You probably have AIR in the channel 'B' line. This may be due to poor priming, a loose fitting, poor quality degassing or a damaged gradient valve channel (check the connections by the low pressure lines near the DCGV and degasser chamber for any loose connections first).

To try and correct the problem, first open up the prime-purge valve to direct the liquid safely to waste.

Next, make sure you have clean, fresh solvent pickup filters installed on your lines (A and B). The filters are consumable items and require replacement, as often as monthly in some cases. If you are running with buffers or any aqueous solutions in the bottle (these should be made up FRESH every day, then filtered before use) the solutions will grow bacteria and other organisms very quickly. This will restrict flow through the lines to the pump and also contaminate the entire flow path of your Agilent 1220-series HPLC system.

Place the clean pickup filters into a clean, fresh beaker filled with 50% Methanol/50% HPLC grade Water (*Note: if you do not have these liquids you may substitute a mixture of 50%/50% HPLC grade IPA/Water). Set the pump to 100% 'A' channel at 5 mL/min and run it for 3 to 4 minutes to waste. Now, switch to 100% 'B' channel and do the same thing. Stop the pump. Disconnect the column and place a restrictor capillary in-line. Direct the restrictor to waste. Set the pump to 100% 'A' at 1.00 ml/min, turn the pump back ON and close the prime purge valve to allow the liquid to flow through solvent lines, through the degasser channel, through the pump head to the restrictor and out to waste. Set the software screen to monitor pressure so you can watch it. Adjust the flow rate at any value which provides ~50 Bars back-pressure [50 - 100 Bars is ideal]. Monitor the flow ripple and see if it stabilizes. Make note of the pressure stability after 5 minutes and if stable, switch over to 100% 'B' channel and do the same thing. This should provide you a way to insure that the system has been properly primed and allow you to measure the pressure stability.

Once the system has been fully primed and checked, you can check the operation of the dual channel gradient valve using a standard gradient test. Example: "Gradient Mixing Test For Your HPLC Pump (Step Gradient)"; https://hplctips.blogspot.com/2014/05/gradient-mixing-test-for-your-hplc-pump.html

PS: Thank you Markus for adding the additional link too.

Hi Asmaa,

for troubleshooting HPLC Pressure Fluctuations I recommend you the following link:

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

Maybe it will help you to fix the problem.

Regards

Markus

You probably have AIR in the channel 'B' line. This may be due to poor priming, a loose fitting, poor quality degassing or a damaged gradient valve channel (check the connections by the low pressure lines near the DCGV and degasser chamber for any loose connections first).

To try and correct the problem, first open up the prime-purge valve to direct the liquid safely to waste.

Next, make sure you have clean, fresh solvent pickup filters installed on your lines (A and B). The filters are consumable items and require replacement, as often as monthly in some cases. If you are running with buffers or any aqueous solutions in the bottle (these should be made up FRESH every day, then filtered before use) the solutions will grow bacteria and other organisms very quickly. This will restrict flow through the lines to the pump and also contaminate the entire flow path of your Agilent 1220-series HPLC system.

Place the clean pickup filters into a clean, fresh beaker filled with 50% Methanol/50% HPLC grade Water (*Note: if you do not have these liquids you may substitute a mixture of 50%/50% HPLC grade IPA/Water). Set the pump to 100% 'A' channel at 5 mL/min and run it for 3 to 4 minutes to waste. Now, switch to 100% 'B' channel and do the same thing. Stop the pump. Disconnect the column and place a restrictor capillary in-line. Direct the restrictor to waste. Set the pump to 100% 'A' at 1.00 ml/min, turn the pump back ON and close the prime purge valve to allow the liquid to flow through solvent lines, through the degasser channel, through the pump head to the restrictor and out to waste. Set the software screen to monitor pressure so you can watch it. Adjust the flow rate at any value which provides ~50 Bars back-pressure [50 - 100 Bars is ideal]. Monitor the flow ripple and see if it stabilizes. Make note of the pressure stability after 5 minutes and if stable, switch over to 100% 'B' channel and do the same thing. This should provide you a way to insure that the system has been properly primed and allow you to measure the pressure stability.

Once the system has been fully primed and checked, you can check the operation of the dual channel gradient valve using a standard gradient test. Example: "Gradient Mixing Test For Your HPLC Pump (Step Gradient)"; https://hplctips.blogspot.com/2014/05/gradient-mixing-test-for-your-hplc-pump.html

PS: Thank you Markus for adding the additional link too.

Thanks William and Markus for your reply.

We followed these steps before but no change!!

Unfortunately, we noticed no solvent flow when running %B, the solvent enters the DCGV-B line but no flow from DCGV to the inlet valve.

This confirm that the problem is in DCGV-B line !!

Hi Asmaa,

have you checked the piston of the pump? Maybe it is broken.

Regards

Markus

Yes we checked it yesterday and it was good.

The problem in line B only. No flow from line B to the pump.

Line A is ok !!

So, we suggest the problem may be in Dual channel Gradient valve line B

Hi Asmaa,

the drive for the piston of channel B is also OK?

Regards

Markus

Yes it is OK

Hi Asmaa,

try rinsing channel B with 100% isopropanol. Isopropanol is more viscous and maybe the stubborn air bubble will be carried along (as Wiliam suspects).

Regards

Markus

Hi Asmaa,

okay, the pump doesn't pull anything. Either you have massive air in the system or the suction valve doesn't open. Take it out and put it in an ultrasonic bath for 30 minutes. If that doesn't help, then install a new ball valve. Or you can swap the AIV's (active inlet valve). If it works then, it's the AIV.

Regards

Markus

You may need a new degasser. I had a similar problem with both a Thermo Accela 600 and 1250 pump. Replacing the degasser fixed this issue. I can't speak for the Agilent system, but the replacement was a fairly easy procedure. Good luck!

Asmaa, you are confused about how the HPLC works. The gradient valve is positioned BEFORE the pump head and after the solvent pickups so no liquid can get to the pump until it has been primed through the lines and arrives at the gradient valve (DCGV). In the picture you supplied, it shows that you DISCONNECTED THE PUMP from the gradient valve (the arrow points to the gradient valve OUTLET which connects to the PUMP inlet). To the right side is the degasser (and MP inlet lines). To the left of the DCGV is the pump head. With the pump offline, you will never get any liquid to appear at the gradient valve (except due to gravity siphoning) as the pump is responsible for "pulling" the liquid through the system and you disconnected it! Your test is illogical.

To troubleshoot these types of problems, you must first have an understanding of the actual flow path of the liquid through the system. New users do not seem to understand that the knowledge needed to properly operate and use an HPLC system takes years to acquire (for a basic level proficiency). You can not learn all that is needed in a day, week or month like you can for other simple instruments like a pH meter, balance or UV/VIS spectrophotometer. HPLC systems take a lot more experience to use, and far more experience to troubleshoot properly. As noted earlier, please flush the two lines separately (100 % A.... 100% B) to purge them (with the pump connected) to remove all air from both lines. Monitor for flow at the prime purge valve (not at the disconnected valve). Check those solvent pickup filters for clogs or restrictions too and perhaps most importantly of all, please get as your teacher for some experienced local help with this.

Hello, Check plunger of pump may be some damage there.

Have someone experienced with the system help you with this. They must understand the flow path and operation of the system for the tests to be valid.

• Connect everything back up and try to purge the two lines separately to waste (do as suggested in my earlier post).
• Confirm (by disconnecting and checking if needed) that when the pump is purging, liquid enters lines A and B solvent pickups and flows to the degasser.
• Confirm (by disconnecting and checking if needed) that when the pump is purging, liquid enters lines A and B solvent pickups and flows to the degasser and to the two gradient valve channels.
• Confirm (by disconnecting and checking if needed) that when the pump is purging, liquid enters lines A and B solvent pickups and flows to the degasser, on the gradient valve and through the pump head out the prime purge valve.
• If liquid is making it to a gradient valve channel, but not exiting when that channel is selected (i.e. 100%), then swap the low pressure lines from A to B going into the DCGV only. If liquid now flows out the channel that it did not come out previously, then the problem is upstream towards the bottles (you would restore the connections and repeat the test on the two degasser channel lines to see what happens. If one degasser channel is obstructed/damaged, that could restrict flow). If the degasser channels are not the restriction then check the upstream low pressure solvent lines and pickup filters.
• If liquid is making it through all of the other parts without restriction and swapping low pressure inlet lines to the gradient valve does not restore flow to the suspect channel, then the specific valve channel may be defective (must replace the DCGV unit). Before condemning the channel, connect a syringe with IPA to the inlet side (set the pump to open the valve to 100% on) and try to gently flush IPA through it. Sometimes that will fix it if it is "stuck".

Thanks all

Dear William, Thanks for your reply. Regarding these points

*Connect everything back up and try to purge the two lines separately to waste (do as suggested in my earlier post).....done

*Confirm (by disconnecting and checking if needed) that when the pump is purging, liquid enters lines A and B solvent pickups and flows to the degasser.........OK

*Confirm (by disconnecting and checking if needed) that when the pump is purging, liquid enters lines A and B solvent pickups and flows to the degasser and to the two gradient valve channels........ Ok

*Confirm (by disconnecting and checking if needed) that when the pump is purging, liquid enters lines A and B solvent pickups and flows to the degasser, on the gradient valve and through the pump head out the prime purge valve..... Even the pump line is connected to the gradient valve.. the solvent does not flow from the gradient valve to the purge valve.. Sometimes flow slowly and backward and then stop

I will follow the next steps.

Thanks

"Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)"; https://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html