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May 9th, 2018, 05:11 AM   #1
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Determining concentration of active drug in unknown sample

We did an experiment (High pressure liquid chromatography) today in class.
I have dropbox links to the method, the data table and 2 excel graphs below:
Method: https://www.dropbox.com/s/jqtdv5n5ea...ethod.PNG?dl=0
Data table: https://www.dropbox.com/s/grg2gf60fk...table.jpg?dl=0
2 excel graphs: https://www.dropbox.com/s/wezo2jbyb4...raphs.PNG?dl=0

Note: you don't need a dropbox account to view these links. I'm not sure how to directly attach these images to this forum without making the images all blurry.

The task requires us to:
Plot the peak area versus concentration
Plot the peak area ratio versus concentration
Obtain the least-squares linear regression for both standard curves (from excel). Use this equation to predict the concentration of your unknown samples.
Note: remember to take into account your initial dilution factor.
Express your result as mg of active material/mL of mixture.


We had a 1:1000 dilution.

I don't know whether it is correct, but my working out for the unknown concentration is below:

From data table 2:
Average peak area for unknown paracetamol = 805761
Average peak area for caffeine = 1249165
Calculate average peak area ratio (paracetamol:caffeine):
805761:1249165 = 0.645
The equation obtained from the excel graph:
y=70.827x + 0.0947
From the graph, x-axis = concentration, y-axis = peak area
substitute y=0.645 into equation to get, x = 0.00777mg/mL
multiply 0.00777mg/mL by dilution factor of 1000 to get 7.77mg/mL

My concern is that I didn't calculate this correctly. Should I have used the peak area only instead of the peak area ratio (which takes into account both paracetamol and caffeine)?
The question asked us to calculate the active material/mL of mixture.
I think the active material in this case is just the paracetamol? I don't think the active material includes paracetamol AND caffeine. I think this is because caffeine is used as the internal standard, but I'm not entirely sure.

What do you all think? Any input would be appreciated. And sorry for super long post!

Last edited by skipjack; May 11th, 2018 at 03:31 AM.
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May 10th, 2018, 11:15 PM   #2
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Your working out seems right. But I know what you mean in that the peak area ratio is the ratio of paracetamol to caffeine.

Both of the paracetamol standard solution and the unknown solution contain caffeine. You did work out the concentration of the unknown. However, i'm not sure whether you need to separate the paracetamol from the caffeine in order to calculate mg of paracetamol/mL.

Sorry i wasnt much help, but hopefully someone else will be able to help you further.
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