jim krol, mark benvenuti, and joe romano chemical analysis … · 2007. 11. 21. · jim krol, mark...
TRANSCRIPT
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LC / Mass Spectrometry Analysis of Anions
(m/z
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©2006 Waters Corporation
Water – Bottle or Tap?
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©2006 Waters Corporation
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)
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The Perchlorate
Issue
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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?
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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LC/MS/MS Strategy for Perchlorate Analysis
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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:
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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™
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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LC/MS/MSPerformance
participated in EPA Method 6850 Collaborative
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©2006 Waters Corporation
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.
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
Detection Limit Perspective“Chasing Zero”
What is a ppb, μg/L or ng/g?
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©2006 Waters Corporation
Detection Limit Perspective“Chasing Zero”
If 1 sec equals 1 μg, then what is a billion seconds?
What is a ppb, μg/L or ng/g?
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©2006 Waters Corporation
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)
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
Detection Limit Perspective“Chasing Zero”
1 ppb equals = 1 sec in ?? years
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©2006 Waters Corporation
Detection Limit Perspective“Chasing Zero”
1 ppb equals = 1 sec in 31.71 years
One second memory of life experiences
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©2006 Waters Corporation
Detection Limit Perspective“Chasing Zero”
1 ppt equals = 1 sec in 31,710 years
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©2006 Waters Corporation
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
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Perchlorate AnalysisUsing
LC/ CID-MS
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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99 > 83
Peak Height Ion Ratiomz 99mz 101= 3.08
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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LC/MS/MSfor
Other Anions of Interest
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©2006 Waters Corporation
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)
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©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
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©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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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
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©2006 Waters Corporation
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