[Zel]

Hi there,
I completed a practical which involved HPLC of two known solutions, and two unknown solutions. From this, we have to calculate the concentration of the unknown solutions.

Paracetamol Stock solution: 20ppm concentration
Average Retention time = 3.3695 min
Average Peak Area = 437630.5

Caffeine Stock Solution = 5ppm
Average Retention time = 4.5125 min
Average Peak Area = 200423.5

Unknown Solution 1 (weight capsule = 0.3516g)
(1) Average Retention time = 3.239 min
(1) Average Peak Area = 592642
(2) Average Retention time = 4.406 min
(2) Average Peak Area = 302820

Unknown Solution 2 (weight capsule = 0.2314g)
Average Retention time = 3.3485 min
Average Peak Area = 484653.5

It can be seen that US 1 has both caffeine and paracetamol, where US 2 has paracetamol.
How do I go to calculate the concentration of each component in each US?
We got each Unknown solution by weight a capsule, dissolving it into a 200mL flask, then diluting in a 1:50 ratio into a 100mL flask, so this has to be taken into consideration when calculating the concentration.
I understand I might need to plot a calibration curve, but what will I use? The ratios? Or just use the concenration vs. averages?
Any help will be appreciated. Thank you.

[Borek]

Signal should be directly proportional to the concentration. You don't need a calibration curve here, although drawing one won't be incorrect - it is just much faster to calculate just using proportions. That would be equivalent to use a calibration curve going through 0,0 point.

Then take care of dilutions.