Instrument experiencing poor recoveries as some of the analyte molecules eluted as “breakthrough” peaks far ahead of their expected retention times.
1. Change starting gradient conditions from 5 or 10% methanol to 100% aqueous mobile phase. Figure 1 shows how the elution and shapes of polar opiates changed by this step.
Figure 1: Gradients start with 95% aqueous + 5% methanol
2. Ensure completed column equilibration with 100% aqueous mobile phase. Figures 2 and 3 show column pressure traces typical of insufficient and sufficient equilibration.
Figure 2: PFP column pressure trace showing insufficient equilibration
Figure 3: PFP column pressure trace showing sufficient equilibration
3. Change autosampler method by introducing an air gap between washes and samples.
4. Ensure that no residual Wash 2 remained in injection ports after washes. Excessive needle gaps during rinses with organic wash caused peak problems.