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Question 17.4: Serum Iron Analysis. Serum iron and standard iron solutions ...

Serum Iron Analysis

Serum iron and standard iron solutions were analyzed as follows:

Step 1  To 1.00 mL of sample, add 2.00 mL of reducing agent and 2.00 mL of acid to reduce and release Fe from transferrin.

Step 2  Precipitate proteins with 1.00 mL of 30 wt% trichloroacetic acid. Centrifuge the mixture to remove protein.

Step 3  Transfer 4.00 mL of supernatant liquid to a fresh test tube and add 1.00 mL of solution containing ferrozine and buffer. Measure the absorbance after 10 min.

Step 4  To establish each point on the calibration curve in Figure 17-9, use 1.00 mL of standard containing 2–9 μg Fe in place of serum.

The blank absorbance was 0.038 at 562 nm in a 1.000-cm cell. A serum sample had an absorbance of 0.129. After the blank was subtracted from each standard absorbance, the points in Figure 17-9 were obtained. The least-squares line through the standard points is

Absorbance = 0.067_{0} × (μg  Fe  in  initial  sample) + 0.001_{5}

According to Beer’s law, the intercept should be 0, not 0.001_{5}. We will use the small, observed intercept for our analysis. Find the concentration of iron in the serum.

 

FIGURE 17-9
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