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?