[meo.dota]

Hi all,

I hope someone may have an idea as to what can be done to make this happen.

I am tasked with developing a new method to identify above mentioned substance which is used in agriculture to suppress branching and unproductive plant growth.
The method used now is cumbersome, involves a soap in the mobile phase and it takes too long as many samples have to be run.
I have several literature methods however replicating them is difficult as I do not have the HPLC columns used there.
My column is an Agilent Zorbax Extend C-18 4.6x150mm with 5um particles size.
I am using either water/acn 95:5 or 90:10 or 93:7 with 0.05 - 0.3% H3PO4. My problem is the peak comes out nicely in all mobile phases yet always either within or really close to the solvent front making integration and therefore quantification difficult.
Right now I am trying a 10:90 water/acn mix with 0.1% H3PO4 but again it looks like the peak is right at the solvent front.
Any suggestion is appreciated.

[discodermolide]

Hi all,

I hope someone may have an idea as to what can be done to make this happen.

I am tasked with developing a new method to identify above mentioned substance which is used in agriculture to suppress branching and unproductive plant growth.
The method used now is cumbersome, involves a soap in the mobile phase and it takes too long as many samples have to be run.
I have several literature methods however replicating them is difficult as I do not have the HPLC columns used there.
My column is an Agilent Zorbax Extend C-18 4.6x150mm with 5um particles size.
I am using either water/acn 95:5 or 90:10 or 93:7 with 0.05 - 0.3% H3PO4. My problem is the peak comes out nicely in all mobile phases yet always either within or really close to the solvent front making integration and therefore quantification difficult.
Right now I am trying a 10:90 water/acn mix with 0.1% H3PO4 but again it looks like the peak is right at the solvent front.
Any suggestion is appreciated.

Why not use normal phase? In a methanol/acetonitrile mixture. This compound (in terms of polarity) may just be at the junction of reverse vs. Normal phase silica for adequate separation.
Try out a few TLC's before.

[meo.dota]

Thanks for the suggestion however I have to use what I have, ie no new columns etc.
The published methods all use the earlier mentioned buffer systems yet different columns than I have.
Also the pKa of this compound is ~5.2-5.3, ie similar to phenols in terms of acidity which is why people usually add H3PO4 to a pH of about 4.3 (or up to 0.3%) to the mobile phase to keep it in the in its form with both protons attached to the nitrogens.

[Arkcon]

So, using your current system and materials, you'd like the peak to come out later.  Have you tried reducing the amount of solvent?  At some point, reducing solvent will widen the peak, which makes the system less ideal, but do go ahead and try a slightly lower concentration of ACN.  Also try switching to methanol, if an ingredient switch that is allowed.  Methanol has less eluting power than ACN.  Are you allowed to change the gradient?  If so, a brief hold at very low solvent, to allow the chromatogram to build some baseline before your program switches to another concentration may help.  You're at the very low end of useful solvent concentration 'tho. 

You really should give discodermolide:'s idea some more consideration.  If the peak comes out very close to the solvent front, you're not really giving the column a chance to separate anything.  Maybe you just need to quantitate, but consider ... is it accurate to analyze a peak that may be part something else?