Chromatography Forum

Hi all,

I am facing some problems with unstable, noisy baseline. Photos in below link for your reference. Not sure if this is indication of presence of air bubbles? I've tried purging the flow line and autosampler several times, and equilibrating my column with initial gradient concentration for an hour or so. MP composition is 92% buffer/8% ACN, ran at flowrate 1mL/min. Pressure observed during baseline check was relatively stable, did not notice much fluctuations. Any idea how i can go about troubleshooting this? Am still new to HPLC and learning. Would appreciate any inputs! Thanks a lot!

https://ibb.co/Sch0pkQ
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Yes, it could be an air bubble problem. Do you use a degasser? What is your flow cell volume? Check your system for extra column effects. Mybe it will help to use 30cm capillary on your detector outlet.

This is a very small disturbance (at ~ 220 nm) and shows cyclical behavior.

At first look, that appears to be temperature related. What is the ambient temperature in your lab? What type of column compartment do you have? Is it COOLED and HEATED ? If it is only heated, then you need to set the temperature to at least 10 to 15C more than ambient for better control. You show 30C set temp. Try setting the temp to a more stable temp (higher).

It could also be due inadequate degassing of mobile phase (which causes the pump to not perform well).

Try getting some help from an experienced professional chromatographer. It takes many years of hands-on experience to reach just a basic level of proficiency. Troubleshooting is hard without experience. Otherwise, here is a link to an easy to understand article on the topic that will present you with many possible reasons for the issue.

"Common Causes of Baseline Noise in HPLC, UHPLC"; https://hplctips.blogspot.com/2014/09/c ... noise.html

It doesn't look like it's anything to do with the pump because it's quite different at 220nm and 245nm. The time-course of the fluctuations is really rather short (these views are only a minute wide) which isn't typical of lab temperature issues. How old is the deuterium lamp in your detector? How clean is the flow-cell? Are you sure this isn't just the weak fluctuation of a detector that's not seeing much light, from a possibly-declining bulb?