no grade put in your notebook as a separate page entitled P

(no grade - put in your notebook as a separate page entitled \"Preparation of Solutions for Tyrosinase Assay\") 1. For this lab we will be using 100mM sodium phosphate buffer at pH 7.0. We will need 500ml for the whole lab. How will you prepare this solution using the following information: pKa2 =6.82, Mw for sodium phosphate monobasic= 119.98, Mw for sodium phosphate dibasic = 141.96. tyrosine; 3,4-dihydroxy-L- phenylalanine) at a concentration of 6mM. This will be prepared in the sodium phosphate buffer prepared above. How would you prepare 100ml of this solution if the molecular weight is 197.19?

Solution

Calculations for preparing the buffer solution can be done by using the Henderson-Hasselbalch Equation

This is a two-component buffer system meaning that the weak acid and its conjugate base are added separately.

pH = pKa + log [A] / [HA]

7.00 = 6.80 + log [A] / [HA]

0.18 = log [A] / [HA]

1.5137 = [A] / [HA]

Since [A] / [HA] = 1.5137,

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2. Calculate the decimal fraction (part/whole) of each buffer component. A = 1.5137 / (1.000 + 1.5137) = 1.5137/ 2.5137= 0.6022, HA = 1.000 / 2.5137= 0.3978

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3. Find the molarity (M) of each component in the buffer by simply multiplying the molarity of the buffer by the decimal fraction of each component. 100 mM = 0.01M,

MA- = 0.01M x 0.6022 = 0.006022M

MHA = 0.01M x 0.3978 = 0.003978M

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4. Calculate the moles of each component in the buffer. Moles = Molarity x Liters of buffer moles of A- = 0.006022M x 0.5L = 0.003011 moles

moles of HA = 0.003978M x 0.5L = 0.001989 moles

5. Calculate the amount of each A and HA required

Amount of A = 0.003011 x 141.96 = 0.42744 g

Amount of HA = 0.001989 x 119.98 = 0.2386g

Where A is sodium phosphate monobasic

Where HA is sodium phosphate dibasic

Mix the required amount of sodium phosphate and make up the solution up to 500 ml. you will get the required buffer solution of pH 7.0

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Amount required for preparing 6 mM solution of L-DOPA is

Amount required will be 0.006 M x 197.19 = 1.183 g for i litre of the solution.

For 100 ml the amount required will be 0.118 g of L-DOPA.

Take 0.118 g of L-DOPA and make the volume to 100ml by using the previously prepared buffer solution.