I am using HPLC, with ELSD (Series 1200) detector. My mobile phase is KH2PO4. For samples of the same batches of piperazine and diethanolamines, I am getting different results. Some samples are showing 5, 7, 11, even 16 peaks. Can anybody guide me? What is reason behind that?
Dear Dr. Teodoro,
Thanks for your answer. What can be the other best choice?
1. Are your samples in the same matrix?
2. Are you ensuring you have enough buffering capacity in your mobile phase to avoid pH changes during your runs? (Assuming you're doing RPC).
If you want get consistent and precise data. You just follow the following points-
1. mobile should simple and should have evaporative in nature,
2. Do not use in-organic buffers solution.
3. pH plays major role on the peak elution, i.e. retention time.
4.Use the buffer solutions of TFA, acetate, propionate buffer, ammonium acetate and formic acid.
Hi, you can not use nonvolatile modifiers in your mobile phase because the detector you used is ESLD. KH2PO4 will leave a significant amount of residue in the system after evaporation which will definitely effect your results. Try other volatile modifiers, such as Trifluoroacetic Acid and Acetic Acid.
Salam, my friend. excuse me are you sure from using ESLD. I thought you used UV detector
In addition to the pH control, I think you should also set a constant temperature of the column using an oven.
Hi , try also to monitor your pressure if it is constant. Mobile phase must always be constant. Try also to run the blank between the samples to see if there is a carry over.
Dear Shaukat,
Please see : http://www.chromacademy.com/images/Ishikawa-diagram-variable-peak-height-area.pdf
You could download the HPLC Troubleshooting Guide, and See : http://www.chromacademy.com/chromatography-troubleshooting-HPLC-variable-peak-height.html
The first factor that should control is leaking sample
valve. Many factors should be checked .
ELSD is usually used for high-boiling, non-volatile samples. When you use KHPO4 as mobile phase modifier, you will get a constant stream of non-volatile salt as the background. Are you sure this is even a suitable condition for piperazine and diethanol amine? Are you trying to replicate literature condition or develop new conditions?
Sometimes, gradual development of small but significant leak of the eluent (which may not be in the form of physical droplets) in the system may also cause this kind of retention time shift.
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