1. Department of Bioengineering Buffering Capabilities of Guinea Pig Blood GROUP MB3 Kim Coughlan, Barry Huang, Laura Michelis, Le Truong

2. Department of Bioengineering Background: Based on acid-base titration lab: wanted to further investigate significance of biological buffers Determined buffer capacity of H 3 PO 4 HCO 3 - 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. Above: Experimental titration curve of H2CO3

3. 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 Na 2 CO 3 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 pK a ’s and buffer capacities. Compare the buffer capacities of the Na 2 CO 3 and guinea pig blood using a two-tailed two sample t-test.

4. Department of Bioengineering Proposed Deliverables/Findings: Comparison of HCO 3 - pK a to accepted values Titration curves for all titrations dB/dpH curves (numerical differentiation) to determine pK a ’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

5. 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 21-28mM 1 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

6. 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 Na 2 CO 3 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)