Chromatography Forum

I'm developing a method to separate 2,4-D esters by LC-PDA. I'm having difficulty separating the 2,4-D, butyl ester from the 2,4-D, butoxyethyl ester using reverse phase chromatography. These compounds have similar pKa and retention on a C-18 column. I've tried a slowing down the flow rates, gradients and adjusting the mobile phase pH on a C-18 column. I've changed to a smaller particle size column (from 5 to 3.5 micron) which didn't improve the resolution. I went to an embedded polar phase (Discovery RP-Amide C-16, 4.6 x 250mm, 5 micron) and could achieve about 40 seconds separation at 0.5 mL/min (which is about 95 % separation between the two). The problem is that the run times are getting to be long (40 mins) and I have to ramp to a gradient to remove the remaining esters. At the curent flow rate, the column takes a long time to equilibrate before the next injection.
Any suggestions or applications for this separation would be greatly appreciated.

Douglas,

If you current approach is not working you can try mix mode chromatography. You can separate all three compounds on anion exchange mixed mode column (Primesep B2, Primesep D).
2,4-D will retain based on reverse phase and anion-exchange mechanism and your esters will retain based on reverse phase mechanism. You can also try to add pi-pi interaction.
See the following interactive graph for comparison of reverse phase, ion exchange and mixed mode separations.

http://www.sielc.com/Technology_2D_Properties.html


We did something like this (2,4-D) while ago; I need to see our archives. I will let you know. If you are interested we can o free screening for you:

http://www.sielc.com/Services_MethodDev ... tForm.html

According to ACD, the 2,4-D butyl ester has a log D of 4.26 and log P of 4.26+/- 0.269. The 2,4-D Butoxyethyl ester has a log D of 3.99 and log P of 3.988+/- 0.531 . I'm not sure that mixed mode is going to work in this case, as the 2-D properties are so close. What do you think?

You can try to add Pi-pi interaction by using phenyl column and MeOH as a solvent (in combination with buffer).
Nature of the buffer might give you enough power to achieve separation
We don't have these two compounds in the lab by if you send us a sample we will screen our phases similar to the following:

Effect of Buffer and Chemistry of Column Stationary Phase on Resolution of Neutral Compounds:

http://www.sielc.com/compound_189.html

These are different compounds but application shows effect of buffer nature and chemistry of the column

A combination of what you have with the suggestion from SIELC TECH might work. I think that you are on the right track by looking at an EPG phase. You may be successful on the EPG by switching from methanol to acetonitrile or vice versa. Also, do not forget to try THF. It sometimes does wonders in cases like this.