Department of Bioengineering

Buffering Capabilities of Guinea Pig Blood

GROUP MB3

Kim Coughlan, Barry Huang, Laura Michelis, Le Truong

Department of Bioengineering

Background:

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

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.

Standardization of HCl with Na2CO3

0.000

2.000

4.000

6.000

8.000

10.000

12.000

0 0.005 0.01 0.015 0.02 0.025 0.03

HCl added (mol)

pH Trial 1

Trial 2

Above: Experimental titration curve of H2CO3

Department of Bioengineering

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.

Department of Bioengineering

Proposed Deliverables/Findings:

dB/dpH vs. pH of buffer

00.005

0.010.015

0.020.025

0.030.035

0.04

0.0450.05

0.000 2.000 4.000 6.000 8.000 10.000 12.000 14.000

pH

dB

/dp

H Trial 1

Trial 2

Trial 3

Standardization of HCl with Na2CO3

0.000

2.000

4.000

6.000

8.000

10.000

12.000

0 0.005 0.01 0.015 0.02 0.025 0.03

HCl added (mol)

pH Trial 1

Trial 2

• 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 differenceBelow: sample titration and dB/dpH curves

Department of Bioengineering

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 possible1 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/EQUIPMENTGuinea 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)