I am experiencing ghost peaks in reverse phase lc applications which I believe are due to potential solvent mixing/outgassing issues during an aggressive fast gradient.
The current gradient I am running is a 100% aqueous buffer solution for 0.5 minutes post injection following by an aggressive ramp to 44.5 % acetonitrile-based buffer at 0.51 min for a 3.5 min hold followed by a ramp to 100% acetonitrile-based buffer over 1 min with a 5 min hold for column cleaning. UV detection is done at 260 nm w/ a bandwidth of 4 nm with a reference wavelength of 360 w/ a bandwidth of 80 nm
Following a 30 min purge at 5 mL/min of all buffer lines (to ensure purging of vacuum degasser as per instrument manual) I have completed multiple system blanks with the following results:
- A run of the normal method resulting in 2 peaks with retention times 3.232 and 3.979, areas 69.4 and 39.4, and heights 5.2 and 1.7 respectively
- A run with the initial buffer b ramp increased to 60 % resulting in 2 peaks with retention times 3.073 and 3.635, areas 56 and 50.4, and heights 5.2 and 2.9 respectively
- A run with the initial buffer b ramp increased to 75 % resulting in 2 peaks with retention times 2.977 and 3.372, areas 38.5 and 38.5, and heights 4.4 and 3.2 respectively
- A run with the column bypassed via capillary tubing with only one NEGATIVE peak at time 1.361, area 26.3, and height 2.2
In summary, runs with the column resulted in a larger second peak as the percentage of the initial increase in acetonitrile-based buffer also increased, leading me to believe that the peaks are due to the aggressive mixing during the protocol.
Peak shapes when the column is attached are consistent with the literature regarding outgassing issues with 2 "shark-fin peak shapes" where the leading edge of the peak is quite sharp with a linear decline back to baseline (although these peaks never return to baseline).
Unfortunately these peaks are not consistent whatsoever across runs so a simple blank subtraction will not suffice for this particular method
I was hoping to get some suggestions as to ways to confirm the proper function of the on-line degasser, add back pressure post column to prevent outgassing if that is in fact the issue, or any other methods that have worked for eliminating ghost peaks in the past.
