I'm doing an experiment on the caffeine content in 'Tonic wine' by GLC. (Tonic wine for those who don't know is a high caffeine content alcohol drink 37.5mg/l and it comes in a 750ml bottle)
I've extracted the caffeine from the wine, that was relatively easy. But I'm having a problem with my standards. I made up a stock solution of caffeine and then from this made a 100ppm solution (diluting to the mark with methanol). I also done the same with the internal standard that I am using, Phenacetin. I then ran these two samples through the GC to give me an idea of where the peaks came off, no issues so far.
So, moving on, I made up 5 more standards in 10ml volumetric flasks:
1. 1ml caffeine stock + 3ml phenacetin stock and made up to the mark with methanol.
2. 2ml caffeine stock + 3ml phenacetin stock and made up to the mark with methanol.
3. 3ml caffeine stock + 3ml phenacetin stock and made up to the mark with methanol.
4. 4ml caffeine stock + 3ml phenacetin stock and made up to the mark with methanol.
5. 5ml caffeine stock + 3ml phenacetin stock and made up to the mark with methanol.
I explained I had done this to my advisor and he didn't say it was wrong, just that it "sounds good".
I went ahead and ran each of these samples 3 times, so that I could take the average peak area of the caffeine peak and phenacetin peak. From this I went on to calculate the peak area ratio and then finally construct a calibration curve.
However, when I was calculating my peak areas the first stock solution gave me a peak area ratio of 4.71, the next was 2.something and so on. So basically, my peak area ratio, rather than increasing, is decreasing. This means when I plot the calibration curve it is going negatively (if that makes sense!)
I'm pretty sur ethis is not right, although I seem to remember doing an experiment some time ago when this was the case.
Can anyone shed any light on this? I was assuming the peak area ratios would increase giving me points to add the trendline too, to give the R squared value and equation of the line and then from this I could work out the caffeine content from the wine extract.
Any help or opinions are appreciated!
Cheers