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# Question 1: The pH of a solution describes its acidity or alkalinity: Describe how pH and H3O+

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.

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, 0.0027M 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

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?

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 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 (in g/mol)

Mass of compound per liter of 10x PBS (in g)

Molar concentration (in mol/l)

NaCl

KCl

Na2HPO4

KH2PO4

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?

Watch the following videos

·        "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?

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

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)

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.

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

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.

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?

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