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Topic: HPLC Spetrum  (Read 6080 times)

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Offline CPC

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HPLC Spetrum
« on: March 16, 2009, 09:20:19 PM »
Hallo,
my compound analyzed by HPLC has an image as shown in the attachment.
I do not understand what is going on with the peak heading the negative direction, which seems to be symmetrical to the one on the other side of the base-line.
Could anyone help with interpretating the phenomenon?
I would appreciate very much.
CPC

Offline alphahydroxy

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Re: HPLC Spetrum
« Reply #1 on: March 17, 2009, 06:49:07 AM »
errrmmm... doesn't look much like a HPLC trace to me...!

Perhaps this is just a phasing issue (dunno if this is the right word when discussing HPLC) ?


Offline CPC

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Re: HPLC Spetrum
« Reply #2 on: March 17, 2009, 09:29:02 AM »
Hi alphahydroxy,
thank for the discussion.
I really do not know what is going on here.
It looks very strange to me.
I do not understand the word "donny" either.
CPC

Offline enahs

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Re: HPLC Spetrum
« Reply #3 on: March 17, 2009, 11:08:21 AM »
What type of detector are you using?

Offline macman104

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Re: HPLC Spetrum
« Reply #4 on: March 17, 2009, 11:25:39 AM »

Offline Arkcon

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Re: HPLC Spetrum
« Reply #5 on: March 17, 2009, 12:39:51 PM »
Often, at the moment of injection, you'll see something like you've drawn -- this is the injection disturbance, as pressure builds, then is released, just for an instant, as the injector rotor turns from sample to column.  If you can tell us a bit more about your application, and how your system is set up, we'll tell you more.  And see if you can't attach the real system output.
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline Squirmy

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Re: HPLC Spetrum
« Reply #6 on: March 18, 2009, 12:40:04 PM »
If this were a Rorshach test, I'd say it reminds me of an IR interferogram, but it really could be a lot of things.

I think you should provide scales for your x- and y-axes...units and numbers would be helpful.

Offline CPC

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Re: HPLC Spetrum
« Reply #7 on: March 22, 2009, 03:42:49 AM »
Hallo,
thank you, all for your discussion.
I have got now the original HPLC spectrum.
I will post it here so that you may help with interpretating it.
I really do not understand this phenomenon.
Look forward to hearing from you.
CPC

Offline DrCMS

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Re: HPLC Spetrum
« Reply #8 on: March 22, 2009, 07:19:41 AM »
It looks to me to be caused of the injection. 
Was the sample made up in the eluent or something different, i'd guess the latter.

Offline Arkcon

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Re: HPLC Spetrum
« Reply #9 on: March 22, 2009, 07:33:26 AM »
There we go, now that's easier to understand.  And thank you for including a spectrum for each peak, that's also helpful.  Negative peaks aren't so uncommon when doing UV detection in HPLC, something is separating out the has less absorbance than initial conditions -- perhaps, some solvent component of your sample, or a contaminant with a very strong absorbance outside of your selected spectrum window.  

There are some tricks you can employ.  For starters, different integration software have different ways of dealing with negative peaks, if you turn on, or turn off, negative peak detection, the system may draw a more correct baseline, for the peak that precedes it.  A gradient, or mobile phase change, for isocratic, may separate the peaks better, as another option.

There's an important consideration many people don't realize.  The knee-jerk response for people developing UV detection is to set the peak detection at lambda max, and that's not always the best way.  Sure, that gives you the most signal, but you're at the mercy of your instrument's accuracy, which may drift.  You'll notice, from your spectra, that if you're outside lambda max by say, 5 nm, the peaks would all be smaller, but the effect of the negative peak on the baseline might be much less.
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline CPC

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Re: HPLC Spetrum
« Reply #10 on: March 23, 2009, 09:22:30 AM »
Hallo,
thank you all, especially Arkcon for the discussion and suggestion.
I see now the problem that might be the cause of the phenomenon.
I will ask to perform also GC-MS analysis so that we can have a clear consideration.
Best regards,
CPC

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