Hi all, I am running reversed phase HPLC using a very shallow 60 min gradient from 10%-30%B about 5min of 95%B and a 10min re equilibration. My mobile phase is a 01.% formic acid/ACN, and recently I have explored basic buffer (5mM ammonium acetate pH 8.0/ACN), my detector is UV-Vis. To start my worklist I have a mobile phase blank acidic buffer which is blank, than I have my sample which shows my peak of interest, immediately after I reinject the mobile phase blank (acidic), which is blank.

The problem now comes when I have an equilibrate condition, my next immediate injection is a mobile phase blank (basic mobile phase), I have a huge signal at the RT of my compound, than I run the same injection same vial mobile phase blank (basic) and it is truly blank this time, no signal. I then run my sample and see signal again as expected, and to finish the worklist I run the same vial mobile phase blank (basic) which is blank.

Can anybody explain this phenomena, how is it possible in a staggered sample set, where a blank is run (acidic) shows blank, than you switch to basic mode, immediately run a blank (see the drug in it), than run some samples, than reinjection of the same blank to see nothing there.

I am using a guard column.

Could it be carryover from a previous injection, even though the sample directly before was the (acidic mobile phase blank) which was blank? It seems the species was somehow retained because now it is eluting in the first blank sample injection using basic buffer system. Or could it be air that was in the needle?

My injections at 20uL using a flow rate of 0.35mL/min, column dimensions are 3um , 2.0x150mm C18, reversed phase gradient is setup.

Thanks for your assistance.