HPLC Agilent 1260 Injector Program


I am trying to run a sample injector program on an HPLC Agilent 1260. I am having difficulty with the beginning of the chromatograms not looking good. I do not have much experience with running injector programs, so I am unsure if a mixing issue is occurring or if something else is incorrect in the injector program. We have installed a 500 uL loop in the seat. We are trying to inject 20 uL of sample followed by 100 uL of diluent. This is repeated 3 times and then an additional 30 uL of sample is injected for a total of 380 uL. The injections look good if we do an injection without using the sampler program. For context, mobile phase is 0.1% phosphoric acid in H20 (A) and  0.1% phosphoric acid in ACN (B). The sample is in ethanol, and the diluent is water. We have also tried switching to diluents more similar to the composition of the mobile phase, but it did not help the issue at the beginning of the chromatogram. The gradient is the following:

The injector program looks like this:

The beginning of the chromatograms look like this:

Would someone be able to tell me what is causing this issue?

  • Hi  

    Not really an answer to your question, but more of a comment on the injector program.  If you have a loop of 500uL in the needle seat and you're pushing 300uL water at maximum speed into the loop, I wonder if you will lose some of your analyte as it may pass all of the way through the loop.

    I wonder if you're introducing air bubbles into the loop with all of the steps.  The pressure trace might show if this is the case.

    It would be good to see the chromatogram without the injector program and to know what wavelength you are monitoring.


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