Biological Buffers Lab
Different species have different optimum ranges of pH, but most organisms depend upon a pH very
close to neutral.
Body pH levels of living organisms can be impacted by many factors, such as food, rain,
decomposition, exercise, and rate of chemical activity (metabolism). These variables may potentially
change the pH to a life-threatening point. In an effort to maintain homeostasis, living organisms must
have specialized tissues that absorb H+ when the H+ concentration begins to rise and to release H+
when the H+ concentration begins to fall.
These tissues or body fluids contain buffers. Buffers are substances that work to prevent severe
changes in pH. Buffers may release or store H+ ions as needed to stabilize pH. This mechanism
ensures that the optimum pH can be maintained in the body. If a buffer is unable to compensate for
the pH shift, or the organism is unable to move to a different environment, or if artificial means (such
as medical intervention) cannot correct the situation, then there is a homeostatic imbalance that may
result in death. The change in pH causes essential biological molecules such as enzymes and DNA to
denature (alter their shape) leading to a loss of function.
The most important buffering system in the body is the blood buffer system, which is composed of
carbonic acid (H2CO3) and bicarbonate ion (HCO3-). This keeps the pH of the blood within a narrow
range of 7.35 to 7.45. Any values outside of this range can be deadly.
To test the buffering ability of the experimental samples, 0.1M hydrochloric acid (HCl) will be added in
50µL increments. pH will be measured with a pH meter. The instructor will provide instruction on how
to calibrate and use the equipment to obtain a pH reading.
High/Good Buffering Capacity = A sample that can maintain its pH value even in the presence of
additional amounts of an acid or a base.
Low/Poor Buffering Capacity = A sample that cannot maintain its pH value in the presence of
additional amounts of an acid or a base.
Question:
Hypothesis:
Null hypothesis:
Prediction:
Independent variable(s):
Dependent variable:
Reference: Adapted from Eric Thiel’s pH & Buffer Lab & used with permission.
http://pleasanton.k12.ca.us/avhaweb/thiel/bio/labs/pH_lab.html
Lab Procedure
1. Using a graduated cylinder, dispense 20mL of each experimental sample into the appropriate
beaker. The samples should be mixed thoroughly before they are poured into the graduated
cylinder. The graduated cylinder must be washed out with water before a new sample is
measured.
2. Obtain the initial pH value of your first sample and record the information in Table 1. Record
the actual value; do not round to the nearest whole number. Continue with the next steps for
the same sample. Do not move on to another sample until you have finished with your current
one.
3. Using a micropipette, add 50µL 0.1M HCl to the sample. Swirl the beaker to mix the contents,
then measure the pH. Record the value in Table 1.
4. Repeat step 3 until a total of 200µL of 0.1M HCl has been added to the sample.
5. Repeat steps 3 and 4 for each sample.
6. Make a line graph of the data recorded in Table 1. Use different colors for each of the
samples. Place the amount of added HCl on the x-axis and the pH value on the y-axis.
7. Transfer the data from Table 1 into a linear regression website (such as
https://www.socscistatistics.com/tests/regression/default.aspx) and determine the rate of pH
change for each sample.
8. Conclusions:
a. Based on the rate of pH change, which sample had the highest buffering capacity?
b. Based on the rate of pH change, which sample had the highest buffering capacity?
c. Do your results support your hypothesis your null hypothesis?
d. Was your prediction correct?
e. Which samples modeled the blood buffer system of the human body?
Reference: Adapted from Eric Thiel’s pH & Buffer Lab & used with permission.
http://pleasanton.k12.ca.us/avhaweb/thiel/bio/labs/pH_lab.html
Table 1: pH of samples
Samples
Initial pH
pH with
50µL
0.1HCl
pH with
100µL
0.1HCl
pH with
150µL
0.1HCl
pH with
200µL
0.1HCl
Rate of
Change
Tap Water
Liver Puree
Chemical
Buffer
Salt Water
Whole Milk
Potato Puree
Egg White
Seltzer Water
(H2CO3)
Sodium
bicarbonate
solution 0.1M
(NaHCO3)
Clean Up:
Rinse the pH probe thoroughly and insert the probe back into the storage container. Be sure
the end of the probe is submerged in the buffering solution
Discard disposable pipettes (if used).
Wash all glassware. Check all glassware to ensure it is thoroughly cleaned and that no
residue remains.
Place a clean, dry paper towel on the tray and place the beakers on the paper towel. Invert
the clean beakers so they can drain and dry.
Reference: Adapted from Eric Thiel’s pH & Buffer Lab & used with permission.
http://pleasanton.k12.ca.us/avhaweb/thiel/bio/labs/pH_lab.html
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