No detection/separation from 2695 and 2487

Hello all,

I have a Waters 2695 i'm using in connection with a 2487 and Empower 2. It was purchased second-hand and as far as i understand the 2695 was purchased in a non working condition; this happened before I was hired, and there are no notes which came from the previous employee.  I am using the USP method for Caffeine [Mobile phase: NaAc/H2O/MeCN/THF - 1.64g/2L/50 mL/40 mL/ pH 4.5 with AcOH; Diluent H2O; C18 column at 1mL/min; 275nm; 15 min runs; 10uL injection volume; ambient temperature] and testing both USP standards (hplc ~99%) and products the company I work for makes as samples. My issue is that I have been unable to produce ANY peaks with the machine (caffeine or otherwise).

As a little background: The machine does not appear to have any leaks, and it appears to hold pressure adequately. I replaced the in-line filter, and the check valves in the pumps. I suspected a bad valve somewhere in the sample delivery system, so I replaced valves 1 and 2 from another 2695 that we have. I have flushed the flow cell of the detector with 6M phosphoric acid and washed with RO water until basic again to clean the flow cell. I have used another machine with the same 2487 which did produce the expected peaks, so I know it works. I have tried sample volume sizes ranging from 10uL to 250uL. I am seeing a large drop in pressure upon injection (anywhere from 1000-1500psi). I have flushed the machine with 100% methanol for several hours to remove any air bubbles, and I wet primed the machine for 20 min. I also have performed 200 injections using methanol as a mobile phase and methanol as the sample diluent to try and remove any air bubbles that might be in the sample injection path. 

I have also added some links which are screen shots from empower which I thought might be insightful.

The first is the detector signal:

The second is the pressure during the run:

The third is zoomed in on the initial pressure, as you can see it has a pretty severe drop:


  • Hi Justin,

    The pressure drop that you see in the chromatogram could mean that you still have some air in the injector somewhere (or could also simply be an injection artifact if the sample diluent and MP are different compositions.)  Have you tried performing an injector Compression Check? 

    250uL injection is pretty big for an Alliance system.  Do you know what size sample loop is installed (yellow coated tubing is 100uL loop which is standard).


  • justinW
    I tried a compression check this morning and the machine failed. What would be the remedy to this? 
    If I do still have air in the injector, what would be the best way to remove it? 
    I do not think it is a problem between diluent and mobile phase, as our other 2690 (which is working) does not see the same drop in pressure (it has some drop in pressure but it is much less). 

    I have added a picture of our sample loop so you can see for yourself. Our other 2695 has the same yellow tubing but it is maybe 1/2 the size so i'm not sure which is which.
  • Compression checks can fail because of leaky syringes, solenoid valves, high pressure seal packs. Syringe leaks are easy enough to spot. Only bad seal pack leaks are easy to spot because they will result in wet vial tops. Solenoids can leak into the 2695 body or, more commonly, into their drain lines making them either very easy or very hard to spot. Removing the waste line from the solenoid makes it easy to spot a leak, if you know when to look.
  • justinW
    Thanks Dan! 

    The tops of the vials are not wet, so we could rule out a leaky seal pack (hopefully). I do not think the syringe is leaking (no wet spots around the area). I cannot find any visible leaks anywhere in the machine. If there were a leak in the body of the machine, how could I go about spotting it? Are there panels which are easier to remove, or parts I could move while keeping the machine running? If a solenoid were leaking into its drain-line, how would I be able to make that determination? If I do remove the drain line, when is the most optimal time to watch for a leak? 
  • The 2695 display shows the status of 4 different valves. Valves 3 and 4 are solenoids that are quite visible once the right-hand panel is removed (2 screws once the column heater is lifted off its pegs, assuming you have one). When those valves are closed, you can pull the drain side and see if anything is passing through them.
    Also, you have to actually watch the syringe cycle to make sure it isn't pulling in any air...
  • MarcNoble
    edited June 2019

    I'd suggest replacing the syringe before worrying about the valves.  Much easier repair and a wearable part that should be replaced at least every year especially if the instrument is rarely used.  Check out these articles from the Knowledge base:

  • justinW
    I closed valves 3 and 4, and tried pulling on the drain side. Nothing came out of either one of them and my syringe (plastic syringe - not part of the machine) produced a small vacuum so I'm assuming they're both good. I have watched the syringe cycle many times and I do not see any leaks or air bubbles being drawn in the syringe. 
    One thing I failed to mention in my initial post: I had filled a vial and weighed the vial before doing some injections, and after doing several 10uL injections I weighed the vial again. The vial had the appropriate weight removed from it according to the number of injections, so I'm fairly certain that the sample is being drawn up from the needle. 

    Any other ideas on what could be the culprit? I'm still seeing a large drop in pressure upon injection and producing no separation according to my detector. Could I have a blockage somewhere? 
  • This really sounds like a Valve 1 issues (the "inject valve").  To test, it with flow, go to the valve/motors page in diagnostics.  Open valve 3 then 2 then close valve 1.  Drive the needle to vial position (with no vial in the carousel carrier).  You should not see a significant pressure drop.  You should also not have any solvent flowing down the outside of the needle - that would indicate a seal pack problem - or out of the needle port.  A significant pressure drop or solvent flowing from the needle indicate a valve 1 failure.

    This is a good test but passing results don't necessarily mean that the valve is in perfect working order.

    Good luck!