Determination of glyphosate and related compounds using HPLC with tris(2,2'-bipyridyl)ruthenium(II) electrogenerated chemiluminescence detection

Tris(2,2'-bipyridyl)ruthenium(II)(Ru(bpy)(3)(2+)) electrogenerated chemiluminescence (ECL) is investigated for the detection of glyphosate (HO2CCH2NHCH2PO3H2, N-(phosphonomethyl)glycine) and several structurally related compounds separated by ion exchange chromatography. These related compounds are iminodiacetic acid (IDA, HO2CCH2NHCH2CO2H), diethanol amine (DEA, HOCH2CH2NHCH2CH2OH), hydroxyethyl glycine (HEG, HO2CCH2NHCH2CH2OH), and glycine (NH2CH2CO2H). Resulting ECL intensities increase in the order: glycine<DEA<HEG<IDA<glyphosate. The study of the effect of pH on chemiluminescence (CL) emission intensity for the compounds investigated showed that this detection method is most sensitive in the alkaline pH range. The detection limit for glyphosate is 0.01 mu M with a linear working range that extends five orders of magnitude. The separation is done using both cation exchange and anion exchange chromatography. For the cation exchange separation, both post-column addition of Ru(bpy)(3)(2+) and adjustment of pH is required. Incorporating Ru(bpy)(3)(2+) in the mobile phase has no benefit since post-column adjustment of pH is still necessary. For the anion exchange separation, studies are done with both post-column addition of Ru(bpy)(3)(2+) and with Ru(bpy)(3)(2+) incorporated in the mobile phase. Including Ru(bpy)(3)(2+) in the mobile phase eliminates the need for any post-column reagent addition, decreasing band broadening and simplifying instrumental set-up. Including Ru(bpy)(3)(2+) in the mobile phase also modifies the stationary phase, which causes retention times of the analytes to decrease. With anion exchange chromatography, separation of the analytes is accomplished using both isocratic and step-gradient elution. Although step-gradient elution gives the best separation of the analytes examined, the method of including Ru(bpy)(3)(2+) in the mobile phase works best when the compounds of interest can be separated isocratically.