washed the column in the reverse direction also but the same problem persists. when flow rate is reduced to 0.8mL/min from 1 ml/min...the pressure reduced to 2800 psi apprx.
what more can be done to comprehend the problem?
When you change the run conditions, you cannot expect the same result. The higher temperature will reduce the viscosity of the mobile phase and lower the back pressure, retention will get shorter and the elution order may change.
Change a column to a connector and check the pressure of the system without a column.
Regards
G
Why are you using such a high concentration of OPA(1000 ppm) in your mobile phase? What are your chromatographic conditions?
From experience, using such concentrations of OPA would usually result in high back pressures. Usually, I would prefer an acetate buffer system to phosphate buffer. If the method is already validated, you can change the buffer composition to sodium acetate/acetic acid and revalidate.
Lowering the OPA concentration should help if using mentioned concentration is not there for a reason. Also replacing acetonitrile with methanol should help.
I have tried without acid, using water methanol but same problem persists.The chromatographic conditions permits to use 0.1% OPA but lower conc. can also be tried. ACN is a better option but resolution get compromised. MeOH shows better separation properties with sample. Emmanuel Orman Bruce Neagle Navid J. Ayon
Grzegorz Boczkaj ....having done....the inline filter was also cleaned. pressure was normal below 100 psi. washed the column in the reverse direction disconnecting from detector with 40 % MeOH/water. but still problem persists. But at 0.8% flow rate...pressure declines and rum smoothly.
Any suggestions from you?
So, what is the pressure of the system without a column for your standard flowrate?
regards,
GB
If you tried without OPA and still have issue, the problem is not from OPA. Remember that the viscosity of water/methanol mixture is not constant as % methanol changes from 0 to 100%. As I remember, the maximum viscosity is about 40-50% methanol. You can test by monitoring back pressure (with column) when run a gradient between 0 to 100% methanol for 15 min. You will notice that the pressure will increase at the beginning (100% water) , max out at the middle and will start to drop when it is getting close to 100% methanol. This is normal.
Thank you very much sir for your suggestion. I have tried this too and found the same i.e maximum pressure at 40-50 % MeOH/water. So raised the column temp and run the method, the pressure dropped (obviously).
My next issue I am not able to repeat my method at previously set conditions.I dont know why? @Narong
When you change the run conditions, you cannot expect the same result. The higher temperature will reduce the viscosity of the mobile phase and lower the back pressure, retention will get shorter and the elution order may change.
Also, you should be able to extra-polate the vulnerability of your analyte to switching the elution profile..in which case an external/internal standard would help to validate.
As mentioned previosly.. slowing down the flow-rate (at risk of broadening your peaks) and also increasing the temperature (at risk of co-elution, if their retention time is too close) can work..or maybe just a bit of both? (totally depent on the chromatographic profile of your analytes). Good luck!
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