Normal Phase Problem

[mant0]

Good afternoon!

Please bear in mind my utter newbie-ness as I ask my question. My problem pertains to the running of oligomannose 5 standard on a tsk amide-80 column. I am using a dionex u3000 and visualization via fluorescence as the mannose is AB labeled. I am using acetonitrile as my eluent A and 50 mM ammonium formate as my B eluent (this is the manufacturers rec and i have also seen this used in some papers). The problem that i am having is that, my sample starts to come off at approx 4 minutes regardless of whether im running a 35% B - 53 % B gradient or a broad 0%-100% B gradient. It would seem like my analyte isnt even interacting with the column and coming right off. The expected time for a column volume at my flowrate should be slightly over 6 minutes. Samples have been injected neat as well as diluted 1:9 in 80% ACN/water.

Thanks in advance,

-mant0

[unmgvar]

what are the dimension of your column and what flow rate do you use?

from there it is possible as well to calculate and confirm if your compound is coming off the column unretained or not.

might be a problem of equilibration of the column at first glance, or maybe your proportionning system is off. what solvents are on lines C and D?
is your pump a low pressure or high pressure type?

[mant0]

column dimensions are 2.0 X 150 mm and my flow rate is 0.075. Based on the time for one CV, it would appear that my analyte is coming off unretained.

[unmgvar]

you are correct. your compound looks like it is coming out unretained.

what type of instrument do you have? a 2 mm ID column requires low dwell volumes. your are in the micro column range and your flow rate is low. is your equipment adapted to such an aplication? if you have a standard analitical LPG U3000 then you are out of the specs of the instrument by far.
otherwise i would look at these:

if your vendor claims that the method should work then get a new column and try it out otherwise you need to change chemistry related parameters.

look at the structural chemistry of your compound
then see if changing the mobile phase will help
or you need to look for a different column chemistry

in order to show that the instrument is not faulty try this it should confirm it:
do what is called a gradient test. ask your dionex rep, for their procedure, do it for all 4 channels.
if the procedure passes then your instrument is fine the problem is chemistry

[Uwe Neue]

Are you sure that the original method was done on AB labelled standards and not on an underivatized oligomannose?

[mant0]

thanks for the awesome responses unmgvar and uwe!
To answer your questions, I am just trying to reproduce the manufacturers CoA chromatographic trace using their standard but a different column, which i have read to be equivalent in papers. The manufacturers believed the problem was in how i made my buffer but overall, i'm still getting inconsistent results. I managed to retain my sample by diluting it in 80% ACN and running a 0-100% formate gradient. However, after remaking the buffer to how the manufacturer recommended, the stuff isnt sticking again. Maybe longer equilibration time?

The LC is working properly as i performed a flow and graident test earlier in teh week

[mant0]

Problem solved! It turned out that it works (after i diluted my samples in 80% ACN/20% water) but the re-equilibration time for the method wasnt long enough which caused it to come off in the flowthrough one run and be retained the next.