Analytes that coelute

[pmflanders]

I'm working with 1,4-benzoquinone and the herbicides chloramben, aminopyralid, and clopyralid. I'm having problems with them eluting too close to the dead time and at the same time. I've tried a bondapak C18, an Atlantis T3, and u bondapak phenyl 25cm columns with 0.24% acetic acid and methanol as the mobile phase. I have the same problem with any ratio of buffer to methanol in isocratic mode (even down to 1% methanol) and any gradients I've tried. The herbicides are carboxylic acids, so I used acetic acid to try to keep them neutrally charged.

This instrument is UV-HPLC, but I also use LC/MS, so I tend to stick with volatile buffers.

Are there any other tricks that I've missed?

[tom jupille]

How about ion-pair chromatography using tetrabutylammonium ?
or one of the mixed-mode columns ?

[Gerhard Kratz]

Just a little google and you will find several applications for herbizides on HILIC columns.

[Vlad Orlovsky]

This is a very good case for mixed-mode chromatography where you can adjust retention by changing 3 variables: amount of organic, buffer concentration and buffer pH. This approach is clearly shown in this graph:
http://www.sielc.com/Technology_2D_Properties.html

and here are few examples for separation of closely related isomers:
http://www.sielc.com/Compound-O-Toluidine.html
http://www.sielc.com/Compound-2-Aminobenzoic-Acid.html
http://www.sielc.com/Compound-2-4-Dihyd ... -Acid.html

if you can provide me with sample we will develop a method for your free of charge (usually takes us couple of hours)

[Camisotro]

I'm working with 1,4-benzoquinone and the herbicides chloramben, aminopyralid, and clopyralid. I'm having problems with them eluting too close to the dead time and at the same time. I've tried a bondapak C18, an Atlantis T3, and u bondapak phenyl 25cm columns with 0.24% acetic acid and methanol as the mobile phase. I have the same problem with any ratio of buffer to methanol in isocratic mode (even down to 1% methanol) and any gradients I've tried. The herbicides are carboxylic acids, so I used acetic acid to try to keep them neutrally charged.

This instrument is UV-HPLC, but I also use LC/MS, so I tend to stick with volatile buffers.

Are there any other tricks that I've missed?

Looking at the structures of these herbicides - what form do these take when neutrally charged? Chloramben and Aminopyralid both have a free primary amine group (quite basic) and a carboxylic acid (quite acidic). Is it reasonable to assume they are both zwitterions? Possibly Clopyralid as well.

Could this account for why they just refuse to retain on a C18 column even under conditions when they should be neutral molecules? This all points to the conclusion that a different retention mechanism on a different column would help you out here. Your analytes are inescapably polar, so you should be taking advantage of that.

It's also worth investigating if other mobile phase additives would have better ion-pairing effects, to help with retention here.

[Bintang]

We do not have any application notes for these compounds but for some fluorinated compounds and amino acids that may give som ideas on where to start.

http://www.sequant.com/files/documents/ ... _Dimer.pdf

http://www.sequant.com/files/documents/ ... _Dimer.pdf

http://www.sequant.com/files/documents/ ... lanine.pdf