Buffering Capabilities of
Guinea Pig Blood
GROUP MB3
Kim Coughlan, Barry Huang, Laura Michelis, Le Truong
Department of Bioengineering
Background:
Standardization of HCl with Na2CO3
12.000
10.000
8.000
pH
Based on acid-base titration lab:
wanted to further investigate significance
of biological buffers
• Determined buffer capacity of H3PO4
• HCO3- is primary biological
buffer in blood
Trial 1
Trial 2
6.000
4.000
2.000
0.000
0
0.005
0.01
0.015
0.02
0.025
Above: Experimental titration curve of H2CO3
Hypothesis & Aims:
This lab aims to examine how well pure bicarbonate buffer serves as
a model for the buffering capabilities of real blood. Specifically, it is
hypothesized that the buffering capacities of guinea pig blood and
pure bicarbonate (at a similar concentration as that found in blood)
will be the same.
Department of Bioengineering
0.03
HCl added (mol)
Methods & Protocol:
• Calibrate the pH meter by following the instructions on the
screen and by using the pH 4, 7, and 10 standard solutions.
• Dilute 100mL samples of 25mM HCl and 25mM Na2CO3
• Titrate a 10mL sample of 25mM Na2CO3 with HCl, recording
the pH every 0.5mL until the pH falls below 2.
• Repeat above titration two times.
• Titrate a 10mL sample of guinea pig blood with HCl, recording
the pH every 0.5mL until the pH falls below 2.
• Repeat above titration two times.
• Create titration curves for all titrations and determine all pKa’s
and buffer capacities.
• Compare the buffer capacities of the Na2CO3 and guinea pig
blood using a two-tailed two sample t-test.
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Proposed Deliverables/Findings:
• Comparison of HCO3- pKa to accepted values
• Titration curves for all titrations
• dB/dpH curves (numerical differentiation) to determine pKa’s and
buffer capacities
• T-test results: It is expected that P<0.05, i.e. no significant difference
Below: sample titration and dB/dpH curves
Standardization of HCl with Na2CO3
dB/dpH vs. pH of buffer
12.000
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
HCl added (mol)
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0.03
dB/dpH
10.000
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
pH
10.000
12.000
14.000
Potential Pitfalls:
• Anticoagulants may affect pH and buffering capacity of guinea pig blood,
e.g. citrate is an ionic form of citric acid
• 25mM of bicarbonate in the blood is an approximation; exact value ranges
from 21-28mM1
• Blood contains other substances and buffering agents that may cause its
buffering capabilities to slightly differ from pure bicarbonate
• Difficulty in storage of blood samples, which may affect its properties
ALTERNATIVES:
• Use artificial blood alternatives (would not truly represent real blood)
• Purchase serum only, which does not contain cells, clotting factors, or
anticoagulants (or purchase plasma or whole blood without anticoagulants,
or with only those anticoagulants known to have no effect)
• Purchase blood as late as possible
1 http://www.edu.rcsed.ac.uk/lectures/lt8.htm
Department of Bioengineering
Equipment/Materials and Budget & Justification:
EQUIPMENT
• pH meter & pH 4, 7, 10 standards
• 50 mL burette and stand
• 200 mL beakers
• Magnetic stirrers/stir bars
SUPPLIES
• 1M HCl standard
• Reagent grade anhydrous Na2CO3
• Gloves
NEWLY PURCHASED SUPPLIES/EQUIPMENT
Guinea Pig whole blood: chosen for price, feasibility (mammilian), and safety
Cost- 1,000mL x $170/100mL = $1,700 (50mL for 20 lab groups)
Supplier- Lampire Biological Laboratories (www.lampire.com)
Department of Bioengineering