I performed three experiments, using different wash solvents, to check for carryover with the following setup.
Mobile phase A - 60:40:0.05, MeOH, H2O, HOAc
Mobile phase B (Wash) - Exp #1-100 % MeOH, Exp #2-50:50, MeOH:ACN, Exp #3 - 100% ACN.
Pump profile: 100 % A, 0 - 7.0 min (analysis)
100 % B, 7.01 - 17.0 min (wash)
100 % A, 17.01 - 60.0 min (equilibrate)
Inj Vol - 100uL, Flow - 0.75 mL/min, Column - Discovery HS F5-3, 150mm x 4.6mm, 3u, Col temp - 35C
Detection - 265 nm
Injection order is 2 blanks, 1 LOQ, 3 Stds, Check Std, 4 Samples, 3 Stds (different vial).

For Exp 1 the last 3 Stds are comparable to the first 3 Stds profile. However, for Exp 2 & 3 I observe a large peak which elutes @ about 4 minutes in a Std injected after the samples. It elutes in the first Std after the samples for Exp2 (MeOH:ACN wash), but the third Std after the samples for Exp3 (ACN wash). In addition, the Std peak area of this injection is significantly reduced when compared to the other 2 Stds injected after the samples.

I struggle to believe it is carryover given the wash/equilibration times for the study, and why would the Std peak area be reduced? Help....

thanks,