I am running catecholamine standards in HPLC using UV/FLD detector. I am getting a constant peak at fix RT (retention time @ 3.5 min, image enclosed) every time, even if I run another standard or buffer (acetate buffer) or inject methanol or acetonitrile. I have washed the column, needle but still getting the peak. How can I correct it? Please advice
Thank you
Enclosed: Image
Looks a bit like a solvent front. Have you tried an injection without the column? If you get the same shape peak (it should be much earlier then, of course) then that may be an explanation. Would then check flow-rate, length of tubing, pump performance as it seems rather late for a solvent front, although that will depend on column size and flow.
it may be a peak attributes to the cut off wavelength of solvent postponed. Calibrate the system with a standard like caffeine and check the performance of the system. Condition and equilibrate the column beforehand.
*We do not "get rid" of real data in chromatography. We determine what it is and why it occurs first to understand it.
**No, the peak observed in not related to any solvent cut-off or equilbration concerns at all. This is a basic training and setup problem.
A few problems are obvious from the chromatogram you included.
(1) First of all, you did not include any information about your HPLC method or analysis and it appears you are using HPLC for the first time without any supervision (please ask for help from someone at your school). *When requesting help, esp on a forum like this one, with any HPLC question, please provide us with all of the basic details first (e.g. Column dimensions and type; flow rate, mobile phase composition and time program, if used; detection settings; injection volume and solution used; sample information). This information is vital to have a basic understanding of what you are doing.
(2) . **PLEASE TURN OFF the Reference Wavelength feature and read this free article;
- "REFERENCE WAVELENGTHS (as used in HPLC UV/VIS)"; https://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html
You have elected to turn ON the "Reference Wavelength" software feature. You selected a reference signal of 360 nm, 100 nm bandwidth to monitor a signal at 296 nm. This means you have instructed the system to collect all data from 310 to 410 nm and then subtract it from any data collected at between 294 and 298 nm, then provide the result as your "296 nm" signal for the chromatogram. This process ignores all basic liquid chromatography guidelines and completely invalidates the data collected and method used.
(3) The peak shown around 3.5 minutes does look like a normally seen "pressure peak" (sometimes called a "solvent peak") which is associated with the injection. However, there is no possible way to confirm this unless you can provide us with the exact column dimensions and flow rate used so we can estimate the Tzero of the method.
- "Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
Hello! You can try this:
When you run a baseline does it happen?
If yes, it could be your solvent. Wash your solvent bottles and try using the same solvent (e.c. methanol) from another manufacturer or lot.
If not, your injector could be contaminated. Wash your injector (probably there's an option on the software where you can do it) and run it again.
Please look at the following below links which may help you in your analysis:
https://web.vscht.cz/~schulzov/HPLC/Troubleshooting%20HPLC.pdf
https://www.phenomenex.com/Info/Page/lcttuk25
Thanks
I agree with others that we need the operating conditions, notably stationary phase, column dimensions and flow rate.
However, it's probably not fair to refer to beginners, because so many researchers necessarily have to use analytical techniques with limited experience.
Your peak looks like unretained materiel, where "unretained" is a somewhat variable concept. It may not be obvious to all that, despite the price, microporous packings are about 80% empty space, with some variation in the almost molecular dimensions of the pores. This was the basis of a refutation we published when HPLC was not so well established in biomedical labs:
DOI: 10.1016/0006-2952(87)90189-4
All the chromatographic conditions are constant & your method is robust then constant peak will get without any trouble but +& - 0.5 mins considerable. Main focus should be accurate method and each procedure.Thanks
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