549.2-1

METHOD 549.2 DETERMINATION OF DIQUAT AND PARAQUAT IN DRINKING 

WATER BY LIQUID-SOLID EXTRACTION AND HIGH 

PERFORMANCE LIQUID CHROMATOGRAPHY WITH 

ULTRAVIOLET DETECTION

Revision 1.0

June 1997

J.W. Hodgeson (USEPA), W.J. Bashe (Technology Applications Inc.), and J.W.

Eichelberger (USEPA) - Method 549.1, Revision 1.0 (1992)

J.W. Munch (USEPA) and W.J. Bashe (DynCorp/TAI) - Method 549.2, Revision 1.0 (1997)

NATIONAL EXPOSURE RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
549.2-2

METHOD 549.2

DETERMINATION OF DIQUAT AND PARAQUAT IN DRINKING WATER 

BY LIQUID-SOLID EXTRACTION AND HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY WITH ULTRAVIOLET DETECTION

1. SCOPE AND APPLICATION

1.1 This is a high performance liquid chromatography (HPLC) method for the

determination of diquat (1,1'-ethylene-2,2'-bipyridilium dibromide salt) and paraquat

(1,1'-dimethyl-4,4'- bipyridilium dichloride salt) in drinking water sources and

finished drinking water (1).

 Chemistry Abstract Services

Analytes Registry Number

Diquat 85-00-7 

Paraquat 1910-42-5

1.2 When this method is used to analyze unfamiliar samples, compound identification

should be supported by at least one additional qualitative technique. The use of a

photodiode array detector provides ultraviolet spectra that can be used for the

qualitative confirmation.

1.3 The method detection limits (MDL, defined in Sect. 13) (2) for diquat and paraquat

are listed in Table 1.

1.4 This method is restricted to use by or under the supervision of analysts experienced

in the use of HPLC. Each analyst must demonstrate the ability to generate

acceptable results with this method using the procedure described in Sect. 9.3.

2. SUMMARY OF METHOD

2.1 A measured volume of liquid sample, approximately 250 mL, is extracted using a C8

solid sorbent cartridge or a C8 disk which has been specially prepared for the

reversed-phase, ion-pair mode. The disk or cartridge is eluted with 4.5 mL of an

acidic aqueous solvent. After the ion-pair reagent is added to the eluate, the final

volume is adjusted to 5.0 mL. Liquid chromatographic conditions are described

which permit the separation and measurement of diquat and paraquat in the extract

by absorbance detection at 308 nm and 257 nm, respectively. A photodiode array
549.2-3

detector is utilized to provide simultaneous detection and confirmation of the

method analytes (1).

2.2 Analysis of diquat and paraquat is complicated by their ionic nature. Glassware

should be deactivated to prevent loss of analytes. The substitution of

polyvinylchloride (PVC) for glass is recommended.

3. DEFINITIONS

3.1 LABORATORY REAGENT BLANK (LRB) -- An aliquot of reagent water or

 other blank matrix that is treated exactly as a sample including exposure to all

glassware, equipment, solvents, reagents, internal standards, and surrogates that are

used with other samples. The LRB is used to determine if method analytes or other

interferences are present in the laboratory environment, the reagents, or the

apparatus.

3.2 FIELD REAGENT BLANK (FRB) -- An aliquot of reagent water or other blank

matrix that is placed in a sample container in the laboratory and treated as a sample

in all respects, including shipment to the sampling site, exposure to sampling site

conditions, storage, preservation and all analytical procedures. The purpose of the

FRB is to determine if method analytes or other interferences are present in the

field environment.

3.3 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or

other blank matrix to which known quantities of the method analytes are added in

the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to

determine whether the methodology is in control, and whether the laboratory is

capable of making accurate and precise measurements.

3.4 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) -- An aliquot of an

environmental sample to which known quantities of the method analytes are added in

the laboratory. The LFM is analyzed exactly like a sample, and its purpose is to

determine whether the sample matrix contributes bias to the analytical results. The

background concentrations of the analytes in the sample matrix must be

determined in a separate aliquot and the measured values in the LFM corrected for

background concentrations.

3.5 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing one

or more method analytes prepared in the laboratory using assayed reference

materials or purchased from a reputable commercial source.
549.2-4

3.6 PRIMARY DILUTION STANDARD SOLUTION (PDS) -- A solution of several

analytes prepared in the laboratory from stock standard solutions and diluted as

needed to prepare calibration solutions and other needed analyte solutions.

3.7 CALIBRATION STANDARD (CAL) -- A solution prepared from the primary

dilution standard solution and stock standard solutions and the internal standards and

surrogate analytes. The CAL solutions are used to calibrate the instrument response

with respect to analyte concentration.

3.8 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of 

known concentration which is used to fortify an aliquot of LRB or sample matrix. 

The QCS is obtained from a source external to the laboratory and different from 

the source of calibration standards. It is used to check laboratory performance with

externally prepared test materials.

3.9 EXTERNAL STANDARD (ES) -- A pure analyte(s) that is measured in an

experiment separate from the experiment used to measure the analyte(s) in the

sample. The signal observed for a known quantity of the pure external standard(s) is

used to calibrate the instrument response for the corresponding analyte(s). The

instrument response is used to calculate the concentrations of the analyte(s) in the

sample.

4. INTERFERENCES

4.1 Method interferences may be caused by contaminants in solvents, reagents,

glassware, and other sample processing hardware that lead to discrete artifacts

and/or elevated baselines in the chromatogram. All of these materials must be

routinely demonstrated to be free from interferences under the conditions of the

analysis by analyzing laboratory reagent blanks as described in Sect. 9.2.

4.1.1 Glassware must be scrupulously cleaned (3). Clean all glassware as soon

as possible after use by rinsing with the last solvent used in it. This should

be followed by detergent washing with hot water and rinses with tap water

and distilled water. It should then be drained dry and heated in a

laboratory oven at 130°C for several hours before use. Solvent rinses with

methanol may be substituted for the oven heating. After drying and

cooling, glassware should be stored in a clean environment to prevent any

accumulation of dust or other contaminants.

4.1.2 Before the initial use of all glassware, the procedure described in Sect.

4.1.1 should be followed. Silanization of all glassware which will come in

contact with the method analytes is necessary to prevent adsorption of the

diquat and paraquat cations onto glass surfaces (7.13).
549.2-5

4.1.3 Plasticware should be washed with detergent and rinsed in tap water and

distilled water. It should be drained dry before use.

4.1.4 The use of high purity reagents and solvents helps to minimize interference

problems. Purification of solvents by distillation in all-glass systems may

be required.

4.2 Interferences may be caused by contaminants that are coextracted from the sample. 

The extent of matrix interferences will vary considerably from source to source. 

Because of the selectivity of the detection system used here, no interferences have

been observed in the matrices studied. If interferences occur, some additional

cleanup may be necessary.

4.3 This method has been shown to be susceptible to interferences from Ca+2 and Mg+2

ions which may be present in hard water samples. These divalent cations can cause

low recovery of method analytes, by interfering with the ion exchange process. Use

of LFM samples can assist the analyst in evaluating the affect of these interferences

in different matrices.

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been

precisely defined. Each chemical compound should be treated as a potential health

hazard. From this viewpoint, exposure to these chemicals must be minimized. The

laboratory is responsible for maintaining a current awareness file of OSHA

regulations regarding the safe handling of the chemicals specified in this method. A

reference file of material data handling sheets should also be made available to all

personnel involved in the chemical analysis.

6. EQUIPMENT AND SUPPLIES

6.1 SAMPLING EQUIPMENT, discrete or composite sampling.

6.1.1 Grab sample bottle -- Amber polyvinylchloride (PVC) high density, 1-L,

fitted with screw caps. If amber bottles are not available, protect samples

from light. The container must be washed, rinsed with deionized water,

and dried before use to minimize contamination.

6.2 GLASSWARE

6.2.1 Volumetric flask -- 5 mL, silanized

6.2.2 Autosampler vials -- 4 mL, silanized
549.2-6

6.3 BALANCE -- analytical, capable of accurately weighing 0.0001 g

6.4 pH METER -- capable of measuring pH to 0.1 units

6.5 HPLC APPARATUS

6.5.1 Isocratic pumping system, constant flow (Waters M6000A HPLC pump or

equivalent).

6.5.2 Manual injector or automatic injector, capable of delivering 200 μL.

6.5.3 Analytical column -- any column which produces results equal to or 

better than those listed below may be used.

6.5.3.1 Phenomenex Spherisorb (3 μ, 100mm X 4.6mm),

Hamilton PRP-1, (5 μ, 150 mm x 4.1 mm), or MICRA

NPS RP-C18 (1.5 μ, 33 mm X 4.6mm) or equivalent.

6.5.3.2 Guard column, C8

 packing

6.5.4 Column Oven (Fiatron, Model CH-30 and controller, Model TC-50, or

equivalent).

6.5.5 Photodiode array detector (LKB 2140 Rapid Spectral Detector or

equivalent). Any detector which has the capability to switch between 

257 nm and 308 nm may be used.

6.5.6 Data system -- Use of a data system to report retention times and peak

areas is recommended but not required.

6.6 EXTRACTION APPARATUS

6.6.1 Liquid solid extraction cartridges, C8, 500 mg or equivalent.

Note: EPA has observed significant variability between brands of C8

 

LSE media, and also between lots of the same brand of C8 LSE media.

Verification of analyte recovery should be performed any time a new brand

or lot of LSE media is used.

6.6.2 Liquid solid extraction system (Baker - 10 SPE, or equivalent).

6.6.3 Liquid solid extraction disks (C-8 Empore, 47 mm, or equivalent).

Note: EPA has observed significant variability between brands of C8 LSE

media, and also between lots of the same brand of C8

 LSE media.
549.2-7

Verification of analyte recovery should be performed any time a new brand

or lot of LSE media is used.

6.6.4 Liquid solid extraction system, Empore, 47 mm, 6 position manifold

(Varian Associates or equivalent).

6.6.5 Vacuum pump, 100 VAC, or other source of vacuum, capable of

maintaining a vacuum of 8-10 mm of Hg.

6.6.6 Membrane Filters, 0.45 μm pore-size, 47 mm diameter, Nylon.

7. REAGENTS AND STANDARDS

7.1 DEIONIZED WATER -- Water which has been processed through a series of

commercially available filters including a particulate filter, carbon bed, ion exchange

resin and finally a bacterial filter to produce deionized, reagent grade water. Any

other source of reagent water may be used provided the requirements of Sect. 9 are

met.

7.2 METHANOL -- HPLC grade or higher purity

7.3 ORTHOPHOSPHORIC ACID, 85% (w/v) -- Reagent grade

7.4 DIETHYLAMINE -- Reagent grade

7.5 CONCENTRATED SULFURIC ACID -- ACS reagent grade

7.6 SODIUM HYDROXIDE -- Reagent grade

7.7 CONCENTRATED HYDROCHLORIC ACID, 12 N -- Reagent grade

7.8 CETYL TRIMETHYL AMMONIUM BROMIDE, 95% -- Aldrich Chemical

7.9 SODIUM THIOSULFATE -- Reagent grade

7.10 1-HEXANESULFONIC ACID, sodium salt, 98%, Aldrich Chemical

7.11 1-HEPTANESULFONIC ACID, sodium salt, 98%, Aldrich Chemical

7.12 AMMONIUM HYDROXIDE, ACS, Concentrated

7.13 SYLON CT -- Silanization solution, Supelco
549.2-8

7.14 REAGENT SOLUTIONS

7.14.1 Conditioning solution A -- Dissolve 0.500 g of cetyl trimethyl ammonium

bromide and 5 mL of concentrated ammonium hydroxide in 500 mL of

deionized water and dilute to 1000 mL in volumetric flask.

7.14.2 Conditioning solution B -- Dissolve 10.0 g of 1-hexanesulfonic acid,

sodium salt and 10 mL of concentrated ammonium hydroxide in 250 mL of

deionized water and dilute to 500 mL in volumetric flask.

7.14.3 Sodium hydroxide solution, 10% w/v -- Dissolve 50 g of sodium hydroxide

into 400 mL of deionized water and dilute to 500 mL in a volumetric flask.

7.14.4 Hydrochloric acid, 10% v/v -- Add 50 mL of concentrated hydrochloric

acid to 400 mL of deionized water and dilute to 500 mL in a volumetric

flask.

7.14.5 Disk or cartridge eluting solution -- Add 13.5 mL of orthophosphoric acid

and 10.3 mL of diethylamine to 500 mL of deionized water and dilute to

1000 mL in a volumetric flask.

7.14.6 Ion-pair concentrate -- Dissolve 3.75 g of 1-hexanesulfonic acid in 15 mL

of the disk or cartridge eluting solution and dilute to 25 mL in a volumetric

flask with the disk eluting solution.

7.15 STOCK STANDARD SOLUTIONS

7.15.1 Diquat dibromide and Paraquat dichloride.

7.15.2 Stock diquat and paraquat solutions (1000 mg/L). Dry diquat and 

paraquat salts in an oven at 110°C for 3 hr. Cool in a desiccator. 

Repeat process to a constant weight. Weigh 0.1968 g of dried diquat 

salt and 0.1770 g of dried paraquat salt and place into a silanized glass or

polypropylene 100-mL volumetric flask. Dissolve with approximately 

50 mL of deionized water. Dilute to the mark with deionized water.

7.15.3 The salts used in preparing the stock standards (Sect. 7.15.2) were taken 

to be diquat dibromide monohydrate and paraquat dichloride 

tetrahydrate (4). The drying procedure described in Sect. 7.15.2 will

provide these hydration levels.

7.16 MOBILE PHASE -- Make mobile phase by adding the following to 500 mL of
549.2-9

deionized water: 13.5 mL of orthophosphoric acid; 10.3 mL of diethylamine; 3.0 g

of 1-hexanesulfonic acid, sodium salt. Mix and dilute with deionized water to a final

volume of 1 L.

8. SAMPLE COLLECTION, PRESERVATION AND STORAGE

8.1 Grab samples must be collected in either amber PVC high density bottles or silanized

amber glass bottles. Conventional sampling procedures should be followed (5). 

Automatic sampling equipment must be free as possible of adsorption sites which

might extract the sample.

8.2 The samples must be iced or refrigerated at approximately 4°C from the time of

collection until extraction. The analytes are light-sensitive, particularly diquat.

8.3 Samples which are known or suspected to contain residual chlorine must be

preserved with sodium thiosulfate (100 mg/L). Samples which are biologically

active must be preserved by adding sulfuric acid to pH 2 to prevent adsorption of

method analytes by the humectant material.

8.4 Analyte stability over time may depend on the matrix tested. All samples must be

extracted within 7 days of collection. Extracts must be analyzed within 21 days of

extraction (6).

9. QUALITY CONTROL

9.1 Minimum quality control (QC) requirements are initial demonstration of laboratory

capability, analysis of laboratory reagent blanks, laboratory fortified matrix samples,

and laboratory fortified blanks. The MDL (2) must also be determined for each

analyte. The analyst should institute quality control practices to ensure that the

brand and lot of LSE media being used show reliable recoveries of method analytes. 

The laboratory must maintain records to document the quality of the data generated. 

Additional quality control practices are recommended.

9.2 LABORATORY REAGENT BLANKS (LRB) -- Before processing any samples,

the analyst must analyze a LRB to demonstrate that all deactivated glassware or

plasticware, and reagent interferences are reasonably free of contamination. In

addition, each time a set of samples is extracted or reagents are changed, a LRB

must be analyzed. If within the retention time window (Sect. 11.4.2) of the analyte

of interest, the LRB produces a peak that would prevent the determination of that

analyte, determine the source of contamination and eliminate the interference before

processing samples.

9.3 INITIAL DEMONSTRATION OF CAPABILITY
549.2-10

9.3.1 Prepare laboratory fortified blanks (LFBs) at analyte concentrations of 100

μg/L. With a syringe, add 25 μL of the stock standard (Sect. 7.15.2) to at

least four 250 mL aliquots of reagent water and analyze each aliquot

according to procedures beginning in Sect. 11.2.

9.3.2 Calculate the recoveries and relative standard deviation (RSD). The

recovery (R) value for each sample, should be within ± 30% of the fortified

amount. The RSD of the mean recovery should be less than 30%. For

analytes that fail this critera, initial demonstration procedures should be

repeated.

9.3.3 For each analyte, determine the MDL. Prepare a minimum of 4-7 LFBs (7

is recommended) at a low concentration. Fortification concentrations in

Table 1 may be used as a guide, or use calibration data obtained in Section

10 to estimate a concentration for each analyte that will produce a peak

with a 3-5 times signal to noise response. Extract and analyze each

according to Sections 11 and 12. It is recommended that these LFBs be

prepared and analyzed over a period of several days, so that day to day

variations are reflected in precision measurements. Calculate mean

recovery and standard deviation for each analyte. Use the standard

deviation and the equation in Section 13 to calculate the MDL.

9.3.4 The initial demonstration of capability is used primarily to preclude a

laboratory from analyzing unknown samples via a new, unfamiliar method

prior to obtaining some experience with it. As laboratory personnel gain

experience with this method the quality of the data should improve beyond

the requirements stated in Sect. 9.3.2.

9.4 The analyst is permitted to use other HPLC columns, HPLC conditions, or HPLC

detectors to improve separations or lower analytical costs. Each time such method

modifications are made, the analyst must repeat the procedures in Sect. 9.3.

9.5 LABORATORY FORTIFIED BLANKS

9.5.1 The laboratory must analyze at least one laboratory fortified blank (LFB)

sample per sample set (all samples extracted within a 24-hr period). The

fortified concentration of each analyte in the LFB should be 10 times the

MDL. If the recovery of either analyte falls outside the control limits

(Sect. 9.5.2), that analyte is judged out of control, and the source of the

problem must be identified and resolved before continuing analyses.

9.5.2 Until sufficient data become available, usually a minimum of results from

20 to 30 analyses, the laboratory should assess laboratory performance
549.2-11

against the control limits in Sect. 9.3.2. When sufficient internal

performance data become available, develop control limits from the mean

percent recovery (R) and standard deviation (Sr) of the percent recovery. 

These data are used to establish upper and lower control limits as follows:

UPPER CONTROL LIMIT = R + 3Sr

LOWER CONTROL LIMIT = R - 3Sr

After each five to ten new recovery measurements, new control limits

should be calculated using only the most recent 20-30 data points. 

These calculated control limits should not exceed the limits established in

Sect. 9.3.2.

9.6 LABORATORY FORTIFIED SAMPLE MATRIX

9.6.1 The laboratory must add a known fortified concentration to a minimum of

10% of the samples or one fortified sample per set, whichever is greater. 

The fortified concentration should not be less than the background

concentration of the original sample. Ideally, the fortified concentration

should be the same as that used for the laboratory fortified blank (Sect.

9.5). Over time, samples from all routine samples sources should be

fortified.

9.6.2 Calculate the accuracy as percent recovery (R) for each analyte, 

corrected for background concentrations measured in the original sample,

and compare these values to the control limits established in Sect. 9.5.2

from the analyses of LFBs.

9.6.3 If the recovery of any such analyte falls outside the designated range, 

and the laboratory performance for that analyte is shown to be in control

(Sect. 9.5), the recovery problem encountered with the dosed sample is

judged to be matrix related, not system related. The result for that analyte

in the original sample is labeled suspect/matrix to inform the data user that

the results are suspect due to matrix effects.

9.7 QUALITY CONTROL SAMPLES (QCS) -- Each quarter the laboratory should

analyze one or more QCS. If criteria provided with the QCS are not met, 

corrective action should be taken and documented.

9.8 The laboratory may adopt additional quality control practices for use with this

method. The specific practices that are most productive depend upon the needs of
549.2-12

 the laboratory and the nature of the samples. For example, field or laboratory

duplicates may be analyzed to assess the precision of the environmental

measurements or field reagent blanks may be used to assess contamination of

samples under site conditions, transportation and storage.

10. CALIBRATION AND STANDARDIZATION

10.1 Establish HPLC operating conditions indicated in Table 1. The chromatographic

system can be calibrated using the external standard technique.

10.2 In order to closely match calibration standards to samples, process standards by 

the following method: Using C8 disks or C8 cartridges conditioned according to

Sect. 11.2.1, pass 250 mL of reagent water through the disk or cartridge and 

discard the water. Dry the disk or cartridge by passing 5 mL of methanol through 

it. Discard the methanol. Pass 4.0 mL of the eluting solution through the disk or

cartridge and catch in a 5 mL silanized volumetric flask. Fortify the eluted solution

with 100 μL of the ion-pair concentrate and with 500 μL of the stock standard and

dilute to the mark with eluting solution. This provides a 10:1 dilution of the stock. 

Use serial dilution of the calibration standard by the same method to achieve lower

concentration standards.

10.3 Analyze a minimum of three calibration standards prepared by the procedure

described in Sect. 10.2 utilizing the HPLC conditions given in Table 1. From full

spectral data obtained, extract the 308 nm chromatographic trace for diquat and 

the 257 nm trace for paraquat. Integrate and record the analyte peak areas. Any

mathematical manipulations performed to aid in data reduction must be recorded and

performed on all sample chromatograms. Tabulate the peak area against quantity

injected. The results may be used to prepare calibration curves for diquat and

paraquat.

10.4 The working calibration curve must be verified on each working day by

measurement of a calibration check standard, at the beginning of the analysis day. 

These check standards should be at two different concentration levels to verify the

calibration curve. For extended periods of analysis (greater than 8 hr), it is strongly

recommended that check standards be interspersed with samples at regular intervals. 

If the response for any analyte varies from the predicted response by more than

+20%, the test must be repeated using a fresh calibration standard. If the results still

do not agree, generate a new calibration curve.

11. PROCEDURE
549.2-13

11.1 SAMPLE CLEANUP -- Cleanup procedures may not be necessary for a relatively

clean sample matrix. The cleanup procedures recommended in this method have

been used for the analysis of various sample types. If particular circumstances

demand the use of an alternative cleanup procedure, the analyst must demonstrate

that the recovery of the analytes is within the limits specified by the method.

11.1.1 If the sample contains particulates, or the complexity is unknown, the

entire sample should be passed through a 0.45 μm Nylon membrane filter

into a silanized glass or plastic container.

11.1.2 Store all samples at 4°C unless extraction is to be performed immediately.

11.2 CARTRIDGE EXTRACTION

11.2.1 Before sample extraction, the C8 extraction cartridges must be conditioned

by the following procedure.

11.2.1.1 Place a C8 cartridge on the cartridge extraction system

manifold.

11.2.1.2 Elute the following solutions through the cartridge in the

stated order. Take special care not to let the column go

dry. The flow rate through the cartridge should be

approximately 10 mL/min.

11.2.1.2.1 Cartridge Conditioning Sequence

a. Deionized water, 5 mL

b. Methanol, 5 mL

c. Deionized water, 5 mL

d. Conditioning Solution A, 5 mL

e. Deionized water, 5 mL

f. Methanol, 10 mL

g. Deionized water, 5 mL

h. Conditioning Solution B, 20 mL

11.2.1.2.2 Retain conditioning solution B in the C8

cartridge to keep it activated.

11.2.2 The C8 cartridges should not be prepared more than 48 hr prior to use. 

After conditioning, the cartridge should be capped and stored at 4°C.
549.2-14

11.2.3 Measure a 250-mL aliquot of the sample processed through Sect. 11.1 in 

a silanized, volumetric flask.

11.2.4 Measure the pH of the sample. If the pH is between 7.0 and 9.0, the

sample can be analyzed without adjustment. If the pH is not in this range,

or if the sample has been acidified for preservation purposes, adjust the

pH of sample to between 7.0 and 9.0 with 10% w/v NaOH (aq) or 10%

v/v HCl (aq) before extracting. 

11.2.5 Place a conditioned C8

 cartridge on the solid phase extraction vacuum

manifold. Attach a 60-mL reservoir to the C8 cartridge with the

appropriate adapter. Put a 250- mL beaker inside the extraction manifold

to catch waste solutions and sample. Transfer the measured volume in

aliquots to the reservoir. Turn on the vacuum pump or house vacuum and

adjust the flow rate to 3 to 6 mL/min. Filter the sample through the C8

cartridge, and wash the column with 5 mL of HPLC grade methanol. 

Continue to draw the vacuum through the cartridge for one additional

minute to dry the cartridge. Release the vacuum and discard the sample

waste and methanol.

11.2.6 Place a silanized 5-mL volumetric flask beneath the collection stem in the

vacuum manifold. Add 4.5 mL of the eluting solution to the sample

cartridge. Turn on the vacuum and adjust the flow rate to 1 to 2 mL/min.

11.2.7 Remove the 5-mL volumetric flask with the extract. Fortify the extract

with 100 μL of the ion-pair concentrate. Adjust the volume to the mark

with cartridge eluting solution, mix thoroughly, and seal tightly until

analyzed.

11.2.8 Analyze sample by HPLC using conditions described in Table 1. 

Integration and data reduction must be consistent with that performed in

Sect. 10.3. 

11.3 DISK EXTRACTION -- The top surface of the disk matrix must remain covered

with liquid at all times. If the disk is exposed to air at any step in the disk cleanup

procedure, the elution procedure should be restarted. Eluants applied to the disk

should be allowed to soak into the disk before drawing them through. Vacuum

should then be applied to draw most of the eluant through the disk, leaving a thin

layer of solution on the top of the disk. Flow rate through the disk is not critical.

11.3.1 Assemble the 47 mm disk in the disk holder or a filter apparatus. Be sure

that the surfaces of the holder are either silanized glass or Teflon coated to

avoid adsorption or decomposition of the analytes.
549.2-15

11.3.2 Measure the pH of the sample. If the pH is between 7.0 and 9.0, the

sample can be analyzed without adjustment. If the pH is not in this range,

or if the sample has been acidified for preservation purposes, adjust the

pH of sample to between 7.0 and 9.0 with 10% w/v NaOH (aq) or 10%

v/v HCl (aq) before extracting. 

11.3.3 Apply 10 mL of methanol to the disk. Apply vacuum to begin elution, then

immediately vent the vacuum when drops of liquid appear at the drip tip. 

Allow the methanol to soak into the disk for a minimum of 1 min, then

reapply the vacuum to bring the methanol to just above the top surface of

the disk.

11.3.4 Draw 2 10-mL aliquots of reagent water through to just above the top

surface of the disk to remove the methanol.

11.3.5 Apply 10 mL of Conditioning Solution A to the disk. As with the

methanol, draw a few drops through, then allow the disk to soak for at

least 1 min. Draw the Conditioning Solution A through the disk to just

above its top surface.

11.3.6 Draw 2 10-mL aliquots of reagent water through to just above the top

surface of the disk.

11.3.7 Apply 20 mL of Conditioning Solution B to the disk. Draw a few drops

through using vacuum and allow the disk to soak for at least 1 min. Draw

the remaining Conditioning Solution B through to just above the top

surface of the disk.

11.3.8 Measure 250 mL of the sample using a polypropylene graduated cylinder. 

Pour the sample aliquot into the filtration apparatus reservoir and apply

vacuum to draw the sample through the disk. Pass the entire sample

through the disk, leaving no liquid on the top of the disk, then vent the

vacuum.

11.3.9 Assemble a graduated collection tube under the drip tip with the tip

descending into the tube slightly to prevent losses of eluants. Be sure 

the tube will hold at least 10 mL of eluate.

11.3.10 With the vacuum vented, drip enough methanol onto the disk to cover it

completely (0.5-1.0 mL). Allow the methanol to soak into the disk for 1

min. Add more methanol as needed to keep the disk covered as it soaks.
549.2-16

11.3.11 Pipet 4 mL of Disk Eluting Solvent onto the disk. Apply vacuum until

drops appear at the drip tip. Vent the vacuum and allow the disk to soak

for 1 min.

11.3.12 Draw the Disk Eluting Solution through to just above the top surface of

the disk. Add 4 mL of Disk Eluting Solution and draw it completely

through the disk. Tap the disk holder assembly gently to loosen 

adhering drops into the collection tube.

11.3.13 Vent the vacuum, disassemble the disk extraction device, and remove the

collection tube. Fortify the extract with 200 μL of the ion-pair

concentrate. Add Disk Eluting Solution to the tube to a final volume of 

10 mL.

11.3.14 Analyze samples by HPLC. Some suggested conditions, which were used

in developing this method, are listed in Table 1. This table includes the

retention times and MDLs that were obtained using the suggested

conditions. 

11.4 IDENTIFICATION OF ANALYTES

11.4.1 Identify a sample component by comparison of its retention time to the

retention time of a reference chromatogram. If the retention time of an

unknown compound corresponds, within limits (Sect. 11.4.2), to the

retention time of a standard compound, then identification is considered

positive.

11.4.2 The width of the retention time window used to make identification should

be based upon measurements of actual retention time variations of

standards over the course of a day. Three times the standard deviation of a

retention time can be used to calculate a suggested window size for a

compound. However, the experience of the analyst should weigh heavily in

the interpretation of chromatograms.

11.4.3 Identification requires expert judgment when sample components are not

resolved chromatographically. When peaks obviously represent more than

one sample component (i.e., broadened peak with shoulder(s) or valley

between two or more maxima), or any time doubt exists over the

identification of a peak in a chromatogram, a confirmatory technique must

be employed. Through the use of the photodiode array detector, full

spectra of the analyte peaks are obtained (Figure 2). When a peak of an

unknown sample falls within the retention time windows of method

analytes, confirm the peak identification by spectral comparison with
549.2-17

analyte standards.

If additional confirmation is required, replace the 1-hexanesulfonic acid salt

with 1-heptanesulfonic acid, sodium salt in the mobile phase and reanalyze

the samples. Comparison of the ratio of retention times in the samples by

the two mobile phases with that of the standards will provide additional

confirmation.

11.4.4 If the peak area exceeds the linear range of the calibration curve, a smaller

sample volume should be used. Alternatively, the final solution may be

diluted with mobile phase and reanalyzed.

12. DATA ANALYSIS AND CALCULATIONS

12.1 Determine the concentration of the analytes in the sample.

12.1.1 Calculate the concentration of each analyte injected from the peak area

using the calibration curves in Sect. 10.3 and the following equation.

Concentration, μg/L = (A) x (VF) 

 (VS)

where: A = Peak area of analyte in sample extract

VF = Final volume of sample extract, in mL

VS = Sample volume, in mL

12.2 Report results as micrograms per liter without correction for recovery data. When

duplicate and fortified samples are analyzed, report all data obtained with sample

results.

13. METHOD PERFORMANCE

13.1 METHOD DETECTION LIMITS -- The method detection limit (MDL) is defined

as the minimum concentration of a substance that can be measured and reported with

99% confidence that the value is above the background level (2). The MDL data

listed in Table 1 were obtained using disks with reagent water as the matrix.

MDL = S t(n-1,1-alpha = 0.99)

where:

t

(n-1,1-alpha = 0.99) = Student's t value for the 99% confidence level with n-1 degrees 

 of freedom
549.2-18

n = number of replicates

S = standard deviation of replicate analyses.

13.2 This method has been tested for linearity of recovery from fortified reagent water

and has been demonstrated to be applicable over the range from approximately 4

x MDL to 100 x MDL.

13.3 Single-laboratory precision and accuracy results at several concentration levels in

drinking water matrices using disks are presented in Table 2.

13.4 This methodology has been shown to be sensitive to brand differences and even

lot differences in C8 LSE media. This is presumed to be due to variations in

manufacturing processes. If the method does not demonstrate performance data

similar to those demonstrated in Sect. 17, C8 LSE media should be obtained

from a different source.

14. POLLUTION PREVENTION

14.1 Only an extremely small volume of an organic solvent is used in this method. A

maximum of 15 mL of methanol is used per sample to condition each cartridge or

disk. Methanol is not considered to be a toxic or hazardous solvent. All other

chemicals used in this method are can be handled in a non-hazardous way when

used in the prescribed manner and amounts.

14.2 For information about pollution prevention that may be applicable to laboratory

operations, consult "Less is Better: Laboratory Chemical Management for Waste

Reduction" available from the American Chemical Society's Department of

Government Relations and Science Policy, 1155 16th Street N.W., Washington,

D.C. 20036.

15. WASTE MANAGEMENT

15.1 There are generally no waste management problems involved with discarding

spent or left over samples in this method since most often the sample matrix is

drinking water. If a sample is analyzed which appears to be highly contaminated

with chemicals, analyses should be carried out to assess the type and degree of

contamination so that the samples may be discarded properly. The Agency

requires that laboratory waste management practices be conducted consistent

 with all applicable rules and regulations, and that laboratories protect the air,

water, and land by minimizing and controlling all releases from fume hoods and

bench operations. Also, compliance is required with any sewage discharge

restrictions. For further information on waste management, consult "The Waste
549.2-19

Management Manual for Laboratory Personnel" also available from the American

Chemical Society at the address in Sect. 14.2.

16. REFERENCES

1. Lagman, L. H. and J. R. Hale, "Analytical Method for the Determination of

Diquat in Aquatic Weed Infested Lakes and Rivers in South Carolina",

Technology Conference Proceedings, WQTC-15, American Water Works

Association, November 15-20, 1987.

2. Glaser, J. A., D. L. Foerst, G. M. McKee, S. A. Quave, and W. L. Budde, "Trace

Analyses for Wastewaters", Environ. Sci. Technol., 15, 1426, 1981.

3. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for

Preparation of Sample Container and for Preservation", American Society for

Testing and Materials, Philadelphia, PA, p. 679, 1980.

4. Worobey, B. L., "Analytical Method for the Simultaneous Determination of

Diquat and Paraquat Residues in Potatoes by High Pressure Liquid

Chromatography", Pestic. Sci 18(4), 245, 1987.

5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for

Sampling Water", American Society for Testing and Materials, Philadelphia, PA,

p. 76, 1980.

6. Hodgeson, J.W., Bashe, W.J. and J.W. Eichelberger, "Method 549.1 -

Determination of Diquat and Paraquat in Drinking Water by Liquid-Solid

Extraction and High Performance Liquid Chromatography with Ultraviolet

Detection", Methods for the Determination of Organic Compounds in Drinking

Water, Supplement II, EPA/600/R-92/129, U.S. Environmental Protection

Agency, Envirinmental Monitoring Systems Laboratory, Cincinnati, Ohio, 45268,

1992.
549.2-20

17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

 CONDITIONS AND METHOD DETECTION LIMITS

 

 Method Detection Limitsa

Analyte (μg/L)

 (disks)

 

Diquat 0.72 

Paraquat 0.68

 

HPLC Conditions:

 Column: Phenomenex Spherisorb, 3μ, 4.6 mm x 100 mm

 Column Temperature 35.0° C

 Flow Rate: 2.0 mL/min., Ion-Pair Mobile Phase

(Sect. 7.16)

 Injection Volume: 200 μL

Photodiode Array Detector Settings:

 Wavelength Range: 210 - 370 nm

 Sample Rate: 1 scan/sec.

 Wavelength Step: 1 nm

 Integration Time: 1 sec.

 Run Time: 5.0 min.

Quantitation

 Wavelengths: Diquat - 308 nm

Paraquat - 257 nm

 

aMDL data were obtained from five samples fortified at 2.5 μg/L diquat and 2.5 μg/L paraquat.
549.2-21

TABLE 2. SINGLE OPERATOR ACCURACY AND PRECISION

 USING DISK (N = 5 FOR EACH TYPE OF WATER)

 

DIQUAT

Type of Fortified 2.5 μg/L Fortified 10.5 μg/L Fortified 52.5 μg/L

 Water Mean % Rec. % RSD Mean % Rec. % RSD Mean % Rec. % RSD

 

RW 90.9 8.4 94.1 5.2 92.1 2.9

TW 91.7 6.5 93.6 3.1 93.0 5.3

GW 91.4 6.4 93.7 3.0 90.2 3.3

 

PARAQUAT

Type of Fortified 2.5 μg/L Fortified 10 μg/L Fortified 50 μg/L

 Water Mean % Rec. % RSD Mean % Rec. % RSD Mean % Rec. % RSD

 

RW 94.7 7.7 92.3 5.5 88.8 4.2

DW 93.5 6.6 89.7 3.6 91.4 6.5

GW 92.0 8.1 89.9 2.5 90.4 2.5

 

RW = Reagent Water

TW = Tap Water (Dechlorinated with sodium thiosulfate)

GW = Ground Water

All samples adjusted to pH 7.