Chromatography Forum

Hello,

I am developing a method using isocratic elution with the following conditions:
mobile phase: %20 ACN/WATER
50mM Ammonium acetate
15mM TBA(Tributylamin)
pH adjust 7. 00 with acetic acid
Dedection wavelenght : 232nm

I injected blank sample and the baseline drifted upwards after the 30min.
Any suggestions as to how to overcome this problem.

We need more information here:

• - did the baseline then return to its original value?
  
  - if you simply monitor the baseline for long periods of time, do you also see basline fluctuations?
  
  - what was your sample dissolved in?

Hello,

The baseline did not return original value.
I inject blank. I see small fluctiations from 0 min. to 24 min. After the 24 min. baseline rise 40mAU almost steepy then it is fixed.

Run time is 120 min.

My sample dissolve in mobile phase.

Does this problem arised from ion pairing reagent (TBA) or my column?

Sorry my english!

Thanks your help.

A few more questions:

1). If you make replicate injections of your blank, does the phenomenon repeat on each injection?

2). Are you working from a single bottle or are you mixing in the pump?

3). If you leave the instrument running overnight and compare the background in the morning to what you observed the night before, is there a significant change in background?

If the answer to the last question is: yes, then there is a good chance that you have a contamination problem with your tributyl amine. What color is your reagent? It's common for amines to become highly discolored upon exposure to oxygen and perhaps this is the root cause of your problem (although this doesn't explain any injection related problems). In this case, I would advise either getting a fresh sample or distilling the amine in order to eliminate the contamination problem. Generally, the salt form of amines are significantly more stable so you might also want to consider using an amine salt rather than the free base has a source of your reagent.

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Hello chris,


1). The phenomenon repeat each injection

2). I am working from single bottle

3). (I don’t understand last guestion exactyl) I don't have enough tributylamine for all my experiment so instead of leaving instrument running overnight I inject blank and observed the baseline 300 min. but I can't comment on this chromatograph. I attach it.



Is this phenomenon normal?

I hope these answers are enough. I will be gratefull for your help.

Thanks.

This is repeatable the next injection "sitting" on top of the previuos, isocratic method? If yes you are apparently injecting immense amounts of dirt with your blank. About the third time you do this your detector should be blacked out. Air injection can look similar if air bubbles get stuck in the flow cell, but these usually leave (sudden drop of baseline) or are slowly diminished by dissolving (downtrend of baseline). I have never seen something like that repeatable, though.

Ebru,

Since you are in the method development stage, can you try another buffer and a mobile phase since I am seeing 2 problems in your mobile phase/buffer?
1. pH 7 is out of the acetate buffering range, isnt it (pKa of acetae is 4.?

2. 120 min run time is way too long, if it is a RP-HPLC method, your compound seems very hydrophobic, so why not increasing ACN % and/or try a gradient method?

Good Luck!

Ananda

Ebru,

The chromatogram that you attached looks to me like a very typical equilibration chromatogram that happens when you are using ion-pairing reagents (it is something like frontal chromatography). However, once this happens, your system should have been stable which means you shouldn't have the same thing again. So I want to be sure that we understand well... if after you notice this effect in the morning, you make another injection, the second chromatogram, does it looks like the one that you attached or not? Another question... do you flash your system with something different before you leave your system overnight or not?

I tend to disagree with HMW as even if you injected large amounts of dirt eventually your base line should go back to the baseline and after 150 minutes I would expect to have a much more "potato like" peak and not as stip drift of your base line. What is your black? Is it your mobile phase or water? If yes, then just to be sure clean throuroghly your injection valve before your next injection...

Hello,

My column did not become saturated with M.P. I leaved running my column one day and overnight. Morning the baseline was very good.

Thank you very much Kostas and Ananda, give me some help.

Well, I eliminated the obvious, because of statements like
"I inject blank. I see small fluctiations from 0 min. to 24 min. After the 24 min. baseline rise 40mAU almost steepy then it is fixed.", which I understood to mean that injection of blank caused the shown chromatogram (first approximation of what was meant).
Now "dirt peaks" several hours long are not that unusual, both baseline equilibration and dirt are mostly noisier after the initial rise in this lab as compared to ebru´s chromatogram.

We seem to be playing guessing games, quite wasteful, actually.