Arthur W. Garrison

Separation of phenoxy acid herbicides and their enantiomers by high-performance capillary electrophoresis

Abstract Capillary electrophoresis conditions in the free solution mode (capillary zone electrophoresis) were established for the separation and detection of 2,4-dichlorophenoxyacetic acid and three optically active phenoxy acid herbicides (dichlorprop, mecoprop and fenoprop). A 50 mM acetate buffer at pH 4.5 gave the best separation, using a 50 cm (to detector) × 75 μm I.D. fused-silica column; the column temperature was 30°C. separation voltage 20 kV and optimum detector wavelength 230 nm. Separation of the four herbicides required less than 15 min under these conditions. Baseline separation of the two enantiomers of each of the three optically active herbicides, separately and in mixtures of the three, was accomplished by the addition of 25 mM tri-O-methyl-β-cyclodextrin to the acetate separation buffer. Di-O-methyl- β-cyclodextrin or α-cyclodextrin (CD) separated enantiomers of dichlorprop and mecoprop, but not those of fenoprop; β-CD provided very little separation and γ-CD gave no separation. Addition of methanol to the separation buffer increased separation, but doubled migration times. Over a variety of sample concentrations and injection times, reproducibilities of migration times of racemates and enantiomers ranged from 1.3 to 4.6% R.S.D.; peak area and peak height reproducibilities ranged from 1.6 to 17.9% R.S.D.

Separation of s-triazine herbicides and their metabolites by capillary zone electrophoresis as a function of pH

The effects of buffer pH on electrophoretic mobility of various s-triazines (chloro-, hydroxy-, methoxy- and thiomethyl-s-triazines) in light of their chemical properties have been studied. Good separation was achieved for each class of s-triazines as cations, except for the chlorotriazines, with buffer pH around the pKa of the herbicides, as well as at a pH of pKa − 2. Migration times were correlated with molecular masses (as a first approximation of molecular size and shape). Possible hydrophobic wall interactions were detected at acidic pH and low voltage. Separation of hydroxytriazine metabolites as anions could also be achieved at a high pH of around 8. The detection level calculated for most of these s-triazines was μg/ml of analyte in the injection solution.

Enantiomer separation of polychlorinated biphenyl atropisomers and polychlorinated biphenyl retention behavior on modified cyclodextrin capillary gas chromatography columns.

Seven commercially-available chiral capillary gas chromatography columns containing modified cyclodextrins were evaluated for their ability to separate enantiomers of the 19 stable chiral polychlorinated biphenyl (PCB) atropisomers, and for their ability to separate these enantiomers from achiral congeners, necessary for trace environmental analysis of chiral PCBs. The enantiomers of each of the 19 chiral PCBs were at least partially separated on one or more of these columns. Enantiomeric ratios of eleven atropisomers could also be quantified on six columns as they did not coelute with any other congener containing the same number of chlorine atoms, so could be quantified using gas chromatography-mass spectrometry. Analysis of a lake sediment heavily contaminated with PCBs showed enantioselective occurrence of PCB 91, proof positive of enantioselective in situ reductive dechlorination at the sampling site.

Capillary electrophoresis in the analysis of humic substances: Facts and artifacts

Abstract Humic substances, extracted as mixtures from soil and surface waters according to their solubility in acids and bases, are relatively high-molecular-mass polyelectrolytes containing aromatic, aliphatic and heterocyclic subunits. The degree of ionization of their phenolic and carboxylic groups is governed by the capillary electrophoresis (CE) buffer pH. In CE, fulvic acids exhibit a consistent and characteristic set of sharp peaks (phenolic acids), extending from a humic “hump” whose average electrophoretic mobility (AEM) depends on humic structure and buffer composition; humic acids give only the “hump”. Special attention must be given to the interpretation of CE electropherograms when fingerprinting humic substances with borate buffers because observed peaks do not necessarily indicate distinct humic fractions, but may be artifacts caused by the interaction of borate ions with 1,2- and 1,3-diols present in the humic mixtures. Depending on the molarity of borate ions in the separation buffer, humic acids exhibit electropherograms with sharp peaks extending from the “humic hump” and corresponding to borate complexes. The potential of capillary zone electrophoresis for the comparison of electropherogram patterns is illustrated for the Suwannee River reference humic substances extracted according to the recommendations of the IHSS compared with a fraction of the same source concentrated with the more recent reverse osmosis (RO) technique. The Suwannee River RO fraction behaved like the extracted Suwannee River humic acid fraction under these experimental conditions.

Application of cyclodextrin-modified micellar electrokinetic chromatography to the separations of selected neutral pesticides and their enantiomers

Abstract The environmental chemistry of chiral pesticides is receiving increased attention — enantiomeric ratios are being measured and enantioselective degradation processes are being reported. The requisite analysis involves separation of the various enantiomers. Mixtures of three classes of chiral pesticides — organophosphorus, DDT congeners and methyl esters of phenoxy acids — were separately tested for separation, first by non-chiral micellar electrokinetic chromatography (MEKC). Generally, the components of each mixture were so strongly adsorbed by the micelles that they coeluted with the micelles. Then, different concentrations of an organic modifier, methanol or acetonitrile, were added. Only the five organophosphorus pesticides were separated efficiently; components of the other mixtures still migrated with the micelles. Each of six cyclodextrins (CD) — α-, β-, γ-, hydroxypropyl-β-, dimethyl-β- and trimethyl-β-CD, were then added to the borate–SDS buffer, with and without the organic modifier, to test for separation of the non-chiral compounds and the enantiomers of the chiral racemates by CD-MEKC. The enantiomers of malathion, ruelene and dialifos were separated by hydroxypropyl-β-CD, β-CD and/or γ-CD, while the enantiomers of isofenfos and fenamifos could not be separated. γ-CD with methanol modifier allowed baseline separation of the three phenoxy acid methyl esters and of the enantiomers of fenoprop methyl ester, but none of the CDs separated the enantiomers of mecoprop and dichlorprop methyl esters. The use of γ-CD with acetonitrile modifier resulted in excellent separation of six DDT congeners, o,p′- and p,p′-DDT, -DDD and -DDE, as well as baseline separation of the enantiomers of the chiral members of this series, o,p′-DDT and -DDD. Finally, attempts were made to separate the four enantiomers of the herbicide metolachlor; three of the enantiomers were separated by γ-CD with methanol.

Acute enantioselective toxicity of fipronil and its desulfinyl photoproduct to Ceriodaphnia dubia

Fipronil is a phenylpyrazole insecticide increasingly used in applications such as rice culture, turf grass management, and residential pest control, with a high probability to contaminate aquatic environments. As a chiral pesticide, fipronil is released to the environment as a racemic mixture (equal amounts of optical isomers called enantiomers). Enantiomers can have different toxicological and biological activity; however, information on these differences, which is necessary for accurate risk assessment of chiral pesticides, is limited. Here we examine the acute toxicity of fipronil enantiomers, the racemate, and its photoproduct (desulfinyl fipronil) to Ceriodaphnia dubia. The 48-h median lethal concentration (LC50) values based on measured concentrations of each compound indicate the (+) enantiomer (LC50 = 10.3 +/- 1.1 microg/L, mean +/- standard error [SE]) was significantly more toxic to C. dubia than either the (-) enantiomer (LC50 = 31.9 +/- 2.2 microg/L) or racemate (LC50 = 17.7 +/- 1.3 microg/L). To account for any potential loss of fipronil through photolysis, tests were performed under light (fluorescent) and dark exposure conditions, and no significant differences in toxicity were observed. Desulfinyl fipronil, the major photodegradation product, which is not chiral, was detected at < 1% of each parent compound in test solutions after 48 h. Separate toxicity tests with desulfinyl fipronil found a > 20-fold higher LC50 (355 +/- 9.3 microg/L) compared to the fipronil racemate, suggesting lower adverse effects to C. dubia as a result of fipronil photolysis. The present results suggest selection of the (-) enantiomer in fipronil production for lower impacts to C. dubia; however, the consistency and relevancy of fipronils enantiomer-specific activity at both acute and chronic levels of concern to additional target and nontarget species needs further consideration.

Microbial Transformation of Triadimefon to Triadimenol in Soils: Selective Production Rates of Triadimenol Stereoisomers Affect Exposure and Risk

The microbial transformation of triadimefon, an agricultural fungicide of the 1,2,4-triazole class, was followed at a nominal concentration of 50 μg/mL over 4 months under aerobic conditions in three different soil types. Rates and products of transformation were measured, as well as enantiomer fractions of parent and products. The transformation was biotic and enantioselective, and in each soil the S-(+)-enantiomer reacted faster than the R-(-) one. Rates of the first-order reactions were 0.047, 0.057, and 0.107 d(-1) for the three soils. The transformation involves reduction of the prochiral ketone moiety of triadimefon to an alcohol, resulting in triadimenol, which has two chiral centers and four stereoisomers. The abundances of the four product stereoisomers were different from each other, but abundance ratios were similar for all three soil types. Triadimenol is also a fungicide; the commercial product is composed of two diastereomers of unequal amounts (ratio of about 4.3:1), each having two enantiomers of equal amounts. However, the triadimenol formed by soil transformation of triadimefon exhibited no such stereoisomer profile. Instead, different production rates were observed for each of the four triadimenol stereoisomers, resulting in all stereoisomer concentrations being different from each other and very different from concentration/abundance patterns of the commercial standard. This result is important in risk assessment if the toxicity of the environmental transformation product were to be compared to that of the commercial triadimenol. Because triadimenol stereoisomers differ in their toxicities, at least to fungi and rats, the biological activity of the triadimenol formed by microbes or other biota in soils depends on the relative abundances of its four stereoisomers. This is an exposure and risk assessment issue that, in principle, applies to any chiral pesticide and its metabolites.

Capillary electrophoresis for the characterization of humic substances

Abstract The potential of high performance capillary electrophoresis (HPCE), especially in the free solution mode (FSCE), is demonstrated for the analysis/characterization of environmental humic substances (HUS). The very high efficiency of HPCE separations allows the production of electropherograms that are more characteristic for HUS than are liquid chromatograms; in addition, HUS can be analyzed by FSCE in their naturally occurring anionic forms. Fulvic and humic acid fractions of HUS extracted from soil and water by procedures of the International Humic Substances Society were analyzed using an uncoated silica column and acetate, borate or phosphate buffer systems to produce electropherograms of the anionic species. Soil fulvic acids exhibit a consistent and characteristic set of sharp peaks, extending from a humic “hump”; migration times range between 5 and 12 min depending on soil source, buffer and pH. Humic acids give only the “hump,” which sometimes has shoulders but little definition. Soil and river humic acids produce only slightly different electropherograms at pH 8.30 in a borate buffer system, and each shows a remarkable peak-sharpening as the pH is increased to just 8.55. The electropherograms of “young” and “old” groundwater fulvic acids show different fingerprints.

Binding of s-triazines to dissolved humic substances: Electrophoretic approaches using Affinity Capillary Electrophoresis (ACE) and Micellar Electrokinetic Chromatography (MEKC)

Abstract Binding studies were conducted between s-triazines and soil and water extracted fulvic and humic acids (FA and HA) using capillary electrophoretic methods. A first approach to estimate simultaneously the affinity of several s-triazines (hydroxyatrazine, ameline, atraton and ametryn) to dissolved humic substances (HS) was done with the affinity capillary electrophoresis (ACE) modus; the limits of the ACE method resulted in the measurement of the electrophoretic mobility of the pesticide-HS complexes. In a second approach, the partition of the s-triazines between the water and the dissolved humic substances was successfully described like in micellar electrokinetic chromatography (MEKC) using the humic substances as micellar phase. Similar to surfactants, humic acids (HA) behaved like ionic micelles in the aqueous running buffer at concentrations higher than a defined “ humic critical micellar concentration ” (HCMC). The low molecular weight acidic fulvic acids (FA) behaved the same but showing higher HCMC. These results confirm the micellar properties of HS and the hydrophobic type of interaction of the s-triazines with hydrophobic sites of humic and fulvic ionic micelles.

Simultaneous Determination of Ionization Constants and Isoelectric Points of 12 Hydroxy-s-Triazines by Capillary Zone Electrophoresis and Capillary Isoelectric Focusing

Capillary zone electrophoresis (CZE) was used to separate and determine simultaneously the pK(1), pK(2), and pI values of 12 environmentally relevant hydroxytriazines (hydroxymetabolites of atrazine, terbuthylazine, simazine, and propazine and four (arylamino)-s-triazines) and observe the effects of the alkylamino and arylamino substituents on the measured values. Capillary isoelectric focusing (CIEF) methods were developed to measure the pI of these compounds and compare those values with the CZE-measured pIs. CZE and CIEF can provide accurate pK and pI values reasonably fast, and pI values measured by the two techniques agree. Knowledge of the pK and pI values of hydroxytriazines is important for an understanding of the binding mechanisms of these molecules in environmental matrices. Because the hydroxytriazines may exist as a myriad of species [Formula: see text] neutral, charged, zwitterionic, and keto-enol tautomeric [Formula: see text] depending on structure and pH, we briefly addressed the existence of these species relative to their electrophoretic analysis.