Chromatography Forum

Hello everyone!

I have a problem with analysis of o-phthaldialdehyde derivatives of sphingolipids. I use a C18 column, mobile phase composition is acetonitril/water (9:1). In each sample (or standard) there are late eluting impurities, which form a 30-minute wide "peak". To elute it faster i tried a 5 min gradient to 100% isopropanol, then the column is flushed with it for 10 minutes. It helped a lot, now I need to flush the column for only 20 minutes (compared to 45 before) before the injection of the next sample. I also tried 100% acetonitril, but it didn't help at all. Everything works fine now, but I wonder if it's save for the column in the long term. What's Your opinion?

It's absolutely safe, assuming you do not have huge backpressures during the flush with isopropanol...

The pressure increases from 30 (acetonitril/water) to about 160 atm on pure isopropanol. It changes gradually over 5 minutes, so I think it's acceptable.

i was having a lot of problems getting the phospholipids off a reverse phase HPLC column. If I used pure acetonitrile, the lysopholipids came off very quick, but the phospholipids eluted as a very broad featureless lump over a 10 minute period.

If I used increasing amounts of methanol with the acetonitrile, the peaks eluted very nicely. Temperature also speeded up their elution. Also after peaks of intereste eluted, I tripled the flow at 100% organic to speed up their elution.

Just had to be careful coming back down, because a mixture of 50/50 methanol/water gave the highest column backpressure, so usually reduced flow to twice the normal flow. After getting to the initial gradient conditions, jumped flow back to the normal.

Worked well and injected several 100's of plasma samples without any problems..

see

http://users.chartertn.net/slittle/default.htm

section on matrix effects..

Thanks for help guys.

what about using THF to do the washing? THF may clean the column quicker without changing the flow and pressure. Onle guess