BIO 1121

Unit 2 Written Assignment

Directions
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
information.

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.
Answer
a)
To calculate the pH of an aqueous solution, it is compulsory to know the concentration of the
hydronium ion in moles per liter (molarity). The pH is then calculated using the expression
pH = - log [H3O+]
b)
Diluting an acid decreases the concentration of H+(aq) ions, which increases the pH level of the
solution towards 7, making it less acidic. when an alkali is diluted with water the concentration
of OH - ions decreases. This causes the pH of the alkali to fall towards 7 which is a neutral pH.

Question 2
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:
 H2PO4- (aq)  ⇆  H+(aq) + HPO42--(aq)

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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?

Answer

In PBS, H2PO4- is a hydrogen-ion donor (acid) and HPO42- is a hydrogen-ion acceptor (base).

Question 3

 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 water?
Answer
The reaction given:

H2PO4-(aq) ⇆ H+(aq) + HPO42-( aq)

Here, HPO42- ion functions as a proton acceptor and H2PO4- ion as a proton donor. Na2HPO4
separates into Na+ and HPO42- in a PBS solution. This molecule then acts as a proton acceptor.
In contrast, KH2PO4 dissociates into K+ and H2PO4-. As H2PO4- is a proton donor, KH2PO4
serves as a proton donor in a PBS solution.                                                                 

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Question 4
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 10 23 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 10 23 atoms
of oxygen (O) will have a mass of 16g whereas 1 mole or 6.02 x 10 23 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/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 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 10 23 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
molecular masses. 

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 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 first
step) 

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

NaCl 58.44 80 1.37

KCl 74.54 2 0.027
Na2HPO4 141.92 14.4 0.101
KH2PO4 136.04 2.4 0.018
 

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, 0.0027M KCl,  pH 7.4   How do they compare to the
concentrations you calculated for 10x PBS?

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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

Answer
The concentrations for the compounds in the 10x solutions are higher than those of solution.
The concentration for NaCl is ten times its concentration in solution (0.137 to 1.37 M). KCl went
from 0.0027M to 0.027M Phosphate went from 0.012M to 0.12M. Ratio of these concentrations
is the same in both the solutions.

Question 6
What is the first thing to do after putting a weighing boat on the balance?
Answer
Zeroing out the balance and recheck it.
 
Question 7
If you have excess reagent on the weighing boat, what should you avoid doing and why?
 Answer
It should avoid putting back an excess of the reagent into the stock to avoid contamination and it
may change the properties and PH of the compound.
Question 8
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)
Answer
The beaker should never be used for accuracy because its marks are approximate and not
accurate.

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Question 9
What should be done before measuring an unknown pH of a solution using a pH meter?
Answer
Before measuring an unknown pH of a solution, a two- or three-buffered calibration must be
done to standardize the measuring.

Question 10
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.

Why do you think the protocol does not say to dissolve compounds directly in 1 liter of water?
Answer
Salt should not be dissolved directly in one liter of water for two main reasons. When compared
to pure water, the volume of the solution can initially be increased by the dissolved chemicals.
The solution's final volume will therefore be greater than 1 liter. Second, a specific amount of
sodium hydroxide or hydrochloric acid must be added to the solution in order to change pH. As a
result, the solution's molarity and buffer strength will change in the final volume. As a result,
components must be dissolved, the pH must be adjusted, and one liter of water must be added.
The buffer's parameters won't change in this scenario.
 
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?
Answer`
As HCl is a strong base and tends to lower pH. If the PBS protocol is to adjust pH to 7.4 with HCl,
this means that the pH of the 10X PBS was greater 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.
Answer
Autoclave is required to sterilize the PBS solution, to avoid any microbiological contamination.

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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 litre of 1x working solution PBS, including which glassware
you would use. Will you need to adjust the pH again?
Answer
To prepare a 1 liter of 1x working solution PBS, we need to put 100 ml of a10x PBS solution into
the graduated cylinder or 1 L volumetric flask. Then, 900 ml of distilled water or adjust the final
volume of the solution up to 1 L. The final solution is 1x PBS. Since water dissociation is very low
and has little effect on the solution's pH, there is no need to modify the pH.

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