BIO 1121 Unit 2 Assignment

Question 1: The pH of a solution describes its acidity or alkalinity: Describe how pH

andH3O+ concentration are related and explain why diluting an acid raises the pH, but
diluting a base lowers the pH.

PH measures the concentrations of ions, If we have high concentrations of H+ ions it means we

have low PH and if concentration of H+ ions is low it means we have high PH so H+ hydrogen
ion is the basis of the PH scale So acid increases the concentration of H+ hydrogen ions and the
base increases concentration of the OH- hydroxide ions .H3O+ have high concentration of H+
hydrogen ions it means it is an acidic solution. 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.

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?

Hydrogen ion is donor (acid) - HPO4 Hydrogen ion is acceptor (base) - H2PO4 H2PO4-(aq) ⇆
H+(aq) + HPO42-(The composition of PBS is 0.137M NaCl, 0.012M Phosphate, 0.027M KCl,
pH 7.4. Below isthe protocol to make 1 liter of 10x concentrate PBS. Combine the following:
80g NaCl, 2g KCl1, 4.4g 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.

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?

NaCl (s) + H2O ⇔ Na + + Cl−

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 making 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.

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 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
1023 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 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 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 of Molar

mass (in compound concentration
g/mol) per litre of (in mol/l)
10x PBS (in g)

NaCl 58.44 80
1.37

KCl 74.55 2
0.0268

Na2HPO4 141.96 14.4

0.101

KH2PO4 136.09 2.4

0.0176
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, pH7.4 How do they compare to the concentrations you calculated for 10x
PBS?

The concentration of NaCl and KCl in 10x PBS is ten times larger than the concentration in
1xPBS. The sum of the concentrations of Na2HPO4 and KH2PO4 in 10x PBS (0.101M +
0.0176M= 0.1186M ≃ 0.12M) is ten times larger than the phosphate concentration in 1x PBS
(0.012M).

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

First Thing to do After putting the boat on the weight press TARE or ZERO to get reading
of0.00 g And after taking the sample from the freezer let it equilibrate to room temperature to not
weight the ice crystals

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

Discarded to prevent contaminating the bottle.

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?

The markings on a beaker are not accurate, the word graduated indicates that the markings are
accurate therefore a beaker should never be used for accuracy since its marking are approximate.

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

pH meter?

When we want to measure unknown PH of any solution we need to calibrate the PH meter with
three butters, first one is with PH of 7 second one should be acidic with PH of 4 and the third one
basic with PH of 10 after we calibrate the PH meter with this three buffers we will be ready to
measure the unknown PH 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.

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

I think it is because when i dissolve compounds in 1 liter will not be calculated 100% to balance
the chemical reactions But when the chemical reaction is completed in 800 ml we can add the
remaining water without effecting the solution
Question 11: The PBS protocol above says to adjust pH to 7.4 with HCl. What does thisi
mply on the pH of 10x PBS before adjusting the pH, would it be greater or smaller
than7.4?

Because HCl is acid so adding HCl to any solution will lower the PH of course.

And if it says to add HCl to adjust the PH to 7.4 it means that the 10x PBS is higher than 7.4 so
to add HCl to lower the PH to 7.4 that we need.

Question 12: The last step in the protocol is to autoclave the 10x PBS solution. Why do you
think this step is important?

We are using autoclave to sterilize the solution and when we use PBS it is important to be
sterilized to avoid any sort of contaminating which could come with the buffer.

Question 13: Taking into account your response to question 5, now that you have made
a10x 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?

We will take a graduated cylinder then measuring 100 ml of my 10x PBS solution after that we
add the 900 ml of water to the solution to make it dilute to 1x PBS. I think we need to measure
the PH again to be in the safe side because large amount of water could lower the PH a little bit,
so we may need to add a very small amount of base to higher the PH to the needed level.