Not a lot of information to go on (what is your mobile phase and flow rate?) with many possible answers, but here are some of the most common reasons for this type of observation:

• Your degasser may not be operating properly (For Waters brand systems, you will often see the error message "Degasser hardware fault"). This would indicate that your degasser needs formal service. *The degasser requires regular maintenance and repair just like the other components so get it fixed, but do not run in RP w/o a properly functioning degasser module. Info on professional degasser repair available at this link: http://www.chiralizer.com/hplc-degasser-repair.html
• The pump solvent compression values could be set wrong for the mobile phase you are using. Please read this article for more info: http://hplctips.blogspot.com/2011/10/hplc-pump-solvent-compressibility.html
• You many have a sticking check valve on the pump (common on Waters brand pumps). This causes cavitation which results in bubbles being introduced into the solvent stream. Remove the valve, sonicate and clean it in a solution which will dissolve any contamination or debris stuck to it.
• Improper purging/flushing of the pump. System not primed. Be sure and correctly prime the pump, and ALL channels connected to it, with clean mobile phase. For RP applications you could begin by flushing the entire pump with IPA (make sure the pump's outlet is directed to WASTE, not your column) followed by pure DH20, pure MeOH and then pure ACN. Run the pump at a high flow rate to waste fro several minutes with each solvent being sure to flush any and all channels. Once flushed, set the composition for 100 %MeOH (or ACN) at a moderate flow rate, monitor for several minutes, then resume testing to see if the problem has been resolved.
• Lack of pump, general HPLC system maintenance or leaks. If your piston seals are leaking or scratched, if buffer crystals have dried out in the system or if one or more valves is contaminated or worn, your baseline may be unstable and your hardware may need maintenance before you can resume using it for analysis methods. Regular maintenance is critical to maintain your entire HPLC system. Check your instrument log book to determine what types of maintenance were most recently performed and if in doubt as to to the performance or condition, run formal PV tests on all of the modules to measure their performance and insure that they meet the manufacturer's full specifications.
• Here is an article which may assist you in troubleshooting . "Air Bubbles Exiting the HPLC Degasser, Reasons Why"; https://hplctips.blogspot.com/2019/08/air-bubbles-exiting-hplc-degasser.html

For Future reference to anyone finding this post, here are two related links to assist you in troubleshooting two popular models of HPLC Vacuum Degassers.

• WATERS brand HPLC Degassers: https://www.hplctools.com/waters_degasser_error_troubleshooting.htm
• AGILENT / HP Brand HPLC Degassers: https://www.hplctools.com/agilent_degasser_error_troubleshooting.htm

BTW: Depending on the exact Waters HPLC system model # you have, you can also run the built-in degasser test too. The test menu for this is usually found under "Other Diagnostics"  Press the Start key to run the test and it will evaluate the vacuum level for you.

For Future reference to anyone finding this post, here are two related links to assist you in troubleshooting two popular models of HPLC Vacuum Degassers.

• WATERS brand HPLC Degassers: https://www.hplctools.com/waters_degasser_error_troubleshooting.htm
• AGILENT / HP Brand HPLC Degassers: https://www.hplctools.com/agilent_degasser_error_troubleshooting.htm

In addition to the answers above, check that fittings are tight as a loose fitting can cause a system to suck air.

Thank you all for your insightful answers.......I think these will be very helpful for me. Williams.... my Mobil Phase is 50:50  MeOH and MTBE with flow rate of 1ml/min, inj. vol: 20mls, Isocratic mode on Waters e2695 Separation Module.

Thank you for the extra information. I think you meant injection volume 20 ul (not ml), but other than that I am confident that you will find the solution to your problem by addressing the points in my earlier post.

William Leters, I beg your pardon, I meant 20ul not 20mls. I am really looking into your suggestions and finding them helpful. Thanks

Adesokan: Your mobile phase of "50:50 MeOH and MTBE" is 100% organic with no water? RP mobile phase mixtures are usually water based. This is a very unusual RP HPLC mixture to use and may be contributing to your problem as well.

I sometimes consult in the food chemistry field and recall that I used this type of mobile phase once before. Are you analyzing β-carotene in carrots or other vegetables? That is one of the few applications that I recall where these types of mobile phases are used, usually in isocratic mode on a C18 column. If so, be sure to wash the column down after each run using a gradient with some trace water and high MeOH/MtBe to remove any accumulated fouling. 

Btw: If you find my answers or responses helpful, please "upvote" them by clicking on the blue up arrow (but not for this one!). Thank you and good luck with your project.

William: Actually I do run analysis of B-carotene in some vegetables and some roots and tubers crops.I find this combination suitable for some of the food samples in isocratic mode on C18. Interesting to know that you have consulted in related project. Have you worked with food samples that contain polar carotenoids like lutein and zeaxanthin where you would require a modifier/buffer to optimize the separation of these compounds? Could you share your experience on this. 

Absolutely. These are rather easy to resolve, but the biggest problem that I recall in separating them is that you really need to add a strong solvent to dissolve them (i.e. Methylene chloride, Butanol, Ethyl Acetate, Chloroform) to the mobile phase which will block much of the UV signal. This is not a problem for all of the compounds as many have UV absorption at much higher wavelengths (VIS) too. The other problem is in extraction, which is very important. You must develop proper extraction and cleanup procedures for your samples BEFORE injecting them into the HPLC or you will quickly foul up the column.

So here are a few tips:

(1) Use a gradient to perform your separations. Do not use isocratic runs. Full gradient analysis is best, being sure to hold and end at at a very high concentration of the strongest solvent to elute of all compounds (or wash off any late eluters you did not separate).

(2) Use a high quality C18 column. E.g. 4.6 x 250mm, 5u will work fine for most of these compounds. Do not use a guard column. Instead, use proper technique and always make sure your samples are fully dissolved in solution, Filter all samples before placing in the HPLC to be safe!

(3) Search the web to find plenty of literature for example methods. When I used to run these we did not have the "Web" as a resource, only libraries with paper journals and abstracts. I am sure you will find hundreds of articles to review with a simple search first.

(4) Some examples of methods I used to separate these types of compounds apart on a C18 Column.

   (a) Step or Linear Gradient of: ACN/1-Butanol/MeCl (70/29/1 to 50/20/30) over maybe 30 minutes, depending on column dimensions and flow rate.

   (b) Step or Linear Gradient of: MeOH/ METB (85/15 to 10/90) over maybe 30 minutes, depending on column dimensions and flow rate.

(5) Great sample prep is the key!

(6) Initially scan all samples with a DAD/PDA detector. Determine the lambda max wavelength for each sample in scanning mode. If it is obscured by the mobile phase, then check for a secondary max peak (often in the VIS spectrum) to use instead. *Optionally, an ELSD or CAD detector can be used in gradient mode for many of these compounds, but UV/VIS is so much easier.

(7) WASH the column off with a mixture of high alcohol (which ever one you used in your method) and MeCl (or equiv) after each run. The column will easily foul and cause retention time problems. Frequent and thorough washing will eliminate the problem.

Good luck to you Adesokan with your project!

Great! thank you for sharing your experience on this.