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Equipment
• Thermo Scientific
™
Dionex
™
UltiMate
™
3000 x2 Dual
Rapid Separation LC (RSLC) system, including:
– DGP-3600RS Dual Ternary RS Pump System with
SRD-3600 Integrated Solvent and Degasser Rack
– WPS-3000TRS RS Wellplate Sampler, Thermostatted,
with a 1000 µL sample loop and a 1000 µL syringe
– TCC-3000RS or TCC-3000SD RS Thermostatted
Column Compartment equipped with one 2–6p valve
– DAD-3000RS RS Diode Array Detector - Semi-Micro
Flow Cell for DAD-3000 and MWD-3000 Series,
SST, 2.5 µL Volume, 7 mm Path Length
• Thermo Scientific
™
Dionex
™
Chromeleon
™
Chromatography Data System software, version 7.1
• Thermo Scientific
™
Orion
™
2-Star Benchtop pH Meter
• Thermo Scientific
™
Target2
™
Nylon Syringe Filters,
0.45 µm, 30 mm (P/N F2500-1)
Reagents and Standards
• Deionized (DI) water, 18.2 M
Ω
-cm resistivity
• Acetonitrile (CH
3
CN), HPLC Grade 99.9%
(Fisher Scientific P/N AC610010040)
• Ammonium Acetate (CH
3
COONH
3
), Crystalline/
Certified ACS (Fisher Scientific P/N 631-61-8)
• Dimethyldichlorosilane (DMDCS),
≥
99% (P/N 75-78-5)
• EPA-549.1 STDS 2COMP, 1 ML, Diquat and
Paraquat Standard
Working Standard Solutions for Calibration
Use the EPA-549.1 Diquat and Paraquat Standard and
dilute with DI water to prepare a stock standard solution
with 1.0 μg/mL of each compound. Prepare six working
standard solutions for the calibration with different
concentrations by adding the correct amount of stock
standard solution and diluting with DI water. The volumes
needed of each solution to make the calibration standards
are shown in Table 1.
Sample Preparation
Tap water samples were collected at the Thermo Fisher
Scientific
™
Shanghai Applications Lab. Pond water
samples were collected at Zhangjiang High Science and
Technology Park located in the Pudong District of
Shanghai, China. Samples were filtered using Target2
nylon syringe filters prior to injection.
Note: Soak all glassware used to prepare paraquat and diquat standard solutions for
calibration or used in sample preparation for at least 8 h in a mixture of CH
3
CN and
DMDCS (9:1, v/v) to avoid loss (adsorption) of diquat and paraquat.
Chromatographic Conditions
On-Line SPE
Column:
Acclaim Trinity P1, 3 µm, Guard Cartridges,
3.0 × 10 mm (P/N 071390) with SST
Guard Cartridge Holder V-2 (P/N 069580)
Mobile Phase:
A: 100 mM Ammonium Acetate
(adjust pH 5.0 using acidic acid)
B: Acetonitrile
C: H
2
O
Gradient:
0–2 min, 10% A, 5% B
2.1–4.5 min, 55% A, 45% B
4.6–10 min, 10% A, 5% B
Flow Rate:
0.7 mL/min
Inj. Volume:
1000 µL onto the on-line SPE cartridge
Separation
Column:
Acclaim Trinity Q1, 3 μm, Analytical,
3.0 × 50 mm (P/N 079716)
Mobile Phase:
35% 100 mM Ammonium Acetate
(pH 5.0)/65% Acetonitrile
Flow Rate:
0.5 mL/min
Column Temp:
30 °C
Detection:
UV absorbance at 260 nm for paraquat and
310 nm for diquat
Valve Position
0 min, 1_2
2.0 min, 6_1
4.5 min, 1_2
2
Volume of Stock Standard
Solution of Paraquat and
Diquat, 1.0 µg/mL Each
(mL)
Volume of DI
Water
(mL)
Final
Volume
(mL)
Final
Concentration
(µg/L)
0.01
9.99
10
1
0.05
9.95
5
0.50
9.50
50
1.00
9.00
100
5.00
5.00
500
10.0
0.00
1000
Table 1. Preparation of calibration curve standards.