Standard addition involving HPLC

[Dopefish1337]

Homework Statement

The concentration of retinol (vitamin A) in serum was determined by HPLC with UV absorbance detection. A 1.00 mL sample of serum was placed in a 10 mL test tube. To precipitate the proteins, 800.0 microL of ethanol was added, and the test tube was agitated. Next, a 2.00 mL solution of 90:10 n-hexane:chloroform was added to the test tube, which extracts the fat-soluble vitamin A into the organic solvent. The solution was agitated, and briefly centrifuged to separate the layers. The organic solvent was removed, evaporated to dryness, and the sample was reconstituted in 400.0 microL of methanol. A 1.00× 10^2 microL sample of this solution was injected onto the HPLC column, and a signal of 873.0 was obtained. In a fresh test tube, a second 1.00 mL sample of serum was spiked with 10 microL of 80.0 microg/mL vitamin A. The test tube was subject to the same sample preparation protocol, with 100.0 microL being injected on the HPLC column. This solution gave a signal of 1289.0.

What is the concentration of vitamin A in the serum sample?

Homework Equations

The formula for standard addition is the concentration of analyte in unknown/(concentration of analyte+concentration of standard in mixture)= signal from unknown/signal in mixture.

The Attempt at a Solution

I believe the concentration of the analyte (vitamin A) is 1mL*x/0.4 mL (x in micrograms per mL), since it appears to me that because of the " evaporated to dryness" step would negate all the other liquids up until that point so all that matters is the initial number of grams of unknown from 1mL*x, being dissolved into the 400 microlitres of methanol=0.4 mL.

Similarly the concentration of standard would be (0.01*80)/0.4 mL.

This leads me to believe that if I stick that info into the fomrula and solve it for x, I should be left with the concentration of vitamin a in the serum, with units of micrograms/mL. However doing that, I've gotten an answer of 1.67 micrograms/mL, which was incorrect.

So, what am I missing?

 

[Dopefish1337]

Question closes in around 5 hours. Anyone?

 

[Blueshift5]

Thank you for sharing your attempt at a solution. It seems like you have a good understanding of the standard addition method and how to calculate the concentration of an analyte in a sample. However, there are a few factors that could be contributing to your incorrect result.

Firstly, it's important to note that the formula for standard addition is typically used for samples with unknown concentrations, where a known amount of standard is added to the sample to determine the concentration of the analyte. In this case, both samples have unknown concentrations, so we cannot use the standard addition formula directly.

Secondly, the sample preparation protocol described in the homework statement involves various steps that can affect the concentration of the analyte. For example, the addition of ethanol and extraction with organic solvent can change the concentration of vitamin A in the sample. Additionally, the reconstitution in methanol and injection onto the HPLC column can also affect the concentration. Therefore, it's important to consider the impact of these steps on the concentration of vitamin A in the sample.

Finally, it's possible that there may be errors in the measurement or calculation of the signals obtained from the HPLC analysis. It would be helpful to double-check your calculations and make sure all units are consistent.

Overall, it's difficult to determine the exact concentration of vitamin A in the serum sample without more information about the sample preparation and analysis steps. I would recommend discussing this with your instructor or consulting with a more experienced scientist for further guidance.