Overlapped Injection with strange results

I am developing a method that uses an injector program to do automatic standard additions. This part of the method works fine. The typical retention time for my analyte is about 2 minutes without overlapped injection. However, I am having trouble getting overlapped injections to work. I have tried setting the timing several ways: 1) autosampler program finishes before the previous run is complete, 2) autosampler program finishes at same time as previous run, and 3) autosampler program finishes after the previous run is complete. In all cases, the retention time for my target analyte is 0.5 min. I don't understand why this is happening. The only thing that makes any sense is that maybe the high ACN part of the gradient is happening during injection. This should not be the case, though, since there is a re-equilibration period at the end of the run.

Maybe I'm missing something about how overlapped injection works. Does it inject immediately after the autosampler program finishes, or does it wait until the previous run is complete? I can't find much documentation on this feature.

The column is C18, 1.8um, 2.1 mm x 50 mm. My gradient is as follows:

Time     %B

0            0

3            75

3.01        0

5             75

My autosampler program for the spiked sample:

Draw 4uL from sample with 100uL/min using default offset

Wash needle in flushport for 3 sec

Draw 8uL from P2-A-2 with 100uL/min using default offset

Wash needle in flushport for 3 sec

Mix 12uL from air with 200uL/min for 5 times

  • Sounds to me like the overlapped injection is starting when there is high AcN proportion in the sample loop.  Is this on a 1260 or 1290?  Can you tell what time the pump pressure returns to the starting pressure?  It's at this point I would look to start the overlapped injection, since this is when you can guarantee that the loop contains starting mobile phase percentage.  It really sounds to me like the injector program is starting when too much ACN is in the sample loop.


  • What is your HPLC flow rate and what are your overlapped injection settings?

  • We are using a 1260. I think you are correct that the problem is that the loop still has high ACN mobile phase when overlapped injection starts.

    I'm wondering if either using "Injection Valve to Bypass for Delay Volume Reduction" or performing an "Eject" at the beginning of the Sampler Pretreatment program would solve the issue. We are trying to increase throughput, so waiting until the gradient gets back to initial conditions is not a great solution. We want to be injecting at that point.

  • It sounds like in the sample loop, there is a higher concentration of ACN than in the starting method parameters. If you have no problems with the sample sitting in the needle and the sample loop for a longer period of time, you can just enable overlapped injection with a flush out factor of 5.0, which will be 5 x(injection volume (12) + seat capillary (depends on which seat) + valve volume (depends on which valve)) and depending on the volume between the point of mixing and the sampler (if this volume is larger than the flush out factor volume), then the sample loop should still have the starting conditions of the gradient. This is all dependent on what types of configuration that you have for the system.

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