EPA Methods Book - Waters Corporation · Waters Quattro micro API Mass Spectrometer The Waters...

EPA Methods Book

Waters® is at the forefront of new developments in liquid chromatography

and mass spectrometry technologies that are used extensively in methods

for environmental regulatory compliance. Waters LC and LC/MS systems

for environmental analysis combine versatile and robust instrumentation

capabilities, a broad range of column and sample prep chemistries, and

powerful software tools to ensure that environmental laboratories meet

increasingly stringent regulatory standards for numerous compound types.

Waters LC and LC/MS systems are appropriate for EPA prescribed LC and

LC/MS methodologies, continually delivering results that will meet or exceed

EPA detection limits.

Visit www.waters.com/environmental for updated information on these methods and Waters solutions for environmental testing.

Waters Alliance HPLC System The Alliance® HPLC systems offer flexibility with easy-to-configure

instrumentation modules that address the needs of multiple

applications. Alliance is built around the 2695 Separations Module,

which offers integrated solvent and sample management. The 2695

Separations Module is designed to work with both MassLynx™ mass

spectrometry and Empower™ 2 chromatography software, the complete

range of Waters HPLC column chemistries and a variety of Waters

high performance detectors, including photodiode array (PDA), multi-

wavelength fluorescence and dual-wavelength absorbance.

Waters Alliance System for Carbamate AnalysisThe Alliance HPLC system for carbamate analysis is a completely

integrated system that detects carbamate at parts-per-trillion levels

necessary for regulatory compliance, and exceeds precision and accu-

racy requirements mandated by the U.S. EPA and AOAC methods. The

analysis of glyphosate can also be performed on the same system.

FEATURED SYST EMS

Waters ACQUITY UPLC System The ACQUITY UPLC® system features a novel liquid chromatography

technology that utilizes 1.7 µm stationary phase pressure-tolerant

particles. When combined with high pressure fluidic modules, a fast

response detector and integrated data analysis software, UPLC® technol-

ogy delivers faster run times, better resolution and greater sensitivity.

Waters Quattro micro API Mass Spectrometer The Waters Quattro micro™ API incorporates the finest high-precision

tandem quadrupole mass analyzer technology in only 15.3 inches

(390 mm) of linear bench space. The mass analyzer has a standard m/z

range of 2 to 2000 and a sensitivity equivalent to systems that are

three times the size.

Waters ACQUITY UPLC System with the TQ DetectorThe ACQUITY TQD is a smaller, easier to use, enhanced capability

tandem quadrupole mass detector specifically designed as an afford-

able, fast MS/MS system compatible with UPLC. Labs will benefit from

robust and reliable performance and walk-up operation. Interactive

IntelliStart™ diagnostics software allows for worry-free system optimi-

zation and performance checks.

HExAvALEnT CHRoMIUM AnALYSIS

Waters Alliance HPLC System EPA method 218.6 describes the use of high performance liquid chroma-

tography (HPLC) with ultra violet (UV) detection for the identification

of hexavalent chromium in drinking, ground, municipal and industrial

waste waters.

ConDIT IonS

Instrument: Waters Alliance HPLC system with UV detector

and Waters post column reaction module

Eluent: 25 mM ammonium sulfate in

10 mM ammonium hydroxide

Column: Waters IC-Pak™ Anion HC

(part no. WAT026770) @ 30 °C

Injection: 100 µL of standard or sample (see below)

Flow rate: 1.5 mL/min

Post column: 0.5 mL/min. Reactor temperature is 30 °C

Detection: UV @ 530 nm

Data: Waters Empower software

ELUEnT P REPARAT Ion

Prepare a stock eluent by dissolving 33.0 grams of ammonium sul-

fate and 6.5 mL reagent ammonium hydroxide in water and dilute to

1 liter. Prepare working eluent by diluting 100 mL of the stock eluent

to 1 liter with water, filter and degas. A pH control buffer is prepared

with 33 grams of ammonium sulfate and 6.5 mL reagent ammonium

hydroxide in water and diluted to 100 mL with water.

PoST CoLUMn REAGEnT P REPARAT Ion

Dissolve 0.25 grams of 1,5-diphenylcarbazide in 50 mL methanol.

Gradually stir 14 mL of concentrated sulfuric acid to 300 mL of water,

(18 M). Add the methanolic diphenylcarbazide solution to the aqueous

sulfuric acid with stirring. Dilute this mixture to 500 mL with water, mix

well then filter and degas.

STAnDARD P REPARAT Ion

Dissolve 0.45 grams of sodium chromate tetrahydrate in water and

dilute to 100 mL. The concentration is 1000 mg/L as chromium (VI).

Dilute as necessary to prepare calibration curve.

SAMPLE P REPARAT Ion

Filter aqueous samples through a 0.45 µm hydrophilic filter and

adjust pH to 9.0 to 9.5 with buffer.

CoMMEnTS

n Post column reagent should be prepared fresh every four days.n A method detection limit (MDL) of 0.15 parts per billion (ppb) was

calculated using this method.

0.00 3.00 6.00 9.00

0.0

5 A

U

Minutes

Figure 1. Hexavalent Chrome at 500 ppb.

218.6

Alliance HPLC System with TQ Detector or Quattro micro APIEPA methods 331 and 6850 both describe the use of high performance

liquid chromatography (HPLC) with single or tandem quadrupole mass

spectrometry for the analysis of perchlorate (ClO4-). EPA method 331.0

is used for the determination of perchlorate in drinking water and EPA

method 6850 is utilized for more complex matrices, namely, surface,

ground, waste and salt water, soils and solid wastes.

LC ConDIT IonS

Instrument: Waters Alliance HPLC and Waters conductivity detector

Eluent: 25 mM ammonium bicarbonate,

pH = 10 in 50% acetonitrile

Column: Waters IC Pak A/HR, 4.6 x 150 mm, 7 µm

(part no. WAT026765) @ 30 °C

Injection: 100 µL


Data: Waters MassLynx™ software

MS ConDIT IonS

Instrument: Waters TQ detector or Quattro micro API mass spectrometer

Ion Mode: Electrospray negative (ESI-)

Mode: Multiple reaction monitoring (MRM). The MRM transi-

tions, cone voltages (CV) and collision energies (CE) are

listed in Table 1.

CoMPoUnD MRM TRAnSITIon Cv CE

Perchlorate (quantification) 99.1>82.7 40 30

Perchlorate (confirmation) 101.1>84.7 40 30

Internal Standard (Cl18O4-) 107.1>88.7 40 30


EPA METHoD SAMPLE MATRIx SAMPLE PREPARATIon

331.0 Drinking water No sample pretreatment. Add internal standard.

6850 Surface, ground, waste and salt water

Add internal standard. Filter sample using a 0.45 µm or 0.2 µm syringe filter.

6850 Soils and solid waste Add reagent water, centrifuge and filter supernatant using a 0.45 µm or 0.2 µm syringe filter. Cleanup with C18 cartridge if necessary.

Table 2. EPA methods 331.0 and 6850 sample matrix and preparation.

CoMMEnTS

n Perchlorate can be determined in High Total Dissolved Solids

(HTDS) water at the sub-ppb levels. HTDS is a synthetic solution

defined as 1000 mg/L each of bicarbonate, chloride and sulfate

prepared in drinking water.n Chromatographic selectivity of the polymethacrylate based Waters

IC-Pak A/HR column, using ammonium bicarbonate and aceto-

nitrile, eliminates the interference from sulfate and the need for

sample prep to remove chloride and sulfate.n Positive perchlorate identification is achieved using a second MRM

transition of 101.1>84.7 for the 37Cl isotope and the ion ratios of

the 35Cl and 37Cl isotopes. Although H34SO4- sulfate may give a

99.1>82.7 response, it will not yield the required Cl isotope ratio.

DET ERMInAT Ion oF PERCHLoRAT E In vARIoUS Env IRonMEnTAL MAT RICES

Table 1. Optimized MRM transition parameters for EPA methods 331.0 and 6850 in ES-.

Figure 2. 0.5 ppb perchlorate detection in three different sample matrices by LC/MS/MS.

Analyzed on consecutive days with fresh eluent.

331.0 & 6850

0.00 4.00 8.00 12.00 16.00 20.00 24.00

Minutes

TIC1 ppb ClO4

HTDS defined as:1000 mg/L each of

HCO3, Cl, and SO4

SO4

Cl

Cations & HCO3

Conductivity ProfileBlank HTDS5

0 µ

S F

S

Figure 1. 1 ppb perchlorate in HTDS.

Cations &Water Dip

ClRegion

SO4Region

Det Limit at 3x S/N < 0.1 ppb

100 µL of0.5 ppb Cl4O

HTDS

MilfordDrinking Water

Reagent Waters

0.0 4.0 8.0 12.0 16.0 20.0

Res

ponse

1.2

3 x

130

Time

Analyzed on consecutive days with fresh eluent.

Waters Alliance System for Carbamate Analysis EPA method 531.2 describes the use of high performance liquid

chromatography (HPLC) for the determination of certain N-methylcar-

bamoyloximes and N-methylcarbamates in finished drinking water.

The following compounds may be analyzed with this method:n Aldicarb n Aldicarb sulfonen Aldicarb sulfoxiden Carbaryln Carbofurann 3-Hydroxycarbofurann Methiocarbn Methomyln 1-Naphtholn Oxamyln Propoxur

ConDIT IonS

Instrument: Waters Alliance system for carbamate analysis

Eluent: Water/methanol/acetonitrile (Table 1)

Column: Waters Carbamate Analysis column

3.9 x 150 mm (part no. 35577) @ 30 °C

Injection: 400 µL of standard (see below)


Detection: Fluorescence, Ex- 339 nm, Em- 445 nm, Gain- 10



Filter and degas through a 0.45 micron filter.

A: Water

B: Methanol

C: Acetonitrile

TIME FLoW %A %B %C CURvE

Initial 1.5 88.0 12.0 0.0 -

5.30 1.5 88.0 12.0 0.0 1

5.40 1.5 68.0 16.0 16.0 5

14.00 1.5 68.0 16.0 16.0 3

16.10 1.5 50.0 25.0 25.0 7

20.00 1.5 50.0 25.0 25.0 6

22.00 1.5 88.0 12.0 0.0 5

30.00 1.5 88.0 12.0 0.0 1

Table 1. Eluent gradient.


Reagent 1: 0.05 n sodium hydroxide

Dissolve 2 grams NaOH in water and dilute to 1 liter,

filter and degas.

Reagent 2: oPA/2-mercaptoethanol

Dissolve 19.1 grams of Borax (Na2B4O7•10H2O) Sodium

borate in 1 liter of water. To this, add a solution of

0.1 grams OPA (o-phthaldialdehyde ) in 10 mL methanol.

Swirl to dissolve, filter and degas. To this, add 1.0 mL of

2-mercaptoethanol, gently swirl to dissolve. Protect from

light. Post column reaction temperature is 80 °C.

CARBAMAT ES

STAnDARD MIx P REPARAT Ion

Pipette 25 µL of AccuStandard mix M-531M and M-531-IS each into

100 mL of preserved reagent water for a final concentration of 25 parts

per billion (ppb) per analyte. Preserved reagent water is prepared by

dissolving 9.35 grams of potassium dihydrogen citrate and 0.2 grams

of sodium thiosulfate (anhydrous) in water and diluting to 1 liter.


Refer to EPA method 531.2 for preparation of field grab samples.

A Waters Oasis® method is also available.

CoMMEnTS

n Injection volumes up to 1000 µL may be used.n N-N Dimethyl-2-mercaptoethylamine may be substituted

for 2-mercaptoethanol, refer to EPA method 531.2 for

more information.n EPA method 531.2 may be found at www.EPA.gov. Also refer to

the Waters publication: Waters Alliance System for Carbamate

Analysis Method Guide (#71500017102rC).n Analytes are also known under various trade names, such as

Baygon for Propoxur.

Minutes2.00 6.00 10.00 14.00 18.00 22.00

1

34

5

6

78

9

10

2

11

12

1500 E

U

Figure 1. Standard chromatogram of 25 ppb for each analyte.

PEAk AnALYTE RETEnTIon TIME (MIn)

DETECTIon LIMIT (PPB)

1 Aldicarb Sulfoxide 3.77 0.019

2 Aldicarb Sulfone 4.66 0.041

3 Oxamyl 5.17 0.050

4 Methomyl 6.03 0.031

5 3-Hydroxy carbofuran 9.83 0.022

6 Aldicarb 11.46 0.022

7 Propoxur 14.35 0.038

8 Carbofuran 14.94 0.028

9 Carbaryl 17.37 0.013

10 1-Naphthol 18.99 0.053

11 Methiocarb 22.02 0.022

12 BDMC* 22.56 0.031

* Internal Standard

Table 2. EPA method 531.2 target analytes.

531.2

Waters Alliance HPLC System EPA method 532 describes the use of high performance liquid chro-

matography (HPLC) with photodiode array (PDA) detection for the

determination of phenylurea pesticides in drinking waters.

ConDIT IonS

Instrument: Waters Alliance HPLC system with 2996 PDA detector

Eluent: A: 25 mM Phosphate, pH 2.4

B: Acetonitrile

Composition: Gradient (Table 1)

Column: SunFire™ C18, 4.6 x 150 mm, 3.5 μm

(part no. 186002554) @ 30 °C

Injection: 20 µL


Detection: PDA UV @ 245 nm



A: 25 mM phosphate

Dissolve 1.7 grams of potassium dihydrogen phosphate (KH2PO4)

and 850 µL phosphoric acid (H3PO4) in 100 mL water. Dilute to

1 liter, then filter and degas. Verify that the pH is approximately 2.4.

B: Acetonitrile

TIME FLoW %A %B CURvE

- 1.5 60 40 -

9.5 1.5 60 40 6

10.0 1.5 50 50 6

14.0 1.5 40 60 6

15.0 1.5 60 40 6



Pipette 100 µL of AccuStandard mix M-532 and 20 µL mix M-532-SS

into 880 µL 1:1 water/acetonitrile (10 ppm analytes plus surrogates).


Sample matrix: Ground and surface water, low concentration.

Sample prep: Solid phase extraction of a 500 mL sample using

Oasis HLBX. Elute with 5 mL 1:1 ACN/methanol,

evaporate and reconstitute in 1.0 mL initial mobile

phase (500:1 sample enrichment).

CoMMEnTS

n Detection limits of <1.0 parts per billion (ppb) can be achieved

using UV detection. n Diuron is also known as Karmex.n Siduron is a mixture of two isomers, A and B.n EPA method 532 may be found at www.EPA.gov.

PHEnYLUREAS

Minutes

3.00 5.00 7.00 9.00 11.00 13.00 15.00

12

3

45

6

7

8

9

10

11

0.4

5 A

U

Figure 1. Standard chromatogram, 10 ppm each analyte.

PEAk AnALYTE

1 Tebuthiuron

2 Thidiazuron

3 Monuron*

4 Fluometuron

5 Diuron

6 Propanil

PEAk AnALYTE

7 Siduron A

8 Siduron B

9 Linuron

10 Carbazole*

11 Diflubenzuron

* Surrogate compounds UV @ 245 nm

532

Waters ACQUITY System with Quattro micro API Mass Spectrometer (or TQ Detector) EPA method 535 describes the use of high performance liquid chro-

matography (HPLC) with tandem quadrupole mass spectrometry for

the analysis of the metabolites of chloroacetanilide and acetamide

herbicide degradates in drinking water matrices.

Compounds that may be analyzed using this method include:n Alachlor oxanilic acid (OA) n Alachlor ethanesulfonic acid (ESA)n Acetochlor OA n Acetochlor ESAn Metolachlor OA n Metolachlor ESAn Propachlor OA n Propachlor ESAn Flufenacet OA n Flufenacet ESAn Dimetheamid OA n Dimetheamid ESA

LC ConDIT IonS

Instrument: Waters ACQUITY UPLC system

Eluent: 5 mM aqueous ammomium acetate/methanol (Table 2)

Column: ACQUITY UPLC BEH C18, 2.1 x 150 mm, 1.7 µm

(part no. 186002353) @ 60 °C

Injection: 50 µL


Data: Waters MassLynx software

MS ConDIT IonS

Instrument: Waters TQ detector or Quattro micro API mass spectrometer

Ion mode: Electrospray negative (ESI-)

Mode: Multiple reaction monitoring (MRM). The MRM transi-

tions, cone voltages (CV) and collision energies (CE)

are listed in Table 1.

CoMPoUnD MRM TRAnSITIon Cv CE

1 Propachlor OA 206.3>134.2 13 11

2 Flufenacet OA 224.3>152.3 13 11

3 Propachlor ESA 256.3>80.0 35 25

4 Flufenacet ESA 274.3>80.0 35 25

5 Dimethenamid OA 270.3>198.3 15 11

6 Dimethenamid ESA 320.3>80.0 40 30

7 Acetachlor OA 264.1>146.2 20 11

8 Alachlor OA 264.1>160.1 20 11

9 Metolachlor OA 278.4>206.3 20 11

10 Alachlor ESA 314.1>80.0 40 25

11 Acetachlor ESA 314.1>80.0 40 25

12 Metolachlor ESA 328.2>80.0 37 25

13 Butachlor ESA* 356.2>80.0 35 25

Table 1. Optimized MRM transition parameters for EPA method 535 in ES-.


A: 5 mM aqueous ammonium acetate

B: Methanol

TIME FLoW %A %B

Initial 0.45 90 10

5.0 0.45 75 25

7.5 0.45 66 34

10.5 0.45 65 35

11.0 0.45 10 90

13.0 0.45 90 10


CHLoRoACETAnILIDE AnD ACETAMIDE HERBIC IDE DEGRADAT ES In DRInkInG WAT ER

535


A 250 mL water sample is extracted using a graphitized carbon SPE

cartridge, dried and made up to 1 mL with 5 mM ammonium acetate.

CoMMEnTS

n The use of UPLC/MS/MS for the analysis of the herbicide metabo-

lites in EPA method 535 has many benefits over traditional

HPLC/MS/MS. The advantages include a 2.5 to 4 times reduction

in total run time, improved resolution for the structural isomers

Acetochlor ethane sulfonic acid (ESA) and Alachlor ESA and a 2 to

4 times increase in sensitivity.n The HPLC/tandem quadupole MS method described in EPA method

535 has a 35 minute run time and the HPLC/ion trap MS method has

a 60 minute run time. Figure 1 illustrates the improved chromatog-

raphy with respect to run time when the analysis is carried out by

UPLC/MS/MS. The total run time is shortened to 14 minutes. n Two of the analytes in this method, Alachlor ESA and Acetochlor

ESA are structural isomers and are monitored using the same

MRM transition (314>80). Therefore, chromatographic separa-

tion is required. The method states that these analytes must be

separated chromatographically with a resolution (Rs) of 1.0 or

greater. The resolution obtained on the UPLC/MS/MS is superior to

that obtained on the HPLC/MS/MS system and well surpasses the

method requirement of Rs>1 for these compounds (see Figure 2).n The 5 mM aqueous ammonium acetate mobile phase should be

replaced at least every 48 hours, preferably every 24 hours.n EPA method 535 may be found at their website www.EPA.gov.

Time0.00 10.00 20.00 30.00

%

0

100

3

4

5

6

7

8

9

10

11

12

13

1

2

Figure 1. UPLC/MS/MS chromatogram for EPA method 535 analytes.

Time

Time

10.00 15.00 20.00

%

0

100

10.00 15.00 20.00

%

0

100

UPLC: RS = 1.5

HPLC: RS = 1.2

Figure 2. Comparison of resolution of Alachlor ESA and Acetochlor ESA obtained on UPLC (above) and HPLC (below).

Waters Alliance System for Carbamate Analysis EPA method 547 describes the use of high performance liquid chroma-

tography (HPLC) with fluorescence detection for the identification and

measurement of Glyphosate (N-phosphonomethyl glycine) in drinking

water matrices.

ConDIT IonS

Instrument: Waters Alliance system for carbamate analysis

Eluent: 0.05% phosphoric acid

Column: Ion Exclusion, 7.8 mm x 150 mm

(part no. 10295) @ 55 °C

Guard: Guard-Pak™ module and Inserts (part no. 88141 and 32472)


Flow rate: 1.5 mL / min

Detection: Fluorescence, Ex- 340 nm, Em- 455 nm, Gain- 10


Dilute 0.5 mL of 85% phosphoric acid (H3PO4) to 1 liter, mix well, filter

and degas.


Reagent 1: Hypochlorite

Dissolve 1.35 grams KH2PO4, 11.6 grams NaCl,

0.4 grams NaOH and 0.2 mL Chlorox Bleach (plain) in

water and dilute to 1 liter, filter and degas.

Reagent 2: oPA

Dissolve 0.8 grams of OPA (o-phthdialdehyde) in 10 mL of

methanol, add this to an aqueous solution of 19.1 grams

of borax Na2B4O7.10H2O. Make to a final volume of 1 liter,

filter and degas. To this, add 2 mL of 2-mercaptoethanol,

swirl gently to mix. Protect from light.

note: Post column flow rate for both reagents is 0.5 mL/min, post col-

umn reaction temperature is 38 °C. Insert second reaction coil in line

before the fluorescence detector.

STAnDARD MIx P REPARAT Ion

Pipette 100 µL of AccuStandard mix (M-547) into 100 mL of acidified

water for a concentration of 100 parts per billion (ppb). Prepare acidi-

fied water by adjusting the pH of HPLC grade water to 3.0 by dropwise

addition of hydrochloric acid (HCL). Use EPA method 547-02 as above

for AMPA (aminomethyl phosphonic acid).


Refer to EPA method 547 for preparation of field grab samples. Filter

through 0.45 µm Acrodisc filters is described.

CoMMEnTS

n EPA method 547 may be found at www.EPA.gov. Also refer to

the Waters publication: Waters Alliance System for Carbamate

Analysis Method Guide (#71500017102rC) for more informa-

tion on setting up the HPLC system.n The retention time of AMPA, a metabolite of Glyphosate,

is approximately 4 times that of the parent compound. If using

short run times for glyphosate alone, AMPA may appear as a ghost

peak after several injections. n A detection limit of 0.22 ppb was calculated for glyphosate.

GLYPHoSAT E

Minutes

2.00 6.00 10.00 14.00 18.00 22.00

1

2

1400 E

U

Figure 1. Standard chromatogram, 100 ppb each analyte.

547

PEAk AnALYTE

1 Glyphosate

2 AMPA

Waters Alliance HPLC System EPA method 549.2 describes the use of high performance liquid

chromatography (HPLC) with photodiode array (PDA) detection 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.

ConDIT IonS

Instrument: Waters Alliance HPLC system with 2996 PDA detector

Eluent: A: 200 mM ammonium formate, pH 3.7

B: Acetonitrile

Composition: 60% A, 40% B

Column: Atlantis® HILIC Silica 3 µm (part no. 186002015),

2.1 x 150 mm @ 30 °C

Injection: 100 µL


Detection: UV @ 257 nm for paraquat, 308 nm for diquat



A: 200 mM ammonium formate

Dissolve 12.62 grams of ammonium formate in 100 mL water.

Dilute to 1 liter. The natural pH of this solution is approximately 6.6.

Adjust to 3.7 with dropwise addition of formic acid, filter and degas.

B: Acetonitrile


Dilute 50 µL of AccuStandard mix M-549.1 (1.0 mg/mL) diquat-

paraquat to 100 mL using 1:1 water/acetonitrile (500 ppb).


Sample matrix: Ground water and surface water, low concentration.

Sample prep: Solid phase extraction of 25 mL sample using Oasis

weak cation exchange (WCX) sorbents. Elute with

1.5 mL ACN/water/TFA, 84:14:2. Evaporate and

reconstitute in 0.5 mL mobile phase (50:1 sample

enrichment).

CoMMEnTS

n Detection limits of <1.0 ppb can be achieved using UV detection.

LC/MS may allow for detection at parts per trillion (PPT) levels.n Use silanized vials to prevent quaternary compounds from sticking

to glass.n For more information on sample prep, see Young et.al.,

Waters publication: Automated tablet processing: Sample prepa-

ration and HPLC with photodiode array detection (950258).n EPA method 549.2 may be found at www.EPA.gov.

DIQUAT-PARAQUAT

549.2

0.0

5 A

U

Wavelength Change

3.00 4.00 5.00

1

2

6.00 7.00 8.00 9.00 10.00

Minutes

Figure 1. Standard chromatogram, 500 ppb each analyte.

PEAk AnALYTE

1 Diquat

2 Paraquat

Waters Alliance HPLC System EPA methods 550.1, 610 and 8310 describe the use of high performance

liquid chromatography (HPLC) with ultra violet (UV) and fluorescence

detection for the identification of polynuclear aromatic hydrocarbons

(PAHs) in drinking water (550.1), ground and waste water (8310), and

municipal and industrial waste water.

Compounds that may be identified with these methods include:n Acenaphthene n Chrysenen Acenaphthylene n Dibenzo(a,h)anthracenen Anthracene n Fluoranthenen Benzo(a)anthracene n Fluorenen Benzo(a)pyrene n Indeno(1,2,3-cd)pyrenen Benzo(b)fluoranthene n Naphthalenen Benzo(ghi)perylene n Phenanthrenen Benzo(k)fluoranthene n Pyrene

ConDIT IonS

Instrument: Waters Alliance HPLC system with PDA

and fluorescence detectors

Eluent: Water/acetonitrile (Table 1)

Column: Waters PAH 4.6 x 250 mm (part no. 186001265) @ 30 °C

Injection: 20 µL of Supelco Standard EPA 610 (#48743)

diluted 1:50 in 40:60 water/acetonitrile


Detection: UV @ 254 nm and fluorescence using timed

programmed wavelengths



Filter and degas through a 0.45 micron filter.

A: Water

B: Acetonitrile


Initial 1.2 40 60 -

12.0 1.2 0 100 9

23.0 1.2 40 60 11




550.1 Drinking water Extract with C18 cartridge, elute with MeCl2

610 Waste water Liquid/liquid extraction with MeCl2

8310 Solid waste leachates MeCl2 extraction

Table 2. EPA method sample matrix and preparation.

CoMMEnTS

n Stable column temperature control is crucial

for retention time stability.n Analyte 2, acenaphthylene, is non-fluorescent.n For specific sample preparation protocols, see the appropriate EPA

method, which can be found at www.EPA.gov.n Using UV detection, 254 nm is a compromise wavelength, Max for

the individual analytes range from 220 to 307 nm. For optimum

PoLYnUCLEAR ARoMATIC HYDRoCARBonS (PAHS)

UV sensitivity, use the optimum wavelength listed in table below.

Another option is to use the UV Maxplot feature, however, any

impurities absorbing in this range will also be enhanced.n Fluorescence detection will yield the optimum detection limits

for all compounds except acenaphthylene. See Table 3 below for

excitation and emission wavelengths.

Minutes

6.00 10.00 14.00 18.00 22.00

0.0

5 A

U

1

2

3

4

5

6

7

8

9

10

11

1213

14

15 16

Figure 1. Standard chromatogram, UV @ 254 nm, 1-20 ppm PAH analytes.

Minutes6.00 10.00 14.00 18.00 22.00

1

3

4

5

6

7

8

9

10 11

12

13 14

1516

8000 E

U

Figure 2. Standard chromatogram, fluorescence/programmed wavelengths, 1-20 ppm PAH analytes.

AnALYTE Uv MAx (nM)

Ex (nM)

EM (nM)

DETECTIon LIMIT (PPB)1

1- Naphthalene 220 277 330 0.14

2- Acenaphthylene 229 NA NA NA

3- Acenaphthene 227 270 323 0.01

4- Fluorene 261 265 310 0.03

5- Phenanthrene 251 252 365 0.02

6- Anthracene 252 250 402 0.01

7- Fluoranthene 236 284 467 0.02

8- Pyrene 240 332 378 0.01

9- Benzo(a)anthracene 287 284 390 0.01

10- Chrysene 267 270 367 0.04

11- Benzo(b)fluoranthene 256 298 436 0.09

12- Benzo(k)fluoranthene 307 303 432 0.01

13- Benzo(a )pyrene2 296 280 410 0.03

14- Dibenzo(a,h)anthracene 297 294 398 0.01

15- Benzo(g,h,I)perylene 299 290 420 0.03

16- Indeno(1,2,3-cd )pyrene 250 305 480 0.49

1Fluorescence mode used for detection limit determination, no pre-concentration. Seven replicates per 40 CFR pt. 136 App. B.

2Regulated compound; action level 0.17 ppb.

Table 3. PAH target analytes.

550.1, 610 & 8310

Waters Alliance HPLC System EPA method 8330 describes the use of high performance liquid chro-

matography (HPLC) with ultraviolet (UV) detection for the analysis of

samples containing or suspected of containing explosive compounds.

method 8330 provides for the detection in parts per billion (ppb) of

explosive compounds in soil, water, and sediments.

The compounds that can be detected and quantified

by method 8330 are listed below:

n C 1,3-dinitrobenzene (DNB)n C 1,3,5-trinitrobenzene (TNB)n C 2-amino-4,6-dinitrotoluene (2AmDNT)n C 2-nitrotoluenen C 2,4-dinitrotoluene (2,4-DNT)n C 2,4,6-trinitrotoluene (TNT)n C 2,6-dinitrotoluene (2,6-DNT)n C 3-nitrotoluenen C 4-amino-2,6-dinitrotoluene (4AmDNT)n C 4-nitrotoluenen C hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)n C methyl-2,4,6-trinitrophenylnitramine (Tetryl)n C nitrobenzenen C octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)

ConDIT IonS

Instrument: Alliance HPLC system with

2487 Dual λ Absorbance detector

Eluent: 10 mM ammonium formate/isopropanol

Column: XTerra® Phenyl, 2.1 x 150 mm, 3.5 µm

(part no. 186001181) @ 40 °C






10 mM ammonium formate/isoprapanol

Dissolve 0.631 grams of ammonium formate in 100 mL water. Transfer

to a 1 liter volumetric flask. Add 200 mL of Isopropanol. Dilute to the

mark with water and mix well. Carefully pH to 3.8 with formic acid, then

filter and degas.


Dilute 100 µL of AccuStandard mix (M-8330-R) to 10 mL with eluent

for a working 10 ppm standard mixture.

SAMPLE P REP RAT Ion

Sample Matrix: Ground water and surface water, low concentration.

Sample Prep: Solid phase extraction using a 500 mL sample

Porapak Reverse Phase Sorbent (RDX), elute

with acetonitrile.

CoMMEnTS

n With sample prep, detection limits of less than 1 ppb can be achieved.n The separation is very dependent on maintaining accurate

column temperature.n EPA method 8330 may be found at www.EPA.gov.

Ex PLoSIv ES

Minutes

2.00 6.00 10.00 14.00 18.00 22.00

0.2

5 A

U

12

3

4

5

6

78

9

10 11

12 13 14

PEAk AnALYTE

1 HMX

2 RDX

3 1,3,5-Trinitrobenzene

4 1,3 Dinitrobenzene

5 Nitrobenzene

6 TNT

7 Tetryl

PEAk AnALYTE

8 2 Amino-4,6 Dinitrotoluene

9 2,4 Dinitrotoluene

10 4 Amino-2,6 Dinitrotoluene

11 2,6 Dinitrotoluene

12 4- Nitrotoluene

13 2- Nitrotoluene

14 3- Nitrotoluene

Figure 1. Standard chromatogram, 10 ppm each analyte.

8330

Waters Alliance HPLC System EPA methods TO11, 554, and 8315 (Options 1 and 2) describe the

use of high performance liquid chromatography (HPLC) with ultra vio-

let (UV) detection for the identification of aldehydes and ketones as

Dinitrophenylhydrazine (DNPH) derivatives in drinking water (554); soil,

air, water, waste or stacks collected by method 0011 (8315 Option 1)

ambient air (TO11), and samples collected from indoor air by method 0100

(8315 Option 2). EPA methods 554 and 8315 Option 1 target the same

12 compounds. Likewise, methods TO11 and 8315 Option 2 target the

same 15 compounds. Several analytes are common to all four methods.

Compounds that may be identified with these methods include:

n Acetaldehyde1,2 n Heptanal1

n Acetone2 n Hexanal (Hexaldehyde)1,2

n Acrolein2 n Isovaleraldehyde2

n Benzaldehyde2 n Nonanal1

n Butanal (Butyraldehyde)1,2 n Octanal1

n Crotonaldehyde1,2 n Pentanal (Valeraldehyde)1,2

n Cyclohexanone1 n Propanal (Propionaldehyde)1,2

n Decanal1 n m-Tolualdehyde2

n 2,5- Dimethylbenzaldehyde1,2 n o-Tolualdehyde2

n Formaldehyde1,2 n p-Tolualdehyde2

1Analyte per EPA methods 554 and 8315 Option 12Analyte per EPA methods TO11 and 8315 Option 2

ConDIT IonS

Instrument: Waters Alliance HPLC system with UV detection

Eluent: Water/tetrahydrofuran/acetonitrile (Tables 1 and 2)

Column: Waters XBridge™ Phenyl, 3.5 µm, 4.6 x 150 mm

(part no. 186003335) @ 35 °C

Injection: 20 µL each of AccuStandard mix (M- 8315-R1- DNPH and

M- 8315-R2- DNPH) diluted 1:5 in 40:60 water/acetonitrile





Filter and degas through a 0.45 µm filter.

A: 90% water, 10% tetrahydrofuran (THF)

Mix 900 mL water and 100 mL stabilized THF.

B: Acetonitrile


Initial 1.5 70 30 -

20.0 1.5 36 64 6

22.0 1.5 36 64 6

22.1 1.5 70 30 6

Table 1. Eluent gradient. For EPA methods 554 and 8315 Option 1.


Initial 1.5 70 30 -

16.0 1.5 53 47 6

21.0 1.5 53 47 6

21.1 1.5 70 30 6

Table 2. Eluent gradient for EPA methods TO11 and 8315 Option 2.

oPT IonS 1 AnD 2, ALDEHYDES AnD kETonES AS DnPH DERIvAT Iv ES BY HPLC



554, 8315 Option 1 Drinking water, solid waste leachate

DNPH reagent added to 100 mL sample, extract with Oasis HLB or use methylene chloride extraction option

TO11, 8315 Option 2 Indoor or ambient air

Use Sep-Pak® DNPH Silica cartridge, backflush cartridge with acetonitrile

Table 3. EPA method sample matrix and preparation.

CoMMEnTS

n California State method 1004 can also be performed using a modi-

fied gradient, contact Waters for details.n Detection limits lower than 20 ppb for all analytes can be achieved

using these methods.

TO11, 554 & 8315

1

1211

1098

76

5

43

2

0.2

0 A

U

Minutes

4.00 8.00 12.00 16.00 20.00

Figure 1. EPA method 554 and 8315-01 analytes, 20 parts per million (ppm) as DNPH analytes.

1- Formaldehyde 7- Pentanal2- Acetaldehyde 8- Hexanal3- Propanal 9- Heptanal4- Crotonaldehyde 10- Octanal5- Butanal 11- Nonanal6- Cyclohexanone 12- Decanal 1

1514

1312

11

1098

7

65

4

3

2

0.2

0 A

U

Minutes

4.00 6.00 12.00 16.00 20.00

1- Formaldehyde 9- Isovaleraldehyde2- Acetaldehyde 10- Pentanal3- Acetone 11- o-Tolualdehyde4- Acrolein 12- p-Tolualdehyde5- Propanal 13- m-Tolulaldehyde6- Crotonaldehyde 14- Hexanal7- Butanal 15- 2-5 Dimethylbenzaldehyde8- Benzaldehyde

Figure 2. EPA method TO11 and 8315-02 analytes, 20 ppm as DNPH analytes.

Waters, Alliance, ACQUITY UPLC, UltraPerformance LC, UPLC, Atlantis, Oasis, Sep-Pak and XTerra are registered trademarks of Waters Corporation. Empower, SunFire, MassLynx, Quattro micro, IC-Pak, Guard-Pak, XBridge and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2007 Waters Corporation. Printed in the U.S.A. June 2007 720002138EN JH-AC

Waters Corporation 34 Maple StreetMilford, MA 01757 U.S.A.T: 508 478 2000F: 508 872 1990www.waters.com

EPA Methods Book - Waters Corporation · Waters Quattro micro API Mass Spectrometer The Waters...

Documents

Transcript of EPA Methods Book - Waters Corporation · Waters Quattro micro API Mass Spectrometer The Waters...