Question

You want to study the oxygen binding ability of hemoglobin at pH 7.21 and pH 7.60. You decide to use a phosphate buffer.

a) How would you make up 500 ml of 50 mM phosphate buffer, pH 7.21, starting with 1.00 M H3PO4 and either 10.0 M HCl or 10.0 M NaOH? 25 ml 1M H3PO4, 3.75 ml 10.0 M NaOH, and 471.25 ml H2O.

b) How would you make up 500 ml of 50 mM phosphate buffer, pH 7.60, starting from your 50 mM phosphate buffer at pH 7.21? (PLEASE SHOW STEP BY STEP HOW TO GET THE ANSWER to mL)

Answer 1

a) The 500 mL of 50 mM phosphate buffer can be prepared with the help of following formula:

M1V1 = M2V2

From the question, we know the molarity of H3PO4 is 1 M.

M1 = molarity of a stock solution of H3PO4 (1M 0r 1000mM)

M2 = molarity required for buffer (50 mM)

V1 = volume of stock buffer solution required (to be calculated)

V2 = volume of buffer (500 mL)

By putting these values in the above formula we can calculate the V2,

1M * V1 = 50 mM * 500 mL

1000 mM * V1 = 50 mM * 500 mL

V1 = 50 *500 / 1000

V1 = 25 mL

So, 25 mL of 1M H3PO4 solution is required to prepare 50 mM phosphate buffer of 500 mL volume.

Water required to add to make 50 mM phosphate buffer can be obtained by subtracting the volume of stock buffer solution (25 mL) from total buffer volume (500 mL).

Required water volume = 500-25 = 475 mL

It is better to make up the final buffer volume (50 mM phosphate buffer) to 490-495 mL then adjust the pH of the buffer.  To achieve a pH of 7.21, gradually add 10 M NaOH (as it is a base so the addition will increase the pH value as due to H3PO4 acidic in nature pH of buffer will be less). Once pH is adjusted make the buffer volume and check the pH again.

b) The molarity of phosphate buffer is the same (50 mM) so the only thing to be adjusted is pH. We have 500 mL of 50 mM phosphate buffer with pH 7.21 and we required a pH of 7.61. The pH 7.61 is more basic than 7.21 so with the addition of base NaOH we can get the pH value of 7.61 with the help of pH meter.