A 4-fold dilution of plasma is assayed spectrophotometrically for cholesterol. Given the following spectrophotometic data, what is the concentration of cholesterol in the diluted plasma in micromol/mL? (don't include units in answer) A500 standard = 0.77 A500 sample = 0.28 C (standard) = 3.9mM I've been given this biochemistry question, and I just keep getting the answer wrong and I don't know why. It would be great if someone could work through the calculation step-by-step to help me see where I'm going wrong.

Expert Answers

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From what is given within the question, it appears that this is a question which requires one to exploit Beer's Law shown below:

A = k c

A is measured absorbable of a sample with concentration c where k is a constant.

Given a standard solution with absorbable As having a known concentration Cs AND a diluted cholesterol solution with absorbance Aunk with an unknown concentration Cunk, the following is true:

Cs / As = Cunk / Aunk = k

This assumes that the measurements were taken at the same wavelengths using a sample cuvette with a path length of 1.0 cm.

Using the give data, we find that ...

3.9 mM / 0.77 = Cunk / 0.28

Cunk = ( 3.9 mM / 0.77 ) • 0.28 = 1.4 mM

Thus, the concentration of cholesterol within the 4-fold dilution that was measured is 1.4 mM. One would then have to scale up this result 4-food to acquire the predicted concentration of cholesterol in the original undiluted sample.


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