I am working on a HPLC lab where we are to determine the concentration of caffeine in two energy drinks. I am trying to figure out the concentrations but don't think I am doing it right. Below is the info from our chromatograms:
Standard Caffeine peak area
blank 0 mg/100mL 0
Std 1 2.5 mg/100mL 3,039,775
Std 2 5 mg/100mL 7,913,698
Std 3 10mg/100mL 14,790,962
Unknwon 1 unknwon 36,474,730
Unknown 2 unknown 9,784,763
unknown 1 was prepared by diluting 2 mL of the unknown in 8 mL of a 20/80 water/methanol mix. Unknown 2 was prepared by diluting 5 mL of the unknown in 5 mL of the same mix.
When I plot the data in excel I get a equation for the line of y = 753789x - 913332. If I try and figure out the concentrations of the 2 unknowns just by using the graph I am getting a totally different answer then if I plug the peak areas for the unknowns in to the equation for the line. I don't know what I am doing wrong.
I am assuming the better way to go would be to use the equation for the line. If not please let me know. If so should I be doing something else beyond just plugging the peak areas for the unknown in to the equation for the line? If so, what and how do I do it? HELP!! I have spend severl hours trying to figure this out.
I didn't plot the data and I can't verify the equation you have for the line; however, just looking at the data suggests that unknown 2 must have a concn of about 7 mg/100 mL [I used 5 mg/100 x (about 15/about 8) = about 9 mg/100 mL) and 10 mg/100 mL x (about 8/about 15) = about 5 mg/100mL with the average between the two of about 7 mg/100 mL). That should be in the ball park anyway. That's the concn on the column since the standards were made from the column. Now unknown 2 was diluted by a factor of 2 (from 5 mL to 10 mL; therefore, the final concn is about 7mg/100 x (10/5) = about 14 mg/100 mL.
For unknown 1, I note that NO standard is as high as unknown 1; therefore, you must extrapolate the line to more than double your highest standard. Frankly, I wouldn't like to do that. If I had a choice I would run another unknown 1 of a different dilution. Since the first dilution gave a value more than double, I would make a new dilution at least 1:10 and perhaps 0.5:10(1 mL up to 20 mL of the mix) would be better.
Awesome. Thank you! I can't run another unknown but when I plug the peak area for unknown 2 in to my equation for the line I get a concentration of 14.19 mg/100mL so I will just plug in the peak area for unknown 1 to get my concentration for that unknown....unless I shouldn't. Please advise if I shouldn't
To determine the concentration of caffeine in the two unknown energy drinks, you can use the equation for the line you obtained from your plot. Using the equation, y = 753,789x - 913,332, where y represents the peak area and x represents the concentration of caffeine, you can solve for x.
To find the concentration of caffeine for "Unknown 1," substitute the peak area of 36,474,730 into the equation:
36,474,730 = 753,789x - 913,332
Now, solve for x:
753,789x = 36,474,730 + 913,332
x = (36,474,730 + 913,332) / 753,789
Calculating this gives you the concentration of caffeine in "Unknown 1."
Repeat the same process using the peak area of "Unknown 2" (9,784,763) to calculate the concentration of caffeine in it.
Keep in mind that using the equation for the line is the correct approach, as it provides a mathematical relationship between the peak areas and the concentration of caffeine.