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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 your textbook –
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
The pH of a solution describes its acidity or alkalinity: Describe how pH and H3O
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 through the following chemical reaction:
(aq) ⇆ H
(aq) + HPO4
The equilibrium arrows depict that the phosphate ion (H2PO4-
) is dissociating further into two
component ions in solution, but at the same time H+ and HPO42-
ions are combining
simultaneously to form phosphate in solution. So, at any given point in time, and under the
appropriate conditions, there is an equal quantity of dissolved ions and combined ions in
solution. There is therefore always a hydrogen ion donor and an acceptor in solution.
Based on the equation above, which ion plays the role of hydrogen-ion donor (acid) and which
ion plays the role of hydrogen-ion acceptor (base) in PBS?
The composition of PBS is 0.137M NaCl, 0.012M Phosphate, 0.0027M KCl, pH 7.4. Below is the
protocol to make 1 litre 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.
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
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 litre.
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
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
If you need more information on moles, please read Encyclopedia Britannica’s Moles website.
Although you may sometimes see it written as g/litre, 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 litre 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 1023 NaCl
molecules ie 1 mole. So, for 3 moles of NaCl you would need to dissolve 175.32g in 1 litre of
water (175.32/58.44 =3) whereas If you only dissolved 29.22g of NaCl in 1 litre of water this
would result in a 0.5 molar solution (29.22/58.44= 0.5)
1. As directed you need to check the periodic table and pick up the atomic masses for each
of the component atoms in the compounds. For example, for NaCl you need to pick the
atomic weight of both sodium and chlorine and then add them to two decimal places to
obtain the molecular mass of NaCl. Be sure to multiply the atomic masses by the number
of individual atoms of the same element present in each compound before finally adding
to the masses of other component atoms of other elements to make up the total
2. From there you can calculate the number of ‘moles’ of each compound by multiplying
the provided weight of compound used in the PBS solution by their respective molar
mass conversion factors (i.e. 1L divided by the molecular mass you have calculated in the
3. Now, the molarity in Mol per Litre (mol/l) is given by the ‘number of moles’ of each
compound (calculated in step 2 above) divided by the given volume of the solution.
For more information on how to calculate morality, refer to wikiHow’s 4 Ways to Calculate
Using periodic table found in your textbook, calculate (to 2 decimal places) the molecular mass
for each of the compounds used to make PBS.
Create the following table and fill it in with the mass of each component required to make 1 litre
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
per litre of
10x PBS (in
As previously stated, the concentration of NaCl, KCl and Phosphate in working strength 1 x PBS is
0.137M NaCl, 0.012M Phosphate, 0.0027M KCl, pH 7.4 How do they compare to the
concentrations you calculated for 10x PBS?
Watch the following videos and answer the remaining questions
• “Using an Electronic Balance” from Bio-Rad tutorials
• “Using a pH Meter” from Bio-Rad tutorials
• “Making a PBS solution” from Community College Consortium for Bioscience Credentials
What is the first thing to do after putting a weighing boat on the balance?
If you have excess reagent on the weighing boat, what should you avoid doing and why?
If you had the choice between a 1-litre beaker and a 1 litre graduated cylinder, which one should
you use to measure volumes with maximal precision when making 1 litre of PBS? (you can
perform an internet search to find this if you are not sure of the answer)
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 litre. 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
Why do you think the protocol does not say to dissolve compounds directly in 1 litre of water?
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?
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.
Taking into account your response to question 5, now that you have made a 10x PBS solution,
describe how you would prepare 1 litre of 1x working solution PBS, including which glassware
you would use. Will you need to adjust the pH again?