LC-UV backpressure

[irene_wu]

Hi, guys:
I hope somebody could help me to solve the backpressure problem:
I am using HPLC (hp1100 model),column aglient zorbax CN 250mmx4.6mmx5um
Mobile Phase:
buffer: 1.4mL of triethylamine in 1L of distilled water. Adjust pH to 2.5 using phosphoric acid.
organic: Acetonitrile
gradient:from 0to 37mins buffer from75 to 0 % then back total time 50mins
Flowrate1ml/min ambient to analyze reaction mixture which including fmoc-argine-oH etc.
Normally the back pressure should be 143bar, but now goes up to 175.now is 280(retention time keep no changing.)
I use IPA to wash column(instruments too)4hr,cleaning pump,but pressure contains the same, changing instrument to shimadzu, it is same and continue going up. Is there some way to solve it .
thanks

[WK]

Why do you wash with IPA and not just acetonitrile?
IPA and water mixing causes large backpressure.

Have you been running OK for a long time before this problem? If so it may be time to replace the column.

WK

[irene_wu]

wk:
thank you for your replying.
the reason which i use IPA is I have already tried 50/60 water /acn ,100%acn to wash, but those tryings did not work well, and even got worse. the column is very old, may be 3years.like you said it could be time to replace the column.

[Judy-Vancouver]

Have you checked the pH range of Zorbax CN column? Usally, it will be 3~8 pH. Hence, pH 2.5 eluant will solve the column packing material. It could cause high backpressure.

[JA]

excuse my elementary suggestion, but the column has been cleaned in a reverse direction? Unless there is a recommendation by the manufacturer not to do so, this is the first thing I try. I can only think of inlet frit blockage or adsorption/absorption onto the stationary phase to cause high backpressure (from the column only). Frit blockage leads to band broadening which should be evidenced by either a loss of efficiency or a change in analyte asymmetry (Anyone know which parameter is more sensitive to this problem?). Also, silica is not sensitive to low pH; the problem is bonded phase hydrolysis which would be be demonstrated by a loss in analyte retention (normalised by the measurement of k if you have this information over the lifetime of an application on a particular column). From a mechanical point of view I'm not certain if this would increase backpressure (Anyone?).

Maybe some interferences in the sample are irreversibly adsorbed onto the silica as the bonded phase is being torn off by the agressive MP. Followed the manufacturers recommended wash procedure for the cyano column?

There's an article on column regeneration in the LCGC archive under "Column Watch".

[irene_wu]

hi, JA & judy:
thank you giving me more informations .and they are much helpful.
I tried all the ways to clean column. but they all did not work.
excuse me for not telling you I used the guard column to protect the column. and now problem is solved which is there is blockage in guard column. because I use a replace column to test , I found the problem was still there. I took out the guard column and test the backpressure . both columns have same low pressure around 34bar for acn. then I changed new guard columns. the total pressure(guard& column)keeps around 38bars for acn.(with the old guard column back pressure is 251bars).

[JA]

positive outcome nonetheless