Hi Kaitlyn,

There can be several reasons for the pressure drops you observe: air could still be present in the system after your recent repair (i.e. between the degasser and the column). You could flush the system again with isopropanol on all channels to get rid of any residual air.

Do you see the pressure drops are more pronounced when running solvent A (or B) only? This could tell you in which pump head the issue resides (which head did you replace the ball valve on?).

There could also be a problem with the active inlet valve; do you have one spare to check that, by any chance?

I would definitely not proceed with any serious analysis as long as the pressure problem remains..

Good luck, hope this helps!

Hi Kaitlyn,

There can be several reasons for the pressure drops you observe: air could still be present in the system after your recent repair (i.e. between the degasser and the column). You could flush the system again with isopropanol on all channels to get rid of any residual air.

Do you see the pressure drops are more pronounced when running solvent A (or B) only? This could tell you in which pump head the issue resides (which head did you replace the ball valve on?).

There could also be a problem with the active inlet valve; do you have one spare to check that, by any chance?

I would definitely not proceed with any serious analysis as long as the pressure problem remains..

Good luck, hope this helps!

Hi

I think that still you have an air bubbles in your system.. then you must check for leak for every part . Also, you must use a syringe for evacuation of the column because it is very important to eliminate the air bubbles from it ( this is a main reason for pressure drop )

Good luck in your work 

Hi Kaitlyn.

You have described change seal in the check valve (ball valve). I think your check valve is blocked, it's possible if the ball remain in a position and the mobile phase it is not flowing. You could remove the check valve (ball valve) and sonicate with methanol for 10 minutes.

Another possible reason is bubble in the system. As Eef said flow isopropanol in system could solve this trouble.

I hope this help you

Dear Kaitlyn , I dont think you have a difficult problem ,  I just to get this machine properly fixed by the  machine BrandTechinician , & only then you run  the samples on it, dont just waste your time.  Dr Abid Aziz Ajaz.

As previously stated by others, please Do NOT analyze any samples with the HPLC unless it is running perfectly. If the baseline is unstable, then your data will be invalid. You must first correct the problem with the instrument before proceeding. - There are literally dozens of possible causes for an unstable baseline. The first place I check is the PTFE Frit inside the 1100's Prime Purge Valve. This disposable Teflon frit is designed to be replaced every month and is $ 5. You should have lots of them on hand and they are designed to protect the HPLC system from the normal bits of pump seal material which enter the flow path over time from entering the injector, column or other expensive parts of the instrument. These inexpensive filters do a fantastic job, but when ignored, can cause strange pressure or baseline disturbances to be observed. The filter is a normal maintenance part and when in good condition should appear snow white in color. Remove it from the valve, flush out the valve, install a new PTFE filter and replace the valve back on the pump (flush it out after installation). *One of the most overlooked maintenance items on the HPLC! Takes ~ 10 minutes to replace.

The Agilent/HP 1100's outlet check valve (you refer to it as a "ball valve") rarely leak or have problems ("cracked seal"? I hope the valve was replaced). Normally a sticking valve can be cleaned with solution and reinstalled w/o a problem. The electronic inlet check valve (called the "Active Inlet Valve" or AIV) is also VERY reliable in these units and rarely fail. However, failure to properly flush and clean the pump head of any buffer salts, additives or particulate matter at the end of each day can over time "gum" things up a bit on any HPLC. Proper flushing with fully degassed mobile phase (through all channels) should take care of the issue 99% of the time. Make sure the liquid you use to flush the pump is HPLC grade, contains no salts or buffers, is fully degassed and contains a high percentage of organic, but not 100% (e.g. MeOH/DH20, 80/20). Check and flush each channel ONE-AT-A-TIME (100% 'A', 100% 'B'...) with the Prime Purge valve open (one turn) and flush to waste. Run 10 mls through each channel in this way FIRST. Once flushed, you can install fresh mobile phase. Make sure the solvent frits are clean, the liquid is fully flushed through the degasser and lines past the pump head (prime purge valve open) to allow 20-30 mls/channel to flow through to waste first. Make sure your column (and column temp) are also clean and stable. Re-equil and monitor.

BTW: What type of HPLC detector are you using? You implied it was not a UV/VIS so that introduces more variables. You also implied that you degass two times? This seems a bit odd. As long as the mobile phase is compatible with your inline degasser, there is no need to degas before "degassing".

For more information on "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)" please refer to the free article link below:

http://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html

"1ml/min the pressure is 43bar" seems to be using of an acetonitrile (AN).   Then, please use methanol or 2-propanol, although the column-inlet pressure may becomes high.   Then, please use the gradient elution method, which is adequate for HPLC separation (see file; PTH Roma), and shorter length of the column may be employed in the gradient elution method since small molecules elute in the order of their hydrophobicity (based upon the mechanism of hydrophobic affinity chromatomatography).   Although glycoprotein case, attachment of N-acetyl-neuraminic acid (NANA) seems to determine the early retention time in RP-HPLC.    

Thank you everybody for the updates! I am assuming that there must be some type of air still in the system. 

Bill - I have not changed the PTFE frit in the system so will do that as well as flush the system. I did replace the check ball valve. We do not use any type of detector. Our method for detection is collecting samples every minute (1ml/sample) and running them through a scintillation counter as we are interested in the levels of radioactivity. Also, I was only degassing "twice" because it was suggested to me that our online degasser may not be working properly but I don't believe that is the case anymore. 

Hopefully all of these great suggestions work!

Thank you for the update. Dirty/clogged frits cause many problems and are a sign of a lack of any maintenance. Be sure and flush out the prime-purge needle valve and re-prime the system "wet" after re-assembling and cleaning.

If you had a detector in-line it would make it much easier for you to troubleshoot the problem (because you would have something to "view" the signal with). Your job is more difficult without one. The pressure ripple of a properly functioning system should be less than 1%. You can use that guideline along with the actual pressure in psi to gauge it. "Drops" should not be tolerated as they will effect composition and flow rate (you can check flow rate with a stopwatch and small graduated cylinder or volumetric flask).

BTW: If your inline HPLC degasser is broken (as most of them are after 5 years of service), then please refer to this web link to send it out for service. Damaged vacuum chambers can leak gas and contaminates into your mobile phase causing all kinds of serious problems. Thank you.

http://www.hplctools.com/Agilent%20Degasser%20Repair.htm

I too have faced such problem with Fischer equipment, but then their application engineer came over and corrected the fault (it was in the software).