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Trace Analysis of Chlorinated Herbicides in Water with Online Enrichment a Simple and Rapid Method with Automated Sample Cleanup

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Trace Analysis of Chlorinated Herbicides in Water with Online Enrichment a Simple and Rapid Method with Automated Sample Cleanup Trace Analysis of Chlorinated Herbicides in Water with Online Enrichment A simple and rapid method with automated sample cleanup Application Note Author Environmental Analysis Srividya Kailasam Agilent Technologies, mAU Picloram 300 Bangalore, India Bentazone 250 Dicamba 200 Acifluorfen 2,4,2-T,P Dichlorprop 2,4-D Chloramben 150 100 50 0 0 1011121314min Abstract This Application Note demonstrates the analysis of eight chlorinated herbicides: picloram, chloramben, dicamba, bentazone, 2,4-D, dichlorprop, 2,4,5,-T,P and aciflour- fen in acidified water by online enrichment. The selected herbicides can be analyzed in less than 2 mL of water within a run time of 15 min. This is achieved with an Agilent ZORBAX SB-Aq column for trapping the analytes, an Agilent ZORBAX SB-C18 column for analysis, and a two-position/six-port valve for column switching. The developed method was applied to prepare a standard curve each for picloram and dichlorporp to show the suitability of the proposed method for the quantification of the selected herbicides in water. Automated sample cleanup eliminates laborious solid phase extraction (SPE) protocols for sample preparation. The sensitivity of the Agilent 1260 Infinity LC system can achieve the detection limits prescribed by the US Environmental Protection Agency (EPA) with less than one tenth of the original sample volume specified by the official method. Introduction • Agilent 1260 Infinity Thermostatted Picloram; chloramben; dicamba; benta- Column Compartment (G1316A) zone; 2, 4-D; dichlorprop; 2,4,5-T,P and Herbicide contamination in water bod- acifluorfen were purchased from Sigma ies is monitored to mitigate adverse • Two-Position/ Six-Port column Aldrich. Stock solutions were prepared health impacts. Sensitive methods for switching valve (5067-4646) in methanol and samples diluted in detecting and quantifying trace levels of • Agilent 1260 Infinity Diode Array 25 mM phosphoric acid. Solutions con- these chemicals are necessary to meet taining 0.5, 1, 1.5, 3, 4.5, 9, 90, 180 and regulatory requirements. Although Detector (G4212B), with Max-Light 10 mm High Sensitivity flow cell 240 ppb of picloram and dichlorprop offline SPE can help enrich target ana- were used to prepared the calibraiton lytes from complex matrices, the meth- • Software: ChemStation B.04.02 curves. A solution of 50 ppb of each of ods use expensive cartridges and the selected herbicides was used to lengthy protocols making them less Chromatographic conditions examine the method reproducibility. suitable for routine use. Columns Enrichment/Cleaning: Agilent ZORBAX RRHT SB-Aq This Application Note describes a sim- 2.1 X 30 mm, 1.8 µm Results ple, sensitive, and rugged LC method (p/n 824700-914) The US EPA Method 5551 describes the Analytical: Agilent ZORBAX for the routine analysis of water for RRHD SB-C18 2.1 x 50 mm, use of online sample enrichment for the quantifying eight chlorinated herbi- 1.8 µm (p/n 857700-902) analysis of chlorinated herbicides in cides. The analytes are initially trapped Mobile phase A = Phosphoric acid 20 mL of water. An earlier Application on an enrichment column fitted on a six (25 mM, in Milli-Q water) Note2 describes in detail the advan- port/two-position switching valve. B = Acetonitrile tages of using sub-2-µm columns for Subsequently, they are eluted from this Injection volume 1800 µL rapid and sensitive analysis of smaller column and transferred to the analytical sample volumes. This Application Note column for separation followed by UV Enrichment/Loading conditions describes a method with automated detection with the sensitive diode array Flow rate 0.5 mL/min sample cleanup for the assay of eight detector (DAD) of the Agilent 1260 Gradient Time(min) % Acetonitrile chlorinated herbicides using less than Infinity LC system. 00 2 mL of water. 8.1 10 Experimental Conditions 9.0 10 An Agilent 1260 Infinity Binary Pump 9.1 0 System was used to load 1800 µL of the sample Stop time: no limit on to the enrichment (or cleaning) col- The Agilent LC system consisted of the Elution and analysis conditions umn with 100% of mobile phase A. following modules, Flow rate 0.61 mL/min Since the maximum volume of sample Gradient Time (min) % Acetonitrile that can be injected per draw using the • Agilent 1260 Infinity Binary Pump 0.0 20 (G1312B) 9.0 20 Agilent 1260 Infinity Autosampler is 100 12.5 80 µL, it was modified with a Multidraw kit • Agilent 1260 Infinity Micro Degasser 13.5 80 and a 900-µL Injection Upgrade kit to 14.0 20 (G1379B) Stop time: 15 min allow rapid large volume injections. With this hardware modification, 900 µL Column temperature 40 °C on both sides • Agilent 1260 Infinity Autosampler of the sample were drawn twice, filled (G1329B) modified with a Multidraw Column switching 0 min: Load/Equilibrate (valve position 1) in the sample loop and subsequently kit (G1313-68711) and a 900 µL injec- 9 min: Elute/Equilibrate injected on the ZORBAX SB–Aq col- tion Upgrade kit (G1363A) (valve position 2) umn, which can withstand large aque- • Agilent 1260 Infinity Thermostat 14 min: Load/Equilibrate ous loads. After a load time of 8 min, (valve position 1) (G1330B) the column was washed with 10% ace- Detection 230 nm, 8 nm BW; Ref: No; tonitrile to prevent the buildup of organ- PW > 0.062 s (80 Hz); Slit Width: 8 nm ic impurities. During this step the ana- lytes were trapped on the loading col- Samples umn while polar matrix constituents were washed away leading to sample cleaning. A two-position/six-port valve was 2 installed on the thermostatted column compartment. After the loading cycle, Pump A Autosampler the valve was switched and the flow from the second 1260 Infinity Binary Pump was pumped through the enrich- 1 6 ment column. The trapped analytes Analytical column were eluted and carried to the ZORBAX 2 5 SB-C18 analytical column for separation prior to detection using the 1260 Infinity Waste 4 Dectector Diode Array Detector equipped with the 3 proprietary Agilent Max-Light flow cell. Position A Valve position and pump flows Cleaning column Figure 1 shows the flow path through- Pump B out the two valve positions. During sample loading (valve position A) the flow from one of the 1260 Infinity Pump A Autosampler Binary Pumps (Pump A) flows through the enrichment column while the flow from the second 1260 Infinity Binary 1 6 Pump (Pump B) flows through the ana- Analytical column lytical column under initial conditions, 2 5 maintaining equilibrium. During the elute/analyze cycle (valve position B), Waste 4 Dectector the flow from the first 1260 Infinity 3 Binary Pump (Pump A) is diverted to waste while the flow from the second Position B 1260 Binary Pump (Pump B) passes Cleaning column Pump B through the enrichment column to elute the trapped analytes to the analytical Figure 1 column for analysis. Valve positions during online enrichment. Sample volume is decreased by a factor of 20 (Table 1) by reducing the column dimensions from 4.6 × 250 mm (the EPA Column dimensions Column bed volume Trace enrichment volume Sample reduction factor method) to 2.1 x 50 mm (present 4.6 x 250 mm 2.50 mL 20 mL na method). Although 900 µL of the sam- ple can theoretically be used, in the 3.0 x 150 mm 0.63 mL 5 mL 4 present study 1800 µL were used to 2.1 x 100 mm 0.21 mL 1.7 mL 11.7 improve the detection limits of the 2.1 x 50 mm 0.10 mL 0.9 mL 20 assay. This is one order of magnitude Table 1 lower than 20 mL, the volume of the Column dimensions and corresponding injection volumes. sample prescribed by the US EPA Method 555. In addition, the smaller particle size of the packing material in the 2.1 × 50 mm column also helps to improve the sensitivity of the method. 3 At the end of the loading cycle, when the flow path is switched, the elute/ mAU Picloram 300 analysis cycle begins. This process Bentazone completes in less than 3 minutes from the start of the second cycle (Figure 2) 250 Dicamba 200 Acifluorfen 2,4,2-T,P The RSD values for the retention times, Dichlorprop 2,4-D areas and peak heights of all the ana- Chloramben lytes obtained from three replicate 150 injections of the sample containing 100 50 ppb of each analyte were all less than 1%. Excellent reproducibility 50 makes this method suitable for the routine analysis of water samples in 0 environmental laboratories. 0 1011121314min The applicability of the method for Figure 2 quantification of the selected herbi- Chromatogram of a sample containing 50 ppb of each of the eight analytes. cides in water was tested by developing standard curves for two of the analytes: picloram and dichlorprop. Figures 3a, 3b and 4a, 4b illustrate that the area Area Rel.Res%(1):25.654 responses correlate well with the con- centrations for both compounds. The 3500 9 Picloram, DAD1A calibration curves prepared on three inde- 3000 pendent days were found to be linear on Area=16.3056846*Amt-2.9207845 8 2500 all occasions with correlation coefficient values being ≥ 0.99. 2000 7 1500 1000 500 6 12345 0 Correlation: 0.99773 0 100 Amount[ng/ul] Figure 3a Calibration curve for picloram. Area 600 Picloram, DAD1A Area=16.3056846*Amt-2.9207845 400 200 6 5 4 1 2 3 0 -200 0 51510 Amount[ng/ul] Figure 3b Zoomed in view of the lower end of the calibration curve. 4 Table 2 shows a set of peak areas at the various concentrations used to con- Area Rel.Res%(5):-1.477 struct the calibration curves for the two compounds.
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