jim krol, mark benvenuti, and joe romano chemical analysis … · 2007. 11. 21. · jim krol, mark...

LC / Mass Spectrometry Analysis of Anions

(m/z

©2006 Waters Corporation

Water – Bottle or Tap?


Do You Know What is in Your Water?

• US EPA regulates several inorganic and organic analytes in environmental matrices– Drinking water– Ground and Surface water– Irrigation water– Wastewater– Soils and sludge

• Anionic analytes of Interest– NOx, CrO4, ClO4– Oxyhalides and disinfection by products– Haloacetic Acids– Perfluorooctanoic acid (PFOA and PFOS)

The Perchlorate

Issue


What is Perchlorate (ClO4)?

• Perchlorate is found naturally occurring in Chilean nitrate fertilizer deposits– Used world wide

• Perchlorate is a prime ingredient in the manufacture of munitions and solid rocket propellant– Worldwide problem– Space Shuttle solid rocket boosters– Fireworks

• Perchlorate contamination has been confirmed in 26 US states– Soil contamination at Military sites and bases– Two Kerr-McGee Nevada manufacturing facilities – Contaminated the Colorado river and ground water aquifers– Supplies irrigation and drinking water to the US southwest


Perchlorate Consequences

• Perchlorate contaminates California vegetables– Lettuce, and other Imperial Valley vegetables and fruit– Alfalfa used as feed stock for dairy cows--Milk contamination?– Millions of $$$$ effect to the local economy– A national problem

• Perchlorate Health Risks– Hypothyroidism, interferes with thyroid iodine uptake– Impaired fetal nervous system development


Perchlorate Drinking Water Standards

• Perchlorate Standards for Drinking Water– 4 ppb in Texas and California– 5 ppb in New York– 1 ppb in Maryland, Massachusetts, and New Mexico– Higher in Nevada, Arizona

• EPA drinking water limit is 24.5 ppb– Based upon NAS/NIH toxic level of > 0.7 μg / Kg / day

For a 70 kg person (150 lb), 49 μg ClO4/dayOr 24.5 μg / L based upon 2 L daily water consumption

– Military wants 200 ppb– Previous assessment suggests 1 ppb

Adapted by several states


Ion Chromatography orLiquid Chromatography

EPA Office of Drinking Water validated two MS methods for perchlorate analysis in 2005

–Method 331 is LC/MS/MS

–Method 332 is IC/MS

What is the difference?


Ion Chromatography / MS

Ion ChromatographyEPA 332

ICEG anion exchange column Suppressor

MS

2ndpump

CondDet

Primarily anion exchange chemistry and principlesAll aqueous KOH eluentsRequires Eluent Generator (EG) to prepare KOH from DIRequires Chemical Suppression to remove K to form waterWill have problem with sample matrix metals

contaminating suppressor efficiencyConductivity Detector?Requires 2nd pump to add AcCN post Conductivity Detector

to assist with MS desolvation


Liquid Chromatography / MS

Liquid ChromatographyEPA 331LC

anion exchange columnMS

EPA 6850

Primarily reverse phase and anion exchange chemistry andLC principles

Combinations of buffer and solvent eluentsany volatile buffer does not require chemical suppressionNH4OH, NH4HCO3, HOAc, etc

Minimal sample matrix effectsNo eluent generator neededNo conductivity detectorNo post column addition of AcCN


Single Column Ion ChromatographyDirect Conductivity Detection

1.7

µS

Column: Waters IC Pak A/HREluent: Borate-Gluconate / 12% AcCNBack Cond: 240 μSFlow Rate: 1 mL/minInj Volume: 100 μL

1 Fluoride = 1 ppm2 Bicarbonate3 Chloride = 24 Nitrite = 45 Bromide = 46 Nitrate = 47 o-Phosphate = 68 Sulfate = 4

0.00 5.00 10.00 15.00Minutes

1

2

3

4

5 6

7 8

LC/MS/MS Strategy for Perchlorate Analysis


LC/MS/MS Alternativefor Perchlorate

• Perchlorate retains onto polymeric anion exchange columns via 2 mechanisms– Anion exchange– Reversed phase

• Main interferent is SO4 , but addition of AcCN causes perchlorate to elute before SO4– Addresses the reversed phase mechanism only– Position perchlorate baseline resolved after Cl and before SO4

• This approach does not require sample preparation

• Quantification and confirmation within 10 mins– MS/MS selectivity and sensitivity– Little interference from TDS or TOC


Perchlorate AnalysisUsing LC/MS/MS

System: Waters 2695 with 432 Conductivity Detector andQuattro micro™ API Mass Spectrometer in series,

or Waters ZQ™ Mass SpectrometerColumn: IC Pak Anion / HR (4.6 x 75 mm, 7 μm)

(Polymeric Anion exchange)Eluent: 25 mM NH4HCO3,pH10 with NH4OH in 50% AcCNFlow Rate: 0.5 mL/minTemp: 30°CBackPres:


Why the Use ofAmmonium Bicarbonate

• Bicarbonate/Carbonate and Hydroxide are classical buffers for anion exchange chromatography– However, sodium and potassium salts are not volatile

Requires chemical suppression for use with MS– But, ammonium salts are volatile

• Bicarbonate/Carbonate buffers at pH 10 have significant eluting strength compared to hydroxide and acetate– Better choice to elute strongly retaining perchlorate and sulfate

• All alkaline eluents will absorb CO2 from the atmosphere and alter the eluting strength of the eluent– Bicarbonate buffers minimize this effect and give enhanced

ruggedness

• Ammonium Bicarbonate minimizes suppression effects– Decomposes at 60°C: NH4HCO3 ⎯⎯→ NH3↑ + CO2↑ + H2O


Why the Use ofAmmonium Bicarbonate

At the MS InterfaceNH4HCO3 ⎯⎯→ NH3↑ + CO2↑ + H2O>60ºC

NH4HCO3 + H2O ⎯⎯→ NH4+ + HCO3- + H2O

pKa of NH4+ ⎯→ NH3 + H+ is 9.24pKa of HCO3- + H+ ⎯→ H2CO3 is 6.4pKa of HCO3

- ⎯→ H+ + CO3-2 is 10.3

In Solution

Buffering Range 6.4 to 10.3pH of a 10 mM Solution is 8 to 8.2


Suggested LC/MS-MS SystemUsing Negative ElectroSpray

Waters 2695Column Temp Control

Seal Wash

2996 PDA or432 Conductivity

Detectors

Quattro Premier™ XE, orQuattro microTandem MS

MassLynx™ 4.1 withTargetLynx™ & ChromaLynx™


Perchlorate AnalysisUsing LC/MS/MS

Tune conditions using LC Quattro micro API Tandem MS

Ionization: -ESP LM 1 Resolution: 13.5Capillary (V): 2.5 HM 1 Resolution: 13.5Cone (V): 45 Ion Energy 1: 1.2Extractor (V): 1 Entrance (V): -1RF Lens (V): 0.3 Collision: 20Source Temp (°C): 125 Exit 1Desolvation Temp: 350 LM 2 Resolution: 15.0Cone Gas (L/hr): 50 HM 2 Resolution: 15.0Desolvation Gas: 650 Ion Energy 2: 3.0GasCell Pressure: 2 x 10-2 mbar Multiplier (V): 650

MRM Function:99>83 @ 500 ms dwell time 101>85 @ 500 ms dwell time

107>89 @ 100 ms dwell time 109>91 @ 100 ms dwell time


Perchlorate in HTDS“High Total Dissolved Solids”

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Minutes

50 μ

S F

S

TIC1 ppb ClO4

HTDS defined as:1000 mg/L each ofHCO3, Cl, and SO4

SO4

Cl

Cations &HCO3

Conductivity ProfileBlank HTDS

LC/MS/MSPerformance

participated in EPA Method 6850 Collaborative


Perchlorate MS Analysis IonsIons Analytes/Isotopes Dwellm/z 83 35ClO4 Prod ion 1 0.5 sm/z 85 37ClO4 Prod ion 2 0.5 sm/z 89 35Cl18O4 IS Prod ion 1 0.1 sm/z 91 37Cl18O4 IS Prod Ion 2 0.1 sm/z 99 35ClO4 Parent 1 0.5 sm/z 101 37ClO4 Parent 2 0.5 sm/z 107 35Cl18O4 IS Parent ion 1 0.1 sm/z 109 37Cl18O4 IS Parent Ion 2 0.1 s

Natural Perchlorate parent ions

Product ion used for quantification and ion ratio perchlorate confirmation.

Internal standard product ion used to compensate for matrix effects.


2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Minutes

TICHTDS Blank

TIC1 ppb ClO4

Perchlorate in HTDS“High Total Dissolved Solids”

35ClO4 = 99.1 Da37ClO4 = 101.1 Da

HTDS Blank SIR @ 99

H34SO4- m/z=99

0.584.7100.720.582.798.71

Dwell Timeseconds

Productm/z

Parentm/z

Channel


Perchlorate in HTDS

0.5 ppb ClO4

“HTDS”

MilfordDrinking Water

Reagent Water

2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0Minutes

Res

pons

e 1.

23 x

103

Cations &Water Dip

ClRegion

SO4Region

Analyzed on consecutive days with fresh eluent


MRM Perchlorate LinearityUsing Internal Standard

ppb

Rel

ativ

e R

espo

nse

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

r2 = 0.9967

Linear, Origin Excluded1/X Weighting

6 data points per concentration Calibration Used forMethod 6850Collaborative


Peak Area for 0.25 ppb139.9 ± 9.4 for 6.7% RSD

LOD LOQ3x S/N 0.05 0.12EPA* 0.05 .*MDL= Std Dev x 3.14, n=7

MRM Perchlorate Detection LimitsUsing Tandem Quad Quattro micro

ClO4 Std Smooth(Mn,2x4)

0.25 ppb25 pg on column

Minutes1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

S/N (RMS) = 25.71

Noise interval

ClO4 Std Raw Data

Signal atHalf height


Detection Limit Perspective“Chasing Zero”

What is a ppb, μg/L or ng/g?



If 1 sec equals 1 μg, then what is a billion seconds?

What is a ppb, μg/L or ng/g?


MRM Perchlorate Response Precision

2.6%34.51 ± 0.88910.04.6%16.11 ± 0.7385.03.9%6.618 ± 0.2602.05.3%3.214 ± 0.1691.07.5%1.70 ± 0.1270.5

22.7%0.756 ± 0.1710.2

%RSDRelative ResponseConcentrationμg/L

Rel Resp = (98.7>82.7 Area + 100.7>85.7Area)(5)(107>89 IS Area)


MassLynx 4.1QuanLynx Chromatograms

Minutes2.0 4.0 6.0 8.0 10.0 12.0 14.0

%

0

100

107.00>89.00

%

0

100

100.70>84.70

%

0

100

98.70 > 82.70Smooth(Mn,2x4)

1.0 ppb ClO4

Ion Ratio = 98.70 > 82.70100.70 > 84.70

Internal Standard@ 5 ppb, 500 pg


Perchlorate Ion RatioTandem Quadrupole Quattro micro

2.4%2.638 ± 0.0610 n = 31.6%2.649 ± 0.045 n = 33.6%2.608 ± 0.091 n = 35.3%2.653 ± 0.140.5 n=10

11.0%2.849 ± 0.310.25 n= 9

%RSDIon Ratiomz 99>83 / mz

101>85

ClO4Concentration

Uncorrected Peak Area

All concentrations, n=41Ion Ratio = 2.727 ± 0.26

%RSD = 9.6%

TheoreticalIon Ratio

3.08

Method Spec±20%

or ±10%

Spec Range2.45 ↔ 3.672.77 ↔ 3.39



1 ppb equals = 1 sec in ?? years



1 ppb equals = 1 sec in 31.71 years

One second memory of life experiences



1 ppt equals = 1 sec in 31,710 years


Environmental Matrices EvaluatedRaw Data Smoothing

Minutes2.50 5.00 7.50 10.00 12.50 15.00

10% Industrial Sludge Extract∼30 μg/Kg

10% Soil Extract∼10 μg/Kg

Saltwater, No Dilution∼1 μg/L

Milford Drinking WaterSpiked at 0.5 μg/L

Minutes2.50 5.00 7.50 10.00 12.50 15.00

Sm, Mean4 scans

2 iterationsPeak width is38 data points

Perchlorate AnalysisUsing

LC/ CID-MS


Perchlorate AnalysisUsing LC/MS

Tune Conditions optimized for Waters ZQ Single Quadrupole

Ionization: -ESP LM Resolution: UseCapillary (V): 3.00 HM Resolution: CalibrationCone (V): 40 or 70 Ion Energy: SettingsExtractor (V): 1 Multiplier (V): 650RF Lens (V): 0.5 Cone Gas (L/hr): 50Source Temp (°C): 125 Desolvation Gas: 650Desolvation Temp: 350

SIR Acquisition:1) 99 and 101@ 800 ms dwell time & 89 and 91@ 200 ms dwell time

or2) 83 and 85@ 800 ms dwell time & 89 and 91@ 200 ms dwell time


m/z80 82 84 86 88 90 92 94 96 98 100 102 104

98.96

101. 06

82.9

84.9

CV = 45CV = 70

Perchlorate AnalysisUsing LC/MS-CID

Single Quadrupole Collision Induced DisassociationFor Perchlorate

ClO4-→ClO3

-

99 > 83

Peak Height Ion Ratiomz 99mz 101= 3.08


SIR Perchlorate Detection LimitsUsing Single Quad ZQ

Are

a R

atio

ppb ClO4

1 5 10

SIR @ m/z 99@ 45V

r2 = 0.99921/X Weighting

Seven injections eachexcept for 10 ppb = 3

Using Int Std Calibration

On Column Mass1.0 ppb (100 pg)0.5 ppb (50 pg)

Minutes4.00 6.00 8.00 10.00 12.00 14.00

Data Smooth (SG, 15 pt)

Res

pons

e 1.

15 x

105


ppb ClO40.5 1 5 10

Are

a R

atio

SIR @ m/z 83CID @ 70V

r2 = 0.9993

Res

pons

e 1.

0 x

103

On Column Mass1.0 ppb (100 pg)0.5 ppb (50 pg)

Data Smooth (SG, 15 pt)

Minutes5.00 6.00 7.00 8.00 9.00 10.00

SIR Perchlorate Detection LimitsUsing Single Quad ZQ

LC/MS/MSfor

Other Anions of Interest


Common and Regulated AnionsMS Analysis Ions

• Mass Spectrometry is applicable to the analysis of common and regulated inorganic anions– This perchlorate method can be modified for increased anion retention

decrease buffer and AcCN concentrations

• Common Anions– Fluoride, Chloride, Bromide, and Iodide– Sulfate, Phosphate– Formate, Acetate, etc.

• Regulated Environmental Anions– Bromate, Chlorate, Perchlorate– Nitrite and Nitrate– Chromate– Haloacetic Acids (HAAs)


m/z20.00 40.00 60.00 80.00 100.00 120.00 140.00

35Cl34.7

37Cl36.7

NO245.9

NO361.8

35ClO382.8

32SO496.8

35ClO498.8

37ClO4100.8

37ClO384.8

79BrO3126.9

81BrO3128.9

52CrO4116.9

53CrO4117.7

50CrO4114.9

Regulated Environmental AnionsLC/MS

MS Infusion ScanStandards in DI


Common Environmental AnionsMS Ions

m/z20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

35Cl34.7

NO245.8

NO361.8

79Br78.9

81Br80.8

H32SO496.9

H34SO498.9

37Cl36.7

MS Infusion ScanStandards in DI

©2006 Waters Corporation Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

NO2

NO3

ClO4

ClO3

BrO3

H34SO4

SIR @ 46

SIR @ 62

SIR @ 127

SIR @ 83

SIR @ 99

Regulated Environmental Anions

Analytes at 100 ppbexcept ClO4 at 10 ppb

25 mM NH4HCO3, pH 10 / 50% AcCN

©2006 Waters Corporation Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Cl

NO2

NO3

SO4

ClO3

No ClO4

NO3 @ m/z 62

NO2 @ m/z 46

ClO3 @ m/z 83

ClO4 @ m/z 99

NO2

Cl @ m/z 35SO4 @ m/z 97

LC/MS/MS Anion ProfileMilford Drinking Water Blank

IC Pak A/HR25 mM NH4HCO3, pH 10 in 50% AcCN0.5 mL / min100 μL injection

Form

ate


1ppm Br

1% ValerianDI Extract

0.5% Valerian +0.5 ppm Br

Spike

Minutes2.00 4.00 6.00 8.00

m/z =81

Minutes2.00 4.00 6.00 8.00

m/z =79

Br

Bromide Analysis inValerian Nutraceutical Capsule


LC/MS/MS Perchlorate MethodConclusions

• This work demonstrates the feasibility for anion analysis in a variety of environmental water matrices– Sample ionic strength has little effect on chromatography– High SO4 and Cl do not interfere with ClO4 quantification

Chromatographic resolution

• No Sample Preparation is required– 50% AcCN in the eluent effectively minimizes TOC column build–up– Internal standard is mandated (107>89 transition)

Cl18O4 and Confirmation in a single


Thank You For Attending Today

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Thank You For Attending Today

jim krol, mark benvenuti, and joe romano chemical analysis … · 2007. 11. 21. · jim krol, mark...

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