How is the pH of a Solution Related to the H3O+

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. Answer The relationship between pH and H3O+ concentration is described by the equation: pH = log10[H3O+ ], where [H3O+ ] is the concentration of the hydronium ions. Consequently, pH decreases when H3O+ concentration increases and a pH change of 1 on the pH scale is equivalent to a 10-fold change in H3O+ concentration. 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) 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. H2PO4 - is the hydrogen-ion donor and HPO4 2- is the hydrogen-ion acceptor 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? Answers. The ions produced are: Na+ , K+ , Cl- , HPO4 2- , H2PO4 - . 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 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 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. 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 molecular masses. 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 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. Table for Mass of Each Component Mass of Compound Compound Molecular Mass per Litre of 10x Molar Concentration Formula (in g/mol) PBS (in mol/l) (in g) NaCl 58.44 80 1.37 KCl 74.54 2 0.0268 Na2HPO4 141.92 14.4 0.101 KH2PO4 136.04 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, 0.0027M KCl, pH 7.4 How do they compare to the concentrations you calculated for 10x PBS? Directions for Questions 6 through 13 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 Credential s Answer. The concentration of NaCl and KCl in 10x PBS is ten times larger than the concentration in 1x PBS. 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? Answer. TARE the balance Question 7 If you have excess reagent on the weighing boat, what should you avoid doing and why? Answer. Excess reagent should never be put back in the stock to avoid cross-contamination; it should be discarded. 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. A graduated cylinder is more precise, so it should be used to measure volumes. Question 9 What should be done before measuring an unknown pH of a solution using a pH meter? Answer. The pH meter should be calibrated. 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 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 too low. Why do you think the protocol does not say to dissolve compounds directly in 1 litre of water? Answer. If the different salts were added to 1 liter of water, the total volume would exceed one liter because the solubilized salts occupy some 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? Answser. The pH has to be higher before adding HCl, because HCl is an acid and the addition of an acid to a solution lowers its pH. 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. Autoclaving the PBS solution ensures that it is sterile, which is important when working with biological material. 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. 1 liter of 1x PBS can be prepared by mixing 100ml of 10x PBS with 900ml of water (ie performing a 1/10 dilution of you 10x concentrate). Volumes should be measured using a graduated cylinder. Mixing can be done in a beaker using a magnetic stirrer.