your EDF.

Perform an experiment that allows you to quantitatively achieve the following tasks:
1. Prepare a model buffer solution that fulfills the following characteristics of the real blood buffer:
• The pH of the blood buffer is normally between 7.35 and 7.45.
• The blood buffer resists the addition of strong acid to the level where 1.0 L of blood will undergo
a change in its pH of 0.2 units when 0.020 moles of H3O+ are added to it.
2. Expose the model buffer solution to the additional acid and base:
This exposure will be performed by adding an acid or base dropwise to the buffer. The data gathered
will be used to calculate both the acid buffer capacity and the base buffer capacity of the model buffer.
This dropwise addition of acids and bases is conducted using titration techniques.
3. Consider the buffering capacities of the model buffer:
The acid and base buffer capacities of the blood buffer fulfill the physiological requirements of the
human body. As noted above, metabolic processes result in a lowering of the blood pH. Is the acid
buffer capacity of blood therefore greater than its base buffer capacity? The model buffer should
allow you to answer this question.
4. Emergency Room Procedure:
Once the pH restoration process has been studied on a 25 mL sample of the buffer, the procedure can
be expanded to mimic the requirements of an acidosis victim whose volume of blood is, for example,
7.0 L.
5. Save the Patient:
Test the devised E.R. procedure to see if you can both predict and apply the correct amount of buffer
component required to restore a model sample of the patient’s blood back to a pH of 7.45.

Note: Throughout the experimental procedure you will see calculations and questions written in GREEN
with (D and a number) after them. The answers to these calculations and questions must be included in
the appendix of your Experiment 12: Lab Report.

EXPERIMENTAL PROCEDURE
General Concepts and Questions Procedure and Points to Consider
Watch the video on How to Set Up the Microlab Set up the Microlab interface with the following
Interface. (You will lose precious time in the lab sensors:
trying to figure this out if you don’t watch before • Counter
the lab). • pH
Experiment 12 Overview
General Concepts and Questions
Calculate what the concentration of [H2PO4-]
(weak acid) and [HPO42-] (the conjugate base)
should be in the phosphate buffer to meet these • whose pH will drop by only 0.2 pH units when
requirements.
Calculate the number of moles of the two buffer
components required.
Calculate the exact mL of the weak acid required
to create your buffer (D1)
Calculation help: Virtual Lab Experiment 12:
Exploring Buffers
This does not have to be done quantitatively, so
which piece of glassware will you use?
Carefully add NaOH from a graduated cylinder
until the pH is roughly 7.35, then start using a
Pasteur pipet to add it very slowly.
Watch the video on Setting up the Microlab
Program and Calibrating the Drop Dispenser
video showing how to add the formula, once you
have calibrated the drop dispenser.
Use the graph properties in the Microlab
program to add a title.
Use the graphs to calculate the acid buffer
capacity (D2) and the base buffer capacity (D3) of
the phosphate buffer. Is the acid buffer capacity
of blood therefore greater than its base buffer
capacity? (D4). (This can be done at home after
the lab)
Page |8
Procedure and Points to Consider
Prepare 100 mL of a phosphate buffer:
• With a pH of 7.45
0.020 moles of H3O+ are added to 1.0 L of the
buffer
• In the lab you will be provided with stock
solutions of 0.50 M H2PO4- and 1.0 M NaOH
with which to prepare your phosphate buffer.
(The NaOH is used to prepare the conjugate
base HPO42- from its weak acid H2PO4-)
Note: Show calculations for the preparation of the
buffer solution (mL of 0.50 M H2PO4- and 1.0 M
NaOH needed) in your EDF.
Transfer the calculated amount of 0.5 M H2PO4- to
a clean 250 mL beaker
Using the pH probe to monitor the pH of the
solution, add 1.0 M NaOH, while stirring, until a pH
of 7.45 is reached
Transfer the buffer solution to a 100 mL
volumetric flask and fill to the calibration mark
with pre-boiled deionized water
Calibrate the drop dispenser and add the formula
to the Microlab program
Pipet 25.00 mL of your buffer into a clean 250 mL
beaker
Fill the drop dispenser with 0.1 HCl and start the
Microlab program
Dispense the HCl dropwise into the buffer solution
until the pH has dropped 1 unit. Stop the program
Title the graph with your name and HCl and print
Repeat the above 4 steps for the 0.1 M NaOH
(Clean out the drop dispenser and fill with the 0.1
M NaOH)
Title the graph with your name and NaOH and
print
Mark up the graphs to display the acid and base
buffer regions. (This can be done at home after the
lab)
Experiment 12 Overview Page |9

General Concepts and Questions Procedure and Points to Consider

The “Patient Trial” blood is a phosphate buffer Pipet 25.00 mL of the “Patient Trial” blood into a
which simulates acidosis. clean beaker. Record the pH.
Clean out the drop dispenser and fill with
Restoration Solution.
Start the Microlab program then add the
This simulates reversing acidosis Restoration Solution to the patient’s blood
dropwise, until a pH 7.45 is restored.
How much Restoration Solution was required? Title and print the graph. Determine how many
(D5). Which phosphate buffer component do you mL of restoration was need to restore the pH
think the Restoration Solution is comprised of?
(D6).
Calculate how much Restoration Solution would
be required to restore a patient’s blood from the
“patient trial blood” back to 7.45, assuming the
patient has 7.0 L of blood? How much restoration
solution is required? (D7) (This can be done at
home)
Test your model by “Saving the Patient”. Use the small bottle of “patient’s blood”
It will not be re-filled if you make a mistake.
Measure the pH and volume of the sample and
predict the amount of Restoration Solution you
think you need to restore the pH to 7.45.
Add the whole sample to a clean beaker and turn
on the magnetic stirrer. Call over your TA
The pH has to be between 7.40 and 7.50 to save Add your calculated amount of Restoration
your patient and gain a bonus mark Solution all at once.

Helping the ER Doctors

The emergency room staff requires a chart to quickly determine how much base needs to be administered
to a patient with acute acidosis. Based on your results from the third titration graph obtained, prepare a
chart similar to that shown below (D8). Assume human blood volume is 100 mL per 1 kg of body mass for
adults. The restoration from pH 7.2 has been included in the example on the next page.

pH Volumes (mL) of Restoration Solution

7.40
7.30
7.20 7000 11200
7.10
7.00
50 kg 55 kg 60 kg 65 kg 70 kg 75 kg 80 kg weight
Experiment 12 Overview P a g e | 10

Example:

To get from pH 7.2 back to 7.45, Titration of 25 mL of Buffer with Restoration

Solution
the titration curve shows that 35 7.
6
mL of Restoration Solution must
be added to 25 mL of the buffer. 7.
5
Multiply this by 4 to simulate
100 mL of blood. We therefore 7.
4
need 140 mL of solution to
7.
restore 100 mL of blood, or 1 kg 3
of body mass. Multiply this

pH
7.
value by 50 kg and see that 7,000 2
mL of Restoration Solution must 7.
be injected into the patient. An 1
80 kg patient will require 11,200 7.
mL. 0
35 mL
6.
9

6.
8 0. 10. 20. 30. 40. 50. 60.
0 0 0 0 0 0 0
Volume of Restoration Solution added (mL)

CLEAN UP
Glassware Rinse all glassware with tap water and return it to
your kit. Report any breakages or missing items to
your TA.
Drop Dispenser Rinse thoroughly with warm water and replace in the
clamp with the stopcock open.
pH probe Make sure it is in the bottle containing “buffer 7”
solution and the solution is clear and colourless

Pre-boiled deionized water bottle

Patient’s Trial Blood bottle Return all 3 bottles to you TA
Patient’s Blood bottle
Chemical disposal Pour down sink with plenty of water
Computer Close down the MicroLAB program on the computer
Experiment 12 Overview P a g e | 11

PRE-LAB CHECKLIST
Make sure to do the following before you do the experiment:
❑ Read through all the components of the experiment
Complete the Virtual lab Experiment 12: Exploring buffers and bring the feedback form to your
❑ Experiment 12 lab session.
❑ Complete the Experiment 12: Pre-lab quiz (see deadlines below)
Complete the pre-lab sections of the EDF listed below (in pen),
• Research question and Hypothesis
• Glassware / equipment and reagents needed
❑ • Procedure - Remember, keep it concise. If you want to bring in additional notes for
your own reference, that is ok, but do not include them in your EDF.
• calculations for the preparation of the buffer solution (mL of 0.50 M H2PO4- and 1.0 M
NaOH needed)

The more you prepare for the lab the easier your experiment will seem. You only have 3 hours to
perform the experiment in the lab. Make sure you familiarize yourself with:
• your own procedure so you are aware of the order the experimental steps are performed
• any techniques in the experiment
• prepare tables to record data collected during the experiment

SUBMISSION DEADLINE
Task Submit (material) Submit (when)
Prepare for lab:
• Read experimental overview
Click here for exact submission deadlines
• Read online material
• Find research article
Bring to lab
Complete before the start of your
Virtual Lab (screenshot or
Experiment 12 lab session*
printout)
Submit in Canvas before the start of
Pre-lab Quiz Quiz
your Experiment 12 lab session*
Complete before the start of your
Complete pre-lab portion of EDF
Experiment 12 lab session*
Complete before the end of your
Complete in-lab portion of EDF Experiment 12 lab session*
Submit with your lab report
Submit one week after your
Complete Lab Report Lab Report Experiment 12 lab session**
Submit to box outside B370
*No late submissions **Late penalties apply (2 marks deducted per hour after the deadline)