Decreasing retention time with increasing concentrations

[Rooke123]

1. as you are using hilic for retaining those quats, TFA (ion pairing agent) may not be really necessary. ion pairing agents are used when u have to retain polar compounds in reverse phase

2. if the quats are ionizable and as they are basic, there could be multiple retention mechanisms involved and that could be a reason for the tailing of your peaks

3. i would suggest to try 1% formic acid in water as aqueous mobile phase (ph will be around 2.5) , so that your analytes would be ionized to have good sensitivity in ms and silanol groups will be neutralized to reduce the tailing.

[bisnettrj2]

Thanks Rooke, still waiting for parts for a flow cell repair to bring the instrument back up to operating conditions. I plan on running a 3:1 acetonitrile:formate buffer once I get back online, but if that is a bust I'll go with just formic acid. Hopefully just formic acid works, as that will be a nice, simple solution. We'll see, and I'll post results when I have them.

[trobs]

Hi bisnettrj2

I spent most of last year doing an honours project developing an LC-MS method for paraquat and diquat.

I tried a lot of different mobile phases, but we got the best peak shape using 50mM ammonium formate with 2% formic acid (40%) and Acetonitrile (60%). It is a lot of formic acid but it gave by far the best peaks. We originally were using 50mM at pH 3.7 but the peaks really sharpened up with the increased acid, perhaps because it ionizes the remaining strongly acidic silanol groups. We were using a fused core bare silica column.

I am now at a different lab and am looking at bringing the method over to here with a few changes so I would be very interested to hear what works well for you. I wasn't completely happy with the method: it look a lot of equilibration to get reproducible retention times.

Stephen

[bisnettrj2]

That's what I stumbled upon - about 60:40 with about 50mM ammonium formate buffer gave good resolution and good peak shape. However, I accidentally murdered my HILIC column late last week, and it might be awhile before I get another one.

Did you see the same issue I was originally having on your fused core column - the decreasing retention times with increasing mass on-column? I wasn't able to re-reun the same injections with the buffered mobile phase before I killed my column, so I don't know if the buffer would have helped the situation or not. Your input might influence my purchase of either a fused-core or a fully porous silica column.

[Vlad Orlovsky]

Why invent the wheel

here are validated methods:
http://www.conffidence.eu/img/enewslett ... _PA135.pdf
http://www.crl-pesticides.eu/library/do ... CrlSrm.pdf

[trobs]

Hi bisnettrj2

To be honest analysing paraquat and diquat was really a pest. I can't remember seeing that severe a shift in retention time with increasing concentrations but certainly the retention times very not as stable as you would expect for almost anything else. We also had trouble with retention time shift if the column was not equilibrated for a very long time and even then we had some random shifts during runs.

If you want I can email you the chromatography development chapter of my thesis for you to flick through, probably not the greatest read but theres some things in there on the impact of buffer concentration, pH, ACN % etc.

I'd love to hear how your method ends up.

Stephen