Question

Following dilution of a concentrated solution, the absorbance is determined to be 3.935 on our BioTek Plate Reader. What, if any, changes should be made to this experiment?

a The sample should be diluted at least two fold

b The sample should be concentrated 2 fold

c The sample should be diluted 100 fold

d The sample should be put into a different microplate.

e There is nothing wrong with this analysis and the 3.935 should be recorded.

Answer 1

According to Lambert-Beer's law absorbance in dependent on concentration and path length of solution, which is represented by formula

A = Cl ................................(1)

where, A = absorbance

   = Molar absorption coefficient

C = concentration of solution

l = Path length of solution

So,

a. When  sample is diluted at least two fold than

    C = C/2

putting this value in (1)

A = Cl / 2, Hence absorbance of new solution is half of absorbance of stock solution

given absorption of stock solution is = 3.935

so absorbance of new solution is = 3.935 / 2 = 1.9675

b. When  sample is concentrated two fold than

    C = 2C

putting this value in (1)

A = 2Cl , Hence absorbance of new solution is double of absorbance of stock solution

given absorption of stock solution is = 3.935

so absorbance of new solution is = 3.935 x 2 = 7.87

c. When  sample is diluted at least 100 fold than

    C = C/100

putting this value in (1)

A = Cl / 100, Hence absorbance of new solution is 100th part of absorbance of stock solution

given absorption of stock solution is = 3.935

so absorbance of new solution is = 3.935 / 100 = 0.03935

d. When  sample is put into a different microplate

In relation (1) we can see that width of microplate is directly proportional to absorbance so if width of new microplate is increased than absorbance is increases and if width of new microplate is decreased than absorbance is decreases.  

e. There is nothing wrong with this analysis and the 3.935 should be recorded it means stock solution was analyzed without changing its concentration or microplate.