Chromatography Forum

Ive had a high pressure issue for over 2 months now with this method im using (waters explosive method). Ive tryed everything and the problem still isnt resolved! I suspect its particles clogging the column from the mobile phase even though i buy 0.2um filtered mobile phase i also filter the mobile phase inhouse and have a guard column and the column still clogs up. We have gone through 3 columns now and the same issue occurs. My filtering technique is good and i have also used a wide range of filters with not much sucess (filters recommended by waters). I have used 0.2um RC,GHP, PTFE and the same pressure still occurs which i find ridiclous. I have had also waters technican pull apart the UPLC and do a full PM but the pressure issue still continues. All my solvents are hplc grade or better. The glass wear i use has only ever been used for this purpose only (uplc). Yesterday i tryed a new column again and same thing happened. I filtered the Methanol with PTFE and it all apeared good when running 100% through the column then i added the water in a gradient (intial gradient of method is 69:31 water/methanol) and the pressure rose from 5000psi to 10000psi and stayed stable for 1 hour and then the pressure starts to rise again!.I stop the flow and start the flow again and i cant even get 0.100ml/min without a psi of 8000psi! I backed flushed the column today and put the column back on the system and managed to get improvement with a psi of 13500psi at my expected flow rate of 0.5ml/min so thats not bad....but its slowly building again very slowly.

My conditions are below

C18 column: uplc BEH C18, 2.1x100mm, 1.7um

flow rate: 0.5ml/min

gradient

time %A %B

intial 69 31

4 69 31

9 40 60

10 40 60

12 69 31


Mobile A: water

Mobile B : Methanol

I have also used a variety of suppliers of my solvents. Im about to give up on this cause i cant see anywhere else i can improve or change!...any advice!

Are you buying your water? Have you tried premixing your mobile phase and then running through a 0.22-micron GHP or hydophilic PTFE filter, then running isocratic while bypassing your mixer and degasser? Have you tried running as usual while bypassing the degasser (if it has one)? This issue was fixed previously by replacing a cable (?) previously, then re-occurred after a service engineer made a repair. I think the wear on the system (as indicated by the engineer and the need for repairs on the system) and its 6 months of idle time might be an indicator of a chemical contamination in the system (even though you have attempted to clean the system prior to use).

http://chromforum.org/viewtopic.php?f=1&t=20672

http://chromforum.org/viewtopic.php?f=1&t=20767

Are you using glass bottles for the water? I've heard from a Merck technician that water, especially ultrapure water might leach silicic acid out of glass which later might precipitate in the HPLC and clog columns with small particle sizes. Commercial HPLC water is usually sold in glass bottles, so you might have an issue there. Maybe you can switch to metal containers and/or use some sort of plastic bottles in the lab. Have to add that I've never had an occurence of this problem (at least I think so ).

BTW I don't think it's a good idea to backflush UHPLC columns. They usually have smaller pore sized inlet frits, so by reversing them you might flush some of the stationary phase out...

Temperature?
Methanol/water is very viscous.
Flow 0.5ml/min is too much for BEH С18 2.1x100mm 1.7mkm

How much pressure do you have with ACN/MeOH:75/25 0.5 ml/min T=40C ?