Hi, There are three probable explanations I can think of if the problem is with the column, salt build up, blocked frit, or a collapsed bed.
But first, check the backpressure of the system with no column on at all, if some of the tubing is damaged or partially blocked you will see a high pressure anyway and the problem isn't the column. If this is the case, try disassebling and reassembling the flow path piece by piece. Often it just clears for no good reason, or you can find the point where the blockage is. If that doesn't work, go back to the column.. If you are using any salt in your separation gradient (or if someone ran size exclusion methods before you) you can sometimes cause it to crash out if you don't flush the column and system out before putting high concentrations of organic phase through. We used to have some success running it backwards at a low flow rate with heated water as the phase (only if your column will survive being in water though - check first). Running it backwards at a fairly low flow rate as your friends recommended can also help with a blocked frit if it isn't too bad. Otherwise, you might have the change the frit itself (although this seems unlikely to be the problem if you filter your samples as you say). If you are very unlucky and the column has had a nasty shock or been packed down by really high pressure, you may have damaged the bed and need a new column. But don't panic, if you use a system and column a lot you can get these odd changes sometimes, especially if it is a shared system used for lots of different methods. So.. check the backpressure without a column, disconnect and reconnect all the flow path, flush out the system with no column on, flush the column backwards, and finally replace the frit (once you have opened a column up to check it, you might as well change it anyway). Good luck! On Mon, Jan 17, 2011 at 5:37 PM, Matthew Connelly <[email protected]>wrote: > > ------------------------------------------- > *From:* [email protected] on behalf of yoginee budhkar[ > SMTP:[email protected] <smtp%[email protected]>] > *Sent:* Monday, January 17, 2011 5:21:35 PM > *To:* [email protected] > *Subject:* HPLC column back pressure > *Auto forwarded by a Rule* > > Hi all, > I am using Waters silica C18 column for my analyses. It recently started > showing a much higher pressure for ACN at 1.0ml/min, around 100 as compared > to normal which is 45. HPLC profiles are not affected though, I am getting > good peaks. > > Some colleagues suggested that I give it a wash while it is fixed upside > down at a higher flow rate. When I did that, it showed me a back pressure > of > 50 at 1.0ml/min. But when I flipped it back to the right orientation, it > still showed me a back pressure of 100 at 1.0ml/min flow rate.... > > Any ideas what could be going on? I am very careful with my column and > don't > ever leave its ferules open, so there is very thin chance of something > going > in. My samples of injection are also always filtered thru a 0.22 micron > filter. > > > Hope to get some answers > > -- > -- > Yoginee Budhkar > <#> > <#> > <#> <#> > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
