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Topic: GLC Question - Can't explain why I'm getting these results!  (Read 3718 times)

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Offline Jamie1916

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GLC Question - Can't explain why I'm getting these results!
« on: March 12, 2012, 12:39:09 PM »
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

Offline Jamie1916

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Re: GLC Question - Can't explain why I'm getting these results!
« Reply #1 on: March 12, 2012, 12:41:44 PM »
Also, someone said something to me about a response factor and that I had to work that out prior to making up standards or something. I can honestly say I have never heard of this and that was confirmed when I googled it and read about it, never heard of it before!

Offline fledarmus

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Re: GLC Question - Can't explain why I'm getting these results!
« Reply #2 on: March 12, 2012, 04:09:47 PM »
How are you calculating your peak ratios? Can you give the raw data for your peak integrations?

Offline Jamie1916

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Re: GLC Question - Can't explain why I'm getting these results!
« Reply #3 on: March 12, 2012, 06:22:45 PM »
I don't have them to hand, I've left all the print outs from the GC in University in my cupboard but the basically what I've done is run all of the 5 standards 3 times and then calculated the average peak area for the caffeine and phenacetin peaks.

From this I've used a basic equation Peak Area Ratio = Caffeine Peak Area/Phenacetin Peak Area

I don't know if I'm calculating it wrong or if something has went tragically wrong with my samples. I've logged everything I've done in my lab book and I can't see where I might have went wrong! Unless my overall procedure when it came to making the 1ml, 2ml etc standards of caffeine was wrong.

Offline Arkcon

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Re: GLC Question - Can't explain why I'm getting these results!
« Reply #4 on: March 12, 2012, 06:34:47 PM »
What we suspect is that the instrument's software is doing some post integration calculations on the data, which is why the raw data would help.  Something to try, pick any one standard, and inject more, and then less, in addition ti the typical amount.  That way, we can see if we get proportionally more or less signal.  Also, look at the chromatograms, are the injections that should be larger, that give a smaller amount in the result, visibly larger or smaller?
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline Jamie1916

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Re: GLC Question - Can't explain why I'm getting these results!
« Reply #5 on: April 12, 2012, 10:22:03 AM »
Completely forgot to come back and update you on this!

There was nothing wrong with the GC or anything like, I simply got my peaks muddled up so when I was calculating the peak area ratio i was dividing my Phenacetin peak area/Caffeine peak area - amazing what you discover when you go back and look over your work!

Now my graph is a decent straight line R^2 value of 0.9952.

I just need to try and figure out how to calculate the actual concentration of caffeine present now! This should be fun!!

Cheers for the replies!

Offline Jamie1916

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Re: GLC Question - Can't explain why I'm getting these results!
« Reply #6 on: April 12, 2012, 11:01:58 AM »
I've just came onto the part where I am calculating the concentration of caffeine in the Tonic wine using the equation from the calibration curve I have plotted.

The equation of the line is y = 0.0019x + 0.0152 R^2 value 0.9952

and the peak area ratio of the caffeine extract from wine sample is 1.26.

(1 ml of the caffeine extract was added to a 10 ml volumetic flask + 3 ml of internal standard and made to the mark with methanol, the same as my previous standards)

Taking that into account, I have substituted 1.26 in for y and solved the equation to get x = 655.16

Now, this is where I'm confused. Is that the final answer or do I have to account for any dilution factors based on;

- when i extracted the caffeine originally from the Tonic wine sample I used 5 ml of Tonic wine + 10 ml of dichloromethane

The value stated on the bottle of Tonic wine is 37.5 mg / 100 ml (the bottle is a 750 ml bottle).

So I was expecting to get an answer of around about 280 mg ... however as you can see that's not what's happened!

Any help is much appreciated as I can't get my head around this one!

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