Chromatography Forum

I require help on over pressure issue + plus precipitate on the on line filter assay for a 2695 Waters alliance system.

We are extracting two antibiotics Rifampicin and Clindamycin from a source material.

We have three instrument failures in which the system has stopped on injection of a sample in which the pressure has hit 4000psi

Our mobile phase is 55% 20 mM sodium phosphate buffer, pH 6.3. 45 % Acetonitrile

Our chromatography conditions are

Column temperature: 25 C + 3 C
Flow rate: 1.0 mL/min
Injection volume: 20 µL
Detector: PDA or UV detection at 205 nm with suitable settings to maintain peaks on scale
Mobile phase: MPA – 20mM sodium phosphate buffer pH 6.3
MPB – acetonitrile
Autosampler tray temperature:
Refrigerated (4 °C +3 °C)
Run time: 20 minutes
Approximate
Retention time: Rifampicin: 5-6 minutes
Clindamycin: 8-9 minutes
Extraction solvent peaks: 9-10 minutes (minor) and 12-16 minutes (major)
System equilibration: Equilibrate system until suitable chromatography is achieved
Detector warm-up: Warm up detector according to the manufacturer requirements
Autosampler vials: Use amber HPLC autosampler vials
Mobile phase: Isocratic with 55%MPA and 45%MPB

Any help would be appreciated


Thanks

What solvent you use for extraction? I would recommend to change to a volatile buffer. Does your 20µl sample has enough time to get from 3 degrees celcius to 25°C? What particle size has your column?

Changing a buffer isnt an option as it is a validated method.
The extraction is 70% Chloroform/ 30 % Methanol

Nobody can tell if your pressure is normal or too high until you tell us what the column dimensions and particle size are!!!

Also, you say that the analytes are in a "source material" and that your filter is clogging. This can't be solved until we understand the nature of your "source material".

Sounds like your sample diluent(Chloroform/Methanol?) is precipitating the phosphate in the mobile phase, the sample is dropping out of solution in the mobile phase or the viscosity increase due to the mixing of the sample diluent and the mobile phase is increasing the back pressure above 4000 when your sample gets to the column/in-line filter.