Chromatography Forum

Hi All,

Post column derivatisation with NaOCl and OPA and FLD detection (the Pickering method) is the conventional method for analysis of glyphosate. But recently i came across a method (J. Agric. Food Chem. 1986, 34, 535-538) by Lennart N. Lundgren. It involves derivatisation with 1-Fluoro-2,4-dinitrobenzene (DNP) and quantified on reverse phase column with UV detection at 405 nm. Has anyone tried this method? Appreciate your comment if you do.

Thanks and regards.

Leon,

You can do direct analysis of glyphosate using Primesep column and ELSD detector:

http://hplcmethods.com/compound_048.php

We do glyphosate by the NaOCl oxidation method. It works well, but requires highly sensitive fluorescence detection.

Why the interest in the other method?

I would also like to hear from anyone with experience.

Evan L. Cooper

Can you automate the Fluoro-DNB reaction? If so, it might be a reasonable alternative to the post-column method. If if is a manual technique, it would be of more interest for a short-term project where you don't want to invest in capital equipment.

ELSD is a good choice for formulation analysis, but about 2 orders of magnitude away from being sensitive enough for residue analysis.

(Disclosure: I used to work for Pickering and supported their post-column method.)

We do glyphosate by the NaOCl oxidation method. It works well, but requires highly sensitive fluorescence detection.

Why the interest in the other method?

I would also like to hear from anyone with experience.

Evan L. Cooper

The use of reverse phase column attracted me. I feel it is easier to handle than a cation ion exchange column. However, with derivatisation and subsequent workup, recovery may not be favourable. Thats why I need to hear comments.

Although the initial cost is high for a glyphosate column, the columns are very robust if handled properly. We use to Waters IC-PAK column, and it lasts for many many injections, more than your averaged reversed phase column. The column is operated isocratically with an entirely aqueous mobile phase. Aside from the initial cost, it seems an overall winner over reversed phase.

ravenwork

Indeed, the cation exchange columns can last a long time. The problem is that the handling requirements are different enough from C18 that many people don't have their intuition developed for ion exchange. To make the column last you should:
* Observe the maximum pressure and flow specifications. Even a brief exposure to excess flow can cause a head space.
* Avoid organic solvents. They can cause a slow pressure buildup.
* Inject clean, filtered samples dissolved in water.
* Avoid surfactants in your sample if possible.
* Use a guard column.
* Take measures agaings iron contamination. It can kill the chromatography, but it is easy to remove.
* Passivate your HPLC system
* Avoid stainless steel filters in your solvent reservoirs. They corrode too easily.

This advice is aimed at the fully sulfonated PS-DVB gel that is typical for glyphosate analysis (BioRad, Pickering, etc.).

One last point. The ion exchange capacity of these columns is large, and the mobile phase has low ionic strength. Equilibration is very slow.

Indeed, the cation exchange columns can last a long time. The problem is that the handling requirements are different enough from C18 that many people don't have their intuition developed for ion exchange. To make the column last you should:
* Observe the maximum pressure and flow specifications. Even a brief exposure to excess flow can cause a head space.
* Avoid organic solvents. They can cause a slow pressure buildup.
* Inject clean, filtered samples dissolved in water.
* Avoid surfactants in your sample if possible.
* Use a guard column.
* Take measures agaings iron contamination. It can kill the chromatography, but it is easy to remove.
* Passivate your HPLC system
* Avoid stainless steel filters in your solvent reservoirs. They corrode too easily.

This advice is aimed at the fully sulfonated PS-DVB gel that is typical for glyphosate analysis (BioRad, Pickering, etc.).

One last point. The ion exchange capacity of these columns is large, and the mobile phase has low ionic strength. Equilibration is very slow.

Hi Mark,

what is passivate?
Can I use peek material for the mobile phase filter?
Should the sample loop and all other tubings be of Peek material?

Leon,
Passivation is a treatment for stainless steel surfaces; it removes soluble metals and leaves behind an oxide coating. Your HPLC vendor will have specific instructions, but a typical procedure is 1) pump water to remove buffers; 2) pump 2-6 molar nitric acid for 15-30 minutes; 3) pump water to remove all the acid. Pump this through the pump, injector and detector but not the column. I like to use the UV detector to tell me when the nitrate is all gone.

PEEK tubing and filters are very good for glyphosate analysis.