Buffering Capabilities
of Guinea Pig Blood
Group MB3
BE 210-001
Buffering Capabilities of
Guinea Pig Blood
BE 210-001
Group MB3
Kim Coughlan, Barry Huang,
Laura Michelis, Le Truong
April 28, 2006
Buffering Capabilities
of Guinea Pig Blood
Group MB3
BE 210-001
Background
The capability to buffer, or resist pH change, is crucial to life. In animals, a
variation of mere tenths of a pH unit in the blood can be fatal, since many of the
chemical reactions that occur in the body, particularly those involving proteins, are pHdependent. Because of the biological significance of buffering, we chose to expand upon
the buffering experiment to further investigate buffering in a true biological context. In the
pilot experiment, carbonic acid (H2CO3) and phosphoric acid (H3PO4), two important
biological buffers, were titrated against standards; from the titration curve, both pKa’s and
buffering capacities (defined as dB/dpH, where B is moles of the base) could be
determined. These values were then compared to the accepted literature values; the
results were all very precise, with over 95% precision for all trials, but errors ranged from
5% to over 20%. This suggests that while the results are highly repeatable within the
experiment, systematic errors and other sources of uncertainty related to the
experimental set-up (e.g. measurement frequency) leads to variation across different
experiments. Even in the literature, values across different sources seldom exactly agree
(though they are usually quite close).
In this subsequent experiment, we will study just how effective the buffer capacity
of blood is. Since bicarbonate (HCO3-) is the primary buffer, we will use the pure
bicarbonate system (HCO3- ↔ H+ + CO32-) as a benchmark against which to compare
blood as a buffer (see Figure 1 for the experimental titration curve of Na2CO3 against
HCl standard). Normal blood pH is 7.4; since pH deviations below 6.8 or above 7.8 can
be fatal, we expect blood to have the highest buffering capacity within half a unit of 7.41.
Objectives and Hypothesis
This lab aims to examine how well a pure bicarbonate buffer serves as a model
for the buffering capabilities of real blood. Specifically, it is hypothesized that the pKa’s
and buffering capacities of guinea pig blood and pure bicarbonate (at a similar
concentration as that found in the blood) will not be significantly different.
1
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Buffer/Buffer.html
Buffering Capabilities
of Guinea Pig Blood
Group MB3
BE 210-001
Equipment
Major equipment
- pH meter with combination glass-silver/silver chloride electrode and
holder
- pH buffer standards (4, 7, 10)
The pH meter is required to measure the potential difference between
two electrodes sensitive to pH change. However, the pH-potential function is
not linear, therefore it must be standardized at room temperature using the
pH buffer standards at values of 4.0, 7.0 and 10.0. Using this
calibration, we measure the pH during the titration of guinea pig blood and
pure bicarbonate.
Lab equipment
- Two 50 mL burettes and dual burette stand
- 200 mL beakers
- Magnetic stirrer and stirring bar
Burettes and stands are necessary for the titration of blood and
bicarbonate. Beakers are required for the titration mixture. The magnetic
stirrer and stirring bar will keep the solution well blended.
Supplies
- 1L of 1M HCl
- Reagent grade anhydrous Na2CO3
- Gloves
Na2CO3 is dissolved to make pure aqueous bicarbonate solution. HCl is used to
titrate both the pure carbonate and Guinea Pig blood. Gloves ensure
experimenter's safety.
Newly purchased equipment
- Guinea Pig whole blood
Blood from guinea pig was chosen for its price, feasibility, and
safety. The cost of the blood is $170 per 100 milliliters. If each of the 20
lab groups requires 50 milliliters, the total price is $1,700, well within
the budget of $2000. Mammalian blood is more feasible and safer to use for
student experiments. The supplier of the blood, Lampire Biological
Laboratories, has been contacted and would be willing to sell for lab use.
Buffering Capabilities
of Guinea Pig Blood
Group MB3
BE 210-001
Methods and Protocol
Much of the procedure of this proposed experiment mimics that of Experiment 2
(Acid-Base Chemistry: Biological Buffers). The specific steps of the procedure are as
follows:
 Calibrate the pH meter as was done in experiment 2. Follow the prompts on the
screen and use the pH 4, 7, and 10 standard solutions.
 Dilute 100mL samples of 25mM HCl and 25mM Na2CO3.
o To prepare the sample of HCl, mix 2.5mL of the 1.0M stock HCl with 97.5mL of
distilled water in a 200mL beaker. Both of these liquids should be measured in
graduated cylinders. Use the stirrer and stir bar to ensure thorough mixing.
o To prepare the Na2CO3 solution, weigh out 2.65g sodium carbonate on the
Mettler balance and place it in a 200mL beaker. Add 100mL of distilled water,
and mix using the stirrer and stir bar.
 Be sure to clean and prepare the burette as was done in experiment 2. Fill the burette
with prepared 25mM HCl and titrate a 10mL sample of 25mM Na2CO3, recording the
pH every 0.5mL until the pH falls below 2. Repeat this titration two more times.
 Perform three titrations of 10mL samples of guinea pig blood with 25mM HCl,
following the same procedure as in the Na2CO3 titrations. Again, record the pH every
0.5mL until the pH falls below 2.
 Create titration curves for all titrations and determine all pKa’s and buffer capacities.
Just as was done in experiment 2, this should be done by plotting dB/dpH. The pKa’s
can be determined by finding the maxima on this graph. The buffer capacity can also
be determined from this graph by finding the mean value for dB/dpH at the pKa’s.
 Compare the buffer capacities of the Na2CO3 and guinea pig blood using a two-tailed
two sample t-test.
 Be sure to leave place the pH meter in standby and ensure that the lab space is clean,
as was done in all BE210 experiments.
Buffering Capabilities
of Guinea Pig Blood
Group MB3
BE 210-001
Anticipated Results
The primary values that are essential for the accurate analysis of the blood are
the pKa values for the 25mM of pure carbonic acid. These values must be within 5% of
the accepted literature values. This degree of accuracy will allow for a precise
calculation of carbonic acid’s buffering capacity and will be a stable point of comparison
for the buffering capacity of the blood samples. The pKa values and buffering capacity of
the carbonic acid can be easily determined using numerical differentiation. (See below
for sample graph).
Standardization of HCl w ith Na2CO3
pKa2 = 10.35
12.000
10.000
pKa1 = 6.33
pH
8.000
Trial 1
Trial 2
6.000
4.000
2.000
0.000
0
0.005
0.01
0.015
0.02
0.025
0.03
HCl added (mol)
Figure 1. Standardization of hydrochloric acid with sodium
carbonate
Figure 1 depicts a titration of carbonic acid with HCI that resulted in pKa values
that were within 5% of the accepted literature values. This is an essential step expected
in the first portion of this experiment.
dB/dpH
dB/dpH vs. pH of buffer
0.05
0.045
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
0.000
Trial 1
Trial 2
Trial 3
2.000
4.000
6.000
8.000
10.000
12.000
14.000
pH
Figure 2. Numerical differentiation of titration of phosphoric acid
Figure 2 is a sample of the graph resulted by numerically differentiating the
titration curve. From this graph, pKa’s (pH at maxima) and buffer capacity at each pKa
(dB/dpH at pKa) can be determined. Note that this sample graph is for phosphoric, not
carbonic acid.
Furthermore, the titration curves for three blood samples performed need to be
created and then used to calculate pKa values and buffering capacity for the blood. The
buffering capacities of the three blood samples can be compared to the three buffering
capacities obtained from the titration of three samples of pure carbonic acid using a twotailed, two-sample t- test. It is expected that the P-value will be below 0.05, i.e. there is
no significant difference in the buffering capacities of pure carbonic acid and blood.
Buffering Capabilities
of Guinea Pig Blood
Group MB3
BE 210-001
Potential Pitfalls and Alternatives
One of the main goals of this lab is to identify the extent of similarity in the
buffering capacities of red blood cells and carbonic acid. It was hypothesized that the
buffering capacities would be very similar because carbonic acid was considered to be
the main buffer in blood. But, the results from this lab may not correlate exactly with
results from a titration of pure carbonic acid due to the effects of other substances that
are present in the blood. For example, blood also contains other buffering agents,
including phosphoric acid and proteins that, by nature, contain carbonyl (-COOH) and
amide groups (-NH2) that can have some buffering effects; hemoglobin, the primary
oxygen-carrying protein found in red blood cells, also has some pH regulating
capabilities. All of these substances may affect the buffering capacity of blood. However,
if the hypothesis is correct, the effects will be minor and the buffering capacity of
carbonic acid will dominate those of the other buffering agents.
Another potential pitfall of this experiment is the presence of anticoagulants in the
blood samples. These anticoagulants are necessary in order to prevent the clotting of
the blood cells, which would prevent the possibility of an accurate titration. Therefore,
anticoagulants are essential to the experiment. However, these anticoagulants may also
confound the results; for instance, one common anticoagulant is citrate, which is an ionic
form of citric acid (- C3H5O(COO)33−). Hence, citrate has its own acid/ base properties
which will inadvertently affect the buffering capacity of the blood being titrated. This will
cause some divergence in the calculated buffering capacity of blood from that of pure
carbonic acid.
These potential pitfalls can be avoided by using blood serum instead of whole
blood. The serum contains no cells, clotting factors, or anticoagulants. Therefore, if the
buffering capacity of whole blood is significantly different than expected, and if this
difference is suspected to be caused by anticoagulants or other buffering agents in the
blood, using serum samples instead could remove this source of error and provide more
accurate results.
Another option would be to use artificial blood alternatives. These samples may
provide enough useful information about the buffering capacity of blood while avoiding
the effects of the acid/base properties of minor proteins present in the “real” blood
samples. Using real blood samples is still preferred, to study true biological significance.
In order for an accurate comparison in buffering capacities between carbonic
acid and blood, an equal amount of carbonic acid must be present in both samples.
Blood is known to contain approx. 21 to 28mM1, but an exact amount is unknown. In this
experiment, it was estimated that 25mM of carbonic acid is present in the blood. Hence,
the buffering capacity of 25mM of pure carbonic acid will first be titrated in order to later
be compared with the buffering capacity of blood. Since 25mM of carbonic acid in the
blood is an estimation and there can be more or less in the actual sample, the
comparison between the buffering capacities of the blood and of the 25mM of pure
carbonic acid will not be completely accurate due to this inherent uncertainty.
Finally, error can arise from the storage of the blood samples. In order for the
blood to maintain its properties, it must be stored correctly. Blood can be refrigerated for
no more than 42 hours, but can be frozen for up to ten years. Therefore, two days prior
to lab session, the blood sample should be removed from the freezer and allowed to
thaw in the refrigerator. These precautions will assure more accurate results and should
be taken in order to prevent errors.
This source of error may also be avoided by purchasing the blood samples as
near to the date of use as possible. By doing this, unexpected changes in blood
properties due to improper or inadequate storage methods can be prevented.
Buffering Capabilities
of Guinea Pig Blood
Group MB3
BE 210-001
Budget
Since this proposed experiment builds on the “Acid-Base Chemistry: Biological
Buffers” experiment performed this semester, all of the equipment and many of the
supplies are already on hand in the BE210 laboratory. The pH meter and pH standards,
the 50 mL burettes and stands, the 200 mL beakers, and the magnetic stirrers and stir
bars were all used in the previously performed experiment, and therefore, do not need to
be purchased. The 1M HCl standard, reagent grade anhydrous Na2CO3, and gloves
were also stocked in the lab this past semester. The only supply that must be purchased
for this proposed experiment is guinea pig blood. Each of the twenty lab groups will be
expected to perform three blood titrations, using 10mL of blood per trial. Although this
only requires 30mL per group, each team will be allotted 50mL of guinea pig blood to
account for any mistakes or poor titrations. The blood will be ordered from Lampire
Biological Laboratories, from which orders are accepted online at www.lampire.com.
From the main website, clicking on ‘Animal Blood Serum & Plasma’ and then on ‘Whole
Blood Products’ leads to a list of animal blood options, where guinea pig blood can be
found under catalogue number B1-090N. The cost of this product is $170 per 100mL;
1000mL will be ordered to provide 50mL for each group, amounting to a total cost of
$1700. The costs of anticoagulants are included in this price. A Lampire sales
representative was previously contacted to assure us that the blood could be purchased
without any special regulatory requirements. Finally, refrigerators and/or freezers
(depending on how far in advance the blood is purchased) will be needed to store the
blood; however, these have previously been used in the lab, and therefore, do not incur
any further costs.
Guinea pig blood was chosen for this experiment because it is the most feasible
basis for gaining a realistic understanding of blood as a biological buffer. Guinea pig
blood, as opposed to the blood of another animal, was chosen for several reasons,
including price, feasibility, and safety. Ideally, we would like to study the blood of a
species as closely related to humans as possible, as humans are the focus of
bioengineering. However, some closely related species, such as monkeys or pigs, are
known to carry dangerous blood-borne pathogens, and therefore are not ideal for use in
the BE210 laboratory. Guinea pigs are mammals, making them a good subject for study
because, like humans, they are warm-blooded. The amount of guinea pig blood needed
for 20 groups to perform this experiment fits within the price range of $2000 and the
blood itself is easily obtainable, as it can be ordered online and shipped to the
laboratory.