Question

Serum ion and standard ion solutions were analyzed according to the following procedure: To 1.00ml of unknown & std samples are added 2.00ml of reducing agent and 2.00ml of acid to reduce and release Fe from transferrin. The serum proteins are then precipitated with 1.00ml of 30% by weight trichloroactic acid. The mixture is then centrifuged to remove the protein. A 4.00ml aliquot of each solution is then transferred to a fresh test-tube and treated with 1.00ml of a solution containing ferrozine and buffer. The absorbance of this solution was then measured after a 10-minute period to develop the color. The following data were obtained:

Blank=0.018 Abs at 562nm in a 1.00cm cell

Std, 1.00ml of 3ug/ml Fe standard solution, treated as the above procedure=0.209 Abs

Unknown serum sample, 1.00ml treated as above=0.169 Abs

Find the concentration of Fe in the sample, in ppm and M. Also, if the complex that is formed is the complex FeL3, calculate the epsilon value for the complex.

Answer 1

Blank=0.018 Abs at 562nm in a 1.00cm cell

Std, 1.00ml of 3ug/ml Fe standard solution, treated as the above procedure=0.209 Abs

Unknown serum sample, 1.00ml treated as above=0.169 Abs

Find the concentration of Fe in the sample, in ppm and M. Also, if the complex that is formed is the complex FeL3, calculate the epsilon value for the complex.

Actual absorbance of standard solution = 0.209-(absorbance of blank) = 0.209-0.018= 0.191

Similarly; Actual absorbance of Unknown serum sample = 0.169 -0.018 = 0.151

Now we can calculate concentration of unknown sample from known concentration and absorbance of standard solution as follows,

A_s = εc_sl

Where, A_s= absorbance of the standard and C_s = is concentration of standard

Similarly, A_u = εc_ul

Where, A_u= absorbance of the Unknown sample and C_u = is concentration of Unknown sample

By substituting values given in the question we can calculate Conc. Unknown C_u = (C_s ×A_u)/ A_s

C_u = (0.151×3 µg /ml )/0.191 = 2.371 µg /ml

4ml of aliquot take after centrifugation contains 2.371 µg /ml of Fe

Hence complete 4 ml will contain 2.371 µg /ml × 4 = 9.84 µg of Fe

We have initially started with 1 ml sample of serum hence from above calculations we can conclude that 1ml of serum sample contains 9.84 µg of Fe

1 ml contains 9.84 µg of Fe

i.e 1 ml contains 9.84 ×10^-3 mg of Fe

Therefore 1000 ml of serum contains 9.84 mg of Fe , this means concentration of iron in serum is 9.84ppm,

Now, 55.84 g in 1000ml = 1M

Therefore 9.84 ×10^-3 g in 1000 ml will be 1.69M

Molarity of iron in serum is 1.69M

A = εcl

ε epsilon value can be calculated from the fact that absorbance will be equal to the epsilon ε at 1Molar concentration,

We have value of absorbance at 3 µg /ml which is 0.191

We are supposed to calculate absorbance when concentration of solution is 1M

that is 55.84 ×10⁶ µg /ml at this A=ε

therefore, x/0.191= 55.84 g/l / 3 ×10^-3 g/l

x = epsilon = 3.55 ×10³ Lmol^-1cm^-1

This is also called as molar absorptivity