High pressure in the HPLC system



for the quantification of amino acid, I am using a C18 column. Mobile phase is 40mM phosphate puffer pH 7.8 (channel A) and acetonitrile/ methanol/ water 45:45:10 (channel B). . The method worked really well, but recently I have problems with too high pressure in the HPLC system (Agilent 1260). I have disconnected my column from the system, and the pressure remained high. I would assume that I have salt precipitation in the system. Therefore, I have used warm water to flush the system without the coulumn. The pressure is still very high.



  • So we had this problem when we first starting using the 1260 systems too. We would add a standard column (100 x 3.0mm), and after less than 100 injections of solvent based material it would give us huge backpressures and stop the injections. We blamed the column manufacturer at first, so switched to another column from Agilent, and it still happened. We changed various tubing, heat exchangers, and frits for the solvent lines, all with only short-term success. 


    We eventually traced it back to needing to filter our mobile phase solutions. We now filter absolutly everything attached to a HPLC line using 0.2um nylon filters. We also added a pre-column filter (0.2 or 0.5um) and a guard column and now we have no pressure issues. We change the filters when we see poor peak shape, or increasing pressures. We also try to flush out the HPLC system every 6 months, there's a HPLC flushing solvent available from Agilent which we use, and I can send you on the protocol we use if you want.


    The ability to extract out the pressure profile for a batch was really helpful in being able to troublsehoot this issue for us. We now look at these profiles as part of our routine batch processing. This method was working for a long time on a 4000QTrap without the need for any filtering, using the same phases and column, and without any pressure issues, so I do consider the 1260 system fussy, but overall our 6470 LCMS is a fantastic instrument. 


    So, filter everything, and clean your HPLC before you try doing more injections, otherwise we only got temporary relief of the pressure buildup. Our systems were brand new when we experienced these issues. 


    Hope this helps!



  • I am interested in your protocol for flushing out the HPLC. 

    Thank you.

  • The steps below will intensively clean the HPLC tubing, degasser, pumps or the whole HPLC system if chemical or biological contamination is suspected.


    Reagents Required (for each HPLC unit to be cleaned)

    • 500mL of 100% Isopropanol (IPA)
    • Agilent HPLC flushing solvent (used directly from the bottle, Catalog No.G1969-85026, located in E311 safety cabinet 4)
    • 500mL of 18mOhm Water
    • Fresh mobile phase solutions (MPA and MPB, composition is method dependent)
    • Freshly prepared needle wash solutions (5% MPB, 100% Acetonitrile (MeCN))
    • 500mL of freshly prepared seal wash solution (10% IPA)


    NOTE: All solutions must be filtered using 0.2µm nylon membrane, except the HPLC flushing solvent which is supplied by Agilent ready to use.


    Materias Required

    • Union piece.
    • Long length of PEEK tubing, or column restrictor.


    Setup the HPLC Unit

    • Before you begin ensure the HPLC unit is setup to use the degasser unit, i.e, lines A1/A2, B1/B2 are connected to the degasser, note this is the default setup of the HPLC.
    • Remove the PEEK tubing from the MS, and place it into a waste container.
    • Remove the analytical column, and replace with a union piece conncected to a long length of PEEK tubing or to a column restrcitor.
    • Remove the inlet filters and bottle tops from every line.


    Cleaning the system

    • Place all lines into 100% IPA, ensure the lines are secured in place and will not slip out of the bottle, a retort stand and cable ties may help you achieve this.
    • Purge and condition A1, B1 when pathway 1 is selected (select pathway in VTC tab), then A2, B2 when pathway 2 is selected using a 50:50 composition to ensure both pumps are taking and delivering the same volume of IPA. Remember to disconnect and reconnect the column restrictor to the correct pathway in use.
    • Pump IPA through the seal wash line, and needle wash lines, S1, S2, and S3 by performing a seal wash, and injection needle washes respectively.
    • Pump 100% IPA through A1/B1 and pathway 1 for 30mins, at 1.0mL/min, then A2/B2 and pathway 2 for 30mins at 1.0mL/min. (Note: Column should be bypassed/removed for this).
    • If the backpressure is >800bar then drop the flow to 0.5mL/min.
    • After 30mins remove lines A1, A2, B1, B2 and place them into the bottle containing the HPLC flushing solvent solution.
    • Purge and condition lines A1, B1 using pathway 1, then A2, B2 using pathway 2 using a 50:50 composition to ensure both pumps are taking and delivering the same volume of HPLC flushing solvent.
    • Pump flushing solvent through A1,B1 and pathway 1 for 60 mins, at 1.0mL/min. Then pump flusing solvent through A2,B2 and pathway 2 for 60 mins (Note, Column should be bypassed or removed for this). Remember to disconnect and reconnect the column restrictor to the correct pathway in use.
    • Place all lines back into 100% IPA, purge and condition lines A1,B1, A2, B2 using a 50:50 composition to ensure both pumps are taking and delivering the same volume of IPA.
    • Pump 100% IPA through A1,B1 and pathway 1 for 30mins at 1.0mL/min. Repeat for lines A2,B2 and pathway 2.
    • Remove the seal wash line from the 100% IPA solution, re-attach the inlet filter and place the line into fresh 10% IPA solution, and perform a seal wash.
    • Replace the inlet filters on all other lines.
    • Place S1 into 5% MPB solution.
    • Place S2 into 100% MeCN solution, perform a needle wash for S1 and S2.
    • Place A1, B1 into MPA, MPB.
    • Place A2, B2 into 100% Water, and 100% MeCN.
    • Purge and condition lines A1, B1, A2, B2 using a 50:50 composition to ensure both pumps are taking and delivering the same volume of mobile phase.
    • Pump at 50% MPA/MPB at 1mL/min for 5mins without the column attached.
    • Re-attach the column, and equilibrate with starting conditions for the method being used.
    • Perform a system suit to determine if the clean has helped with any previous problems.


    Thank you!  I will give it a shot!

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