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2004 vol. 3

mize residual silanols and metal ions on the

packing particles, which could interact with the

analytes and cause tailing and unwanted (and

sometimes unpredictable) retention.

The reagent solution we use in the mobile

phase, Ultra Quat Reagent Solution (cat.#

32441), alters the chemical nature of the ana-

lytes as perceived by the column and mobile

phase. It reduces the ability of water to solvate

the analytes and hydrogen bond with them, forc-

ing the charged complexes into the stationary

phase and improving retention.

Unlike ion pairing techniques, our new approach

requires only water, Ultra Quat Reagent Solution,

and acetonitrile (which cannot form hydrogen

bonds) to accomplish the separation. For highest

sensitivity, we monitor for paraquat at 257nm

and for diquat at 308nm. Using the new column,

mobile phase, and conditions, the detection limit

for either herbicide is 6ppb in the final sample

extract—a detectable amount of 0.12

nanograms on column. Data are summarized in

Table 1. Using the solid phase extraction proce-

dure in Table 2, which concentrates samples 200-

fold (1L to 5mL), the detection limit is 0.03ppb—

a significant improvement over current method-

ology. Analyte concentrations can be increased

by modifying the solid phase extraction proce-

dure or by increasing the injection volume, to

improve quantification and detection limits.

Figure 2 overlays chromatograms of paraquat

and diquat reference standards at a range of

concentrations (20µg/mL–100µg/mL); resolu-

tion, retention times, and peak symmetry are

highly consistent. Concentrations up to

100µg/mL are consistent with linear detector

responses.

Note that glassware used to prepare and ana-

lyze samples and reference materials for this

analysis must be deactivated (e.g., with

dimethyldichlorosilane—DMDCS, cat.# 31840).

EPA Method 549.2 requires retesting of all sam-

ples if the response for the reference standards

changes by more than 20% over the time of the

analysis. We found all reference standards

showed degradation after only 1 hour in

untreated glassware, with the lowest concentra-

tions being the most affected. 30% losses in

response were not uncommon; a diquat refer-

ence standard of 6ppb in water became unde-

tectable.

When you perform the challenging

paraquat/diquat analysis, our new Ultra Quat

column, Ultra Quat Reagent Solution and

Paraquat/Diquat Calibration Mix, and extraction

procedure will give you the edge you need to

obtain the most accurate and consistent infor-

mation.

Ultra Quat HPLC Column

Physical Characteristics:

particle size: 5µm, spherical

pH range: 2.5 to 7.5

temperature limit: 80°C

Sample Injection

Limit of

Volume Volume Detection Quantification

(mL)

(µL)

(ppb)

100

0.06

0.2

250

0.024

0.08

1000

0.006

0.02

100

1.2

100

0.012

0.04

250

100

0.0048

0.016

1000

100

0.0012

0.004

200

0.6

100

200

0.006

0.02

250

200

0.0024

0.008

1000

200

0.0006

0.002

Table 1

Approximate detection/quantification

limits for paraquat and diquat, using an

Ultra Quat column.

On column limit of detection (LOD): 0.12ng

On column limit of quantification (LOQ): 1.2ng

In Summary

Highly polar paraquat and diquat can’t be

separated on a reversed phase HPLC column

without adding ion pair modifier to the mobile

phase, but the ion pair reagent in current

methodology does not provide optimum

resolution and does not permit detection

below 0.7µg/mL. We have developed a column

and a mobile phase modifier for rapid, com-

plete resolution of paraquat and diquat, with

detection to concentrations as low as

0.5µg/mL—an improvement of 30%.

Sample Extraction

SPE Tubes:

Restek WCX, weak cation exchanger, 3mL/500mg, cat.# 26062.

Samples:

1 liter deionized water containing 50µg each of diquat and paraquat.

Samples spiked with 20µL 549.2 Calibration Mix, cat.# 32437, diluted with HPLC grade water.

Conditioning: 3mL acetonitrile, then 3mL deionized water, applied sequentially.

Do not allow adsorbent bed to dry before applying sample.

Extraction:

Pass 1 liter water samples through SPE tubes at a rate of 5-10mL/min.

Arrange 5mL collection vessels under extraction tubes.

Place 1mL acidic elution solution* in each tube, draw into bed, allow to stand for up to 1 min.

Pass solution at a slow (drop-wise) rate through SPE tubes into collection vessels.

Repeat with 2 x 2mL acidic elution solution.

Correct final volume in collection vessels to 5mL with acidic elution solution.

Analysis:

Neutralize eluates with approximately 20µL concentrated ammonium hydroxide, then analyze by

HPLC. Adjust amount of ammonium hydroxide used to assure each sample is neutral (test with pH

indicating paper).

*1mL 85% H

diluted to 1 liter with deionized HPLC grade water (0.1%).

Results

Analyte Recovery (%) RSD (%)

diquat

99.0

0.89 (n=5)

paraquat

96.3

1.59 (n=5)

Table 2

Solid phase extraction of diquat and paraquat from aqueous samples.

5µm Column, 4.6mm ID

cat.#

price

150mm

9181565

$356

4.0mm ID

Length

cat.#

price

10mm

918150210

$129

20mm

918150220

$129

Ultra Quat Guard Cartridges

Ultra Quat Reagent Solution

Each

10-pk.

In water, 20mL/ampul

32441

$50

32541

$450

Extracted samples stored and analyzed in Silcote

™

CL7 deactivated

autosampler vials (cat.# 24671). Polypropylene vials and inserts

(e.g., cat.# 24651) also may be used.

diquat dibromide

paraquat dichloride

Paraquat & Diquat Calibration Mix

Each

1,000µg/mL each in water, 1mL/ampul

32437

$25

w/data pack

32437-500

$35

Dimethyldichlorosilane (DMDCS)

Each

5-pk.

Neat, 20mL/ampul

31840

$20

31840-510

$90

WCX Solid Phase Extraction Tubes

3mL/500mg, 50-pk., cat.# 26062,

$130

Limited-time introductory price—

order now!