8. Serum ion and standard iron solutions were analyzed according to the followin

Question

8. Serum ion and standard iron solutions were analyzed according to the following procedure: To 1.00 ml of unknown & std samples are added 2.00 ml of reducing agent and 2.00 ml of acid to reduce and to release Fe from transferrin. The serum proteins are then precipitated with 1.00 ml of 30% by weight trichloroactic acid. The mixture is then centrifuged to remove the protein. A 4.00 ml aliquot of each solution is then transferred to a fresh test-tube and treated with 1.00 ml 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 562 nm in a 1.00 cm cell Std, 1.00 ml of 3 ug /ml Fe standard solution, treated as the above procedure 0.209 Abs Unknown Serum Sample, 1.00 ml 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 FeLs, calculate the Evalue for the complex. (10 pts) ml sample of blood was treated with trichloroacetic acid to precipitate proteins. After centrifugation, the resulting solution was brought to pH of 3 and extracted with two 5.0 ml portions of methyl isobutyl ketone (MIBK) that also contained the organic Pb complexing agent APCD. The MIBK extract was analyzed, giving an absorbance of 0.210 at 283.3 nm. 5.0 ml aliquots of 2 standards containing 0.300 ppm and 0.500 ppm Pb were treated in the same way, yielding absorbances of 0.396 and 0.599. Plot this data and perform a linear regression on this line. A 1.00 cm cuvette cell was used.

Explanation / Answer

8)

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,

As = csl

Where, As= absorbance of the standard and Cs = is concentration of standard

Similarly, Au = cul

Where, Au= absorbance of the Unknown sample and Cu = is concentration of Unknown sample

By substituting values given in the question we can calculate Conc. Unknown Cu = (Cs ×Au)/ As

Cu = (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 = cl

epsilon value can be calculated from the fact that absorbance will be equal to the epsilon 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=

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

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