[food]

Hey guys. I am new here.

I am working on a lab here. We used HPLC to determine the contents of a variety of different soft drinks. Specifically, aspartame, benzoic acid, and caffeine. I know how to do every calculation, and standardization (using 5 external standards that we analyzed with HPLC). However I am stuck at the actual interpretation of the chromatogram.

First, I am not sure about determining the dead time, or retention time for the mobile phase. Each of my chromatograms has a peak that shows up first, and I figured I could use the retention time for these peaks as the dead time. However the retention times for each of these peaks varies - and I am not sure if I can subtract these from the raw retention times of each of the peaks of interest, to determine the corrected retention time. Is it likely that different chromatograms obtained within the same hour will have different dead times? Also - the dead times that I am observing are insanely high - nearly a minute (for a 25 cm column) - I dont know if this sounds right.

Anyway - here is a bigger problem. I have determined the retention times for the 3 components of interest, using chromatograms of pure standards that I took a week ago. The retention times of caffeine and benzoic acid are within 0.1 minutes of each other. So when I try to locate these retention times on the external standards (which contain standardized concentrations of each component) - I can't find them. I am guessing that the caffeine and benzoic acid peaks are overlapping.

It's really annoying working with an HPLC that is having problems (there was a pressure overload a couple times), and on top of this, the chromatograms I get show ridiculous numbers of peaks - either indicating that the standards are impure, or have degraded into many products.

Has anyone had an issue with this type of experiment before? It would be helpful if anyone remembers having trouble with peaks overlapping with caffeine and benzoic acid. I'm hoping it's just the specific HPLC we used. I've got a couple more days, and I'm going to see if I can convince the TAs to give me better data - because I cannot possibly do a data analysis with this - unless I am clearly overlooking something. Which is why I posted here...in case I am being stupid and ignored an essential aspect of HPLC analysis...

thanks

[Arkcon]

Well, you've got some problems, but it's hard to understand without a chromatogram.  Here's some tips. 

You'll have to determine dead time, it can be no later (and often a little earlier) than the time it takes your particular flow rate to sweep through an empty column -- pi*radius in cm*length in cm.  Dead time often, but not always shows up as a small peak like you observed, it's caused by the flow disturbance caused by the injection. 

Try to build a table, of dead time (we call that V_o), and your peaks, and their retention times.  If you feel like it, compute average, and std dev of the retention times, so you can clearly see, how good or bad your results are.  Maybe, a really bad outlier, will prompt you to realize, "Wait, I've been misidentifying all along."

[Arkcon]

Urm ... pi*(radius in cm)^2*length in cm.  Heh.   You probably would have figured that out on your own, but just to correct my own mistake.

[NP1957]

Have a look at
http://www.chemistry.adelaide.edu.au/external/soc-rel/content/expts/hplcexpt.htm

There is "Experimental procedure for determination of caffeine in soft drinks by HPLC".

Hope this is useful.
Cheers,
NP1957    

[Arkcon]

Have a look at
http://www.chemistry.adelaide.edu.au/external/soc-rel/content/expts/hplcexpt.htm

There is "Experimental procedure for determination of caffeine in soft drinks by HPLC".

Hope this is useful.
Cheers,
NP1957    

Oh.  Nice link, the parent has lots of useful info as well.  Have one on me.  B)