BIO 1121 Unit 2 Assignment

Accurately measuring the volume of liquids, weighing chemicals, and adjusting the pH of solutions are routine
procedures in a working laboratory environment. This assignment is designed to provide you with an
overview of the general skills and knowledge you would need to perform such tasks.
Before completing this assignment you should ensure you have read chapter 2 of your text book –
particularly the section entitled pH, Buffers, Acids, and Bases. Answers should be concise and well written.
Make sure you correctly explain your thought process and provide all the necessary information.
The questions are available on the following worksheet:

Question 1: The pH of a solution describes its acidity or alkalinity: Describe how pH and H 3O+ concentration
are related and explain why diluting an acid raises the pH, but diluting a base lowers the pH.
Phosphate Buffered Saline (PBS) is a commonly used buffer for experiments in biology, because its pH and
ion concentrations are similar to those in mammalian organisms. It works in a similar fashion to the blood
plasma buffer mentioned in the textbook, but using dihydrogen phosphate ions and hydrogen phosphate ions
for buffering.

A: H3O+ is the hydronium ion in an aqueous solution and acidity is the amount of it, we use PH that is a scale
of how to measure this concentration. It is measured in moles per liter and it can vary. A pH of 1 is acidic, a
pH of 14 is quite basic. If the H+ concentration is higher than that of OH-, the solution it is acidic.

Question 2: Based on the equation below, which ion plays the role of hydrogen-ion donor (acid) and which
ion plays the role of hydrogen-ion acceptor (base) in PBS?
H2PO4-(aq) H+(aq) + HPO42-(aq)
The composition of PBS is 0.137M NaCl, 0.012M Phosphate, 0.027M KCl, pH 7.4. Below is the protocol to
make 1 liter of 10x concentrate PBS.
Combine the following:

 80g NaCl
 2g KCl
 14.4g Na2HPO4 (dibasic anhydrous)
 2.4g KH2PO4 (monobasic anhydrous)
 800mL distilled H2O

1. Adjust pH to 7.4 with HCl

2. Add H2O to 1L
3. Autoclave for 20 minutes on liquid cycle. Store at room temperature.
A: H+(aq) plays the role of hydrogen-ion donor (acid) and HPO42-(aq) plays the role of hydrogen-ion acceptor.

Question 3: Which ions are being produced by this process, assuming that each of the chemical
compounds dissociate into their constituent parts once they are dissolved in
water?

Preparation of the correct buffer is key to any good biological experiment and it is important that you
understand how to calculate the mass of each chemical required to make that buffer and what the resulting
concentration of those constituents will be in moles per liter.
Your text book explains that moles are just a way to express the amount of a substance, such that one mole is
equal to 6.02 x 1023 particles of that substance. These particles can be can be atoms, molecules, ions etc, so 1
mole of water is equal to 6.02 x 1023 water molecules, or 1 mole of Na+ is equal to 6.02 x 1023 Na+ ions. Since
different chemicals have different molecular weights (based on the number of protons and neutrons each
atom contains) 1 mole or 6.02 x 1023 atoms of oxygen (O) will have a mass of 16g whereas 1 mole or 6.02 x
1023 atoms of sodium (Na) will have a mass of 23g
You can read more about the definition of moles here if you wish https://www.britannica.com/science/mole-
chemistry

Although you may sometimes see it written as g/liter, the concentration of solutions is more often described
in term of molarity since it better defines the chemical properties of a solution because it is proportional to
the number of molecules or ions in solution, irrespective of molecular mass of its constituents. However, it is
not possible to measure moles on a laboratory balance, so in the first instance chemicals are measured by
mass (milligrams, grams, kilograms etc) and the number of moles is calculated using the known molecular
mass (often called molecular weight and abbreviated to M.W.) of the chemical. As indicated earlier, the
molecular mass of a chemical is based on the number of protons and neutrons that is contained in each atom
(eg NaCl is made up of one molecule of Na, M.W. = 22.99g and one molecule of Cl, M.W. = 35.45g, so the
M.W. of NaCl is 58.44g). These values can be found in the periodic table however the molecular mass of
chemicals is generally provided by any vendors of the products and so can also be found on various suppliers’
websites.

When the concentrations of solutions are as described as ‘molar’, this refers to number of moles per
liter eg a 3 molar solution of NaCl will contain 3 moles of NaCl in 1 litre of water. As indicated above, the
M.W. of NaCl is 58.44g, so in 58.44g there are 6.02 x 10 23 NaCl molecules ie 1 mole. So for 3 moles of NaCl
you would need to dissolve 175.32g in 1 liter of water (175.32/58.44 =3) whereas If you only dissolved 29.22g
of NaCl in 1 liter of water this would result in a 0.5 molar solution (29.22/58.44= 0.5)

Question 4: Using this webpage below calculate (to 2 decimal places) the molecular mass for each of the
compounds used to make PBS (the values here differ slightly from the periodic table found in your text
book due to rounding errors).

http://stedmansonline.com/webFiles/Dict-Stedmans28/APP19.pdf
Create the following table and fill it in with the mass of each component required to make 1 liter of 10 x PBS
(the recipe for 10x PBS is below question 2) and their final molar concentration in the buffer calculated as
described above.

Compound formula Molecular mass (in Mass of compound Molar concentration (in
g/mol) per liter of 10x PBS mol/l)
(in g)

NaCl 58.43

KCl 74.54

Na2HPO4 141.89

KH2PO4 136.02

Question 5: As previously stated, the concentration of NaCl, KCl and Phosphate in working strength 1 x PBS
is 0.137M NaCl, 0.012M Phosphate, 0.027M KCl, pH 7.4 How do they compare to the concentrations you
calculated for 10x PBS?
Watch the following videos

 "Using an Electronic Balance" from Bio-Rad tutorials:

http://www.bio-rad.com/webroot/web/html/lse/support/tutorial_electronic_balance_wndw.html

 “Using a pH Meter" from Bio-Rad

tutorials: http://www.bio-rad.com/webroot/web/html/lse/support/tutorial_using-a-ph-
meter_wndw.html

 "Making a PBS solution" from Community College Consortium for Bioscience

Credentials: https://youtu.be/S6XspSNnhWg

Question 6: What is the first thing to do after putting a weighing boat on the balance?

A: When the reading has steadied set the balance to zero.

Question 7: If you have excess reagent on the weighing boat, what should you avoid doing and why?

A: Do not put the excess back on the bottle because it may cause contamination.
Question 8: If you had the choice between a 1 liter beaker and a 1 liter graduated cylinder, which one
should you use to measure volumes with maximal precision when making 1 liter of PBS? (you can perform
an internet search to find this if you are not sure of the answer)
A: Graduated cylinders are more accurate instrument for measuring volume, Beakers have only 5 %
accurancy.

Question 9: What should be done before measuring an unknown pH of a solution using a pH meter?

The recipe for PBS says to dissolve compounds in 800 ml of water, adjust the pH to 7.4, then add water up to
1 liter. The final pH should still be 7.4, because the pH of buffer solutions remains stable when they are
diluted as long as the concentration of its constitutive acid and base is not too low.

A: We should remove the protective cape and calibrate the PH meter, PH 4, 7 and 10 are the standard.

Question 10: Why do you think the protocol does not say to dissolve compounds directly in 1 liter of
water?

A: Because on addition of some solution we could pass the required volume.

Question 11: The PBS protocol above says to adjust pH to 7.4 with HCl. What does this imply on the pH
of 10x PBS before adjusting the pH, would it be greater or smaller than 7.4?

Question 12: The last step in the protocol is to autoclave the 10x PBS solution. Why do you think this step
is important? Look up the definition of autoclave if you are unsure what it means.

A: To perform sterilization.

Question 13: Taking into account your response to question 5, now that you have made a 10x PBS solution,
describe how you would prepare 1 liter of 1x working solution PBS, including which glassware you would
use. Will you need to adjust the pH again?