Chromatography Forum

I have been trying to get my HPLC system back up and running for a few months now. I have read through the forum and haven’t seem to found a solution. Any help will be appreciated.

System: Waters 2695 Seperations Module with Waters 2465 Electrochemical Detector

Trouble shooting steps taken:
- Tested injection (weighed vial, multiple injections, weighed vial and calculated efficiency) – 101%
- System Bandspread Test (99uL)
-Pressure in system is constant, flushed out system, free of air bubbles, adjusted seals, flow rate is fine
Electrochemical Detector:
- Dummy cell test, changed spacer, cleaned working and reference electrode


PROBLEMS:
Performance Qualification:
Acetone 20 µL/ml and Acenapthene 1.6mg/ml
1vial, 10 injection, 20 µL
Mobile Phase: 60 Acetonitirile : 40 Water
Flow rate: 1ml/min
Isocratic

See changing peak area

Dopamine Standards:
Dopamine: 1mg/ml
Mobile Phase: Sodium Acetate (50mM), Citric Acid (20mM), Sodium Octyl Sulfate (2mM), EDTA (100uM), Di-N-Butylamine (1mM)
Flow rate: 1ml/min
Isocratic

Peak shifting to longer RT (same protocol used in the past led to Dopamine consistently coming off at 5.5 minutes) , broad peaks

Re the retention time shift, look at both tR and t0 (if you can't see t0 on the ECD then run a standard that has two peaks). If the times change by the same percentage, you have a flow problem. If the percentage changes are different, you have a chemistry problem.

If it's chemistry, it had to be one of three things: temperature, solvent, or column.

If the times shift around at random, then the most likely problem is your solvent proportioning system; you can confirm by pre-mixing your solvents. If that doesn't make any difference, then temperature is the next suspect.

If the times shift gradually during the day, but are back to their original value the next morning (same jug of mobile phase) then it is almost certainly a temperature problem. If the times shift gradually and continuously but come back to their original value when you prepare fresh mobile phase, it's a solvent problem.

If they don't recover with fresh solvent, it's a column problem.