Dear everybody and thanks in advance to whom will answer.
We have HPLC Thermo Scientific Dionex UltiMate 3000 and we are using Acclaim OA 5UM column (Analytical 4 x 250mm) with Na2SO4:H20 100mM mobile phase.
We are examing samples of standard lactic acid in order to create a calibration curve and we are registering noisy baseline and no sharp peaks detection about sample.
We are trying to wash with acetonitrile, the problem seems to be solved, but after some sample runs, the problem rises up again.
Could you suggest us any potential action to solve the issue?
Thank you.
Not enough information to make any good suggestions. Lactic acid is very hard to detect by UV analysis (is that what you are using?). No selectivity available at such low wavelengths (i.e. ~ 200 - 210nm). Maybe your issue is related to column fouling, poor washing, lack of proper degassing or loss of pump prime ? Really need to start with the actual method details and chromatogram(s) with scales... You must first establish that the instrument is clean, operating properly and meets all performance specs (purged, no leaks, passes high-pressure leak testing on ALL channels etc). Do this w/o any samples so you can demonstrate a nice clean baseline, injecting only mobile phase through the fresh clean HPLC column. When the system is running with good reproducibility, run-after-run, then evaluate the method and instrument settings used to make sure they meet good chromatography fundamentals. Next, ask yourself: Is the method 'selective' for the sample (it needs to be)? Is the sample being retained on the column with a sufficient K prime? This process save time and money by insuring the method and system are suitable for the planned analysis.
**For UV analysis, use a new column with FRESH mobile phase solution (make up aqueous mobile phase fresh EVERY day), use a narrow wavelength bandwidth (e.g. 8nm), FULLY Degassed mobile phase (note: For best results, pre-mix mobile phase for isocratic methods).
- "Common Causes of Baseline Noise in HPLC, UHPLC"; https://hplctips.blogspot.com/2014/09/common-causes-of-baseline-noise.html
- "Modern HPLC Method Development Tips (PART I)": https://hplctips.blogspot.com/2016/07/modern-hplc-method-development-tips.html
- "Modern HPLC Method Development Tips (PART II)": https://hplctips.blogspot.com/2016/08/modern-hplc-method-development-tips.html
- "Do your HPLC Methods Meet Good Chromatography Fundamentals? HPLC Training: RETAIN, SEPARATE and RESOLVE"; https://hplctips.blogspot.com/2019/12/do-your-hplc-methods-meet-basic.html
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