gc ms course

Gas Chromatography/Mass Spectrometry Analysis(GC/MS)

Fundamentals and Special Topics

Zbigniew Bernie Wilk, Ph.D.Russell Confer, M.S.

Office of Quality AssuranceNew Jersey Department of Environmental Protection

Gas Chromatography/Mass SpectrometryGC/MS Overview50 min.the nuts and bolts of how GC/MS works

Break10 min.

GC/MS Analysis Special Topics50 min.Chromatograms & Peak IntegrationTICs & MS Libraries, Interferences Break 10 min.

Dioxins and PCB Analyses50 min.GC/High Resolution Mass Spectrometry

Gas Chromatography/Mass SpectrometryIntroductionOrganic Analysis OverviewHistoryThe wide world of Mass SpectrometryHow it all worksTuning/Calibration

BreakGas Chromatography/Mass Spectrometry (GC/MS)From Chromatograms to final reportMass Spectrometry Libraries and Compound Identification (TICs)Proper and Improper Peak Integrations - Manipulating ResultsDealing with Interferences

Break

- Dioxin and PCB Analyses Using GC/High Resolution Mass Spectrometry Review of EPA MethodsWhy High Resolution Mass SpectrometryHigh Resolution Mass Spectrometry FundamentalsDioxin and PCB Analysis Methods HighlightsComparing PCB Methods 1668A to 8082 - Aroclors or Congeners.

Gas Chromatography/Mass Spectrometry(GC/MS)GasChromatographyMassSpectrometryGas Chromatography -Mass Spectrometry=Identifies (detects) pollutant moleculesbased on their molecular weight or mass A Chemical Analysis Technique combining two instruments to provide for powerful separation and identification capabilities Separates mixture of pollutants so each can be identified individually

Gas Chromatography/Mass Spectrometry

Historical Timeline of GC/MS190020001942First CommercialMass Spectrometer1971USEPA Purchases 6Finnigan GC/Mass Specs 1906Sir J.J. ThompsonNobel Prize fordiscovery of electron1952Martin and SyngeNobel PrizeChromatography1979USEPA PublishesWastewater MethodsUnder Clean Water ActGC/MSLC/MSICP/MSEPA Born

Dates of Historical Note1906 - Sir J.J. Thomson (Cambridge) gets Nobel Prize for the discovery of the electron.1930 - Aston uses MS to study isotopes1942 - first commercial magnetic mass spectometer1952 - Martin and Synge win Nobel Prize for Chromatography1959 - Gas Chromatography interfaced to Mass Spectrometer1968 - Finnigan Corp. delivers first Quadrupole GC/MS1969 - Finnigan Corp. delivers first Quadrupole GC/MS with computer1970 - USEPA is born1971 - USEPA purchases 6 Finnigan GC/MS systems1972 - Federal Water Pollution Control Act (CWA) is passed1976 - Hewlett Packard introduces fully computerized GC/MS system1976 - RCRA Enacted1979 - USEPA publishes wastewater methods under CWA1983 - Development of LC/MS interface by Vestal et. al.

Various Forms of Mass SpectrometryA whole range of possibilities/permutations Sample Introduction Ionization Mass Separator Detector

Gas ChromatographyEI (electron impact) Quadrupole channeltron

Liquid ChromatographyCI (chemical ionization) Ion Trap discrete dynodeElectrosprayNCI negative CI Time-of-Flight(TOF) photo-opticalFAB(fast atom bombardment) Sector(BE, EB, EBE) image currentAPI (atmospheric pressure) FTMS (MSn)LIMS (laser ionization) Ion Mobility FI/FD (field desorption) Triple Stage Quadrupoles (MS/MS)MALDI (matrix assisted laser Hybrid Combinations (Q-TOF, BEQ) desorption ionization)Particle Beam (PB/LC/MS Interface)Thermospray (TSP/LC/MS Interface)Atmospheric Pressure Ionization (API/LC/MS)ETC.

GC/MSGreat For the Analysis of Organics

Gas Chromatography Analysis RequirementOrganics to be analyzed must be VOLATILE or at least Partially VOLATILE .

First 30 years of EPA have concentrated on relatively volatile organics

Next 30 years? Polar and Non-Volatiles?LC/MS?

Broad Range of Organic Compounds(How many are there?)Chemical Abstracts Service 16, 000, 000(based on CAS #s as of 1998)

NIST Organic MS Database approx. 150,000

Federal Pollutant Database approx 700

e.g.Most Organic Analyses:approx. 10 to 80 compounds in one analysis

Classification of Organic CompoundsBoiling PointPolarity * Technique

IonichighhighHPLC, HPLC/MS

NonVolatileshighhighHPLC, HPLC/MS

SemiVolatiles mediumlow-mediumGC; GC/MS; HPLC

Volatiles low low-medium GC; GC/MS

* Increasing polarity = Increasing solubility in water

Survey of GC/MS Methods(by Program)SDWAEPA 500 seriese.g. 524.2, 525

Clean Water ActEPA 600 and 1600 seriese.g. 624, 625, 1624, 1625, 1666

RCRA (Solid and Hazardous Waste)EPA 8000 seriese.g. 8260, 8270

CERCLA (Superfund)OLMO contracts

Clean Air ActTO (Toxic Organics) seriese.g. TO-14, TO-15, TO-17

(Some ASTM and Standard Methods are also EPA approved)

Principles of Gas Chromatography Mass SpectrometryAdvantages

- high sensitivityexcellent detection limits. Typically low ppb to high ppt- high selectivityidentification is based on two parameters not one(retention time and mass spectrum must match standard)selects analyte of interest with very high confidence- Speedtypical analysis takes from 1/2 hour to approx. 1 houranalysis can contain upwards of 80 and more pollutants

Disadvantages

- higher capital cost (approx. $ >85 K vs. $15 K for GC)- higher maintenance (time, expertise and money)- for optimum results requires analyst knowledgeable in both chromatography and mass spectrometry

The Analytical Process - GC/MS is Last StepSample SiteContaminated SiteMonitoring WellPermittee EffluentDrinking Water FacilityData Received by DEP5.6 ppbBenzene

Laboratory SideSamplePreparationDeterminative Step

Gas Chromatography (GC)

Gas Chromatography/Mass Spectrometry (GC/MS)

High Pressure Liquid Chromatography (HPLC)SampleClean-Up

(optional)Sample Analysis

The Analytical Process(It all starts with Sample Preparation)SamplePreparationDeterminitive Step

Gas Chromatography (GC)

Gas Chromatography/Mass Spectrometry (GC/MS)

High Pressure Liquid Chromatography (HPLC)Sample AnalysisPurge and TrapLiquid-Liquid ExtractionSonicationSolid Phase Extraction (SPE)Soxhlet Extraction

(not an exhaustive listing)SampleClean-Up(optional)

Sample Preparation TechniquesPreparation - v.v. important first step

1) used to separate organic contaminants from their environmental matrix (e.g. groundwater or soil)2) used to concentrate the contaminants

Typical Preparation Techniques include:Purge and Trap, LLE, Soxhlet, LSE (Sep Paks, Cartridges)

Purge and Trap(Aqueous and Soils / Volatiles Preparation)Courtesy of Environmental Conservation Laboratories, Inc.

Liquid/Liquid Extraction(Separatory Funnel) (Aqueous Samples / Semivolatiles Analysis)Courtesy of Environmental Conservation Laboratories, Inc.

Sonication(Soils, Solids / Semivolatiles)Courtesy of Environmental Conservation Laboratories, Inc.

Solid Phase Extraction(Aqueous / Semivolatiles)Courtesy of Stanford LaboratoryCartridgeSample

Soxhlet Extraction(Soils, Solids / Semivolatiles)Courtesy of Environmental Conservation Laboratories, Inc.

Sample Clean-Up TechniquesClean-Ups - used if interferences are a problem

stand alone methods are availablealso procedures written into some methodsthese are often optional and choices often rest with analyst and is dependent on the sample

Examples of typical clean-up procedures include:

Alumina, Silica, Flourisil, Gel Permeation Chromatography, Acid Wash etc.

Sample Clean-Up Techniques from SW-846(stand alone methods strictly for cleanups)Analytes of Interest Methods

Aniline & aniline derivatives 3620Phenols 3630, 3640, 8041aPhthalate esters 3610, 3620, 3640Nitrosamines 3610, 3620, 3640Organochlorine pesticides & PCBs 3610, 3620, 3630, 3660, 3665Nitroaromatics and cyclic ketones 3620, 3640Polynuclear aromatic hydrocarbons 3611, 3630, 3640Haloethers 3620, 3640Chlorinated hydrocarbons 3620, 3640Organophosphorus pesticides 3620Petroleum waste 3611, 3650All base, neutral, and acid 3640priority pollutants

Gas Chromatography Mass Spectrometry(Operational Description)Introduction System - Gas Chromatography Ionization Mass Separation Mass DetectionData SystemMass SpectrometerIonization SourceMass AnalyzerParticleDetectorGas ChromatographyDedicatedData SystemVacuum System - approx. 10-6 torr

Gas Chromatography

Powerful Analytical Chemistry technique used to separate and identify organic compounds from mixtures.

One requirement:organics must be volatile or semivolatileany very polar, non volatile or ionic compounds in sample will not be detected

Gas Chromatography

Columns Packed CapillaryCross section

THE CHROMATOGRAPHIC PROCESS - PARTITIONING (gas or liquid)MOBILE PHASESTATIONARY PHASE

SampleoutSamplein(solid or heavy liquid coated onto a solid or support system)

Temperature Control Isothermal GradientParameters Affecting SeparationColumn Type (Phase)Polar (DB-1701 NonPolar (DB-1)Phase ThicknessColumn Dimensions

Chart1

40

40

90

140

190

240

240

240

Temp

Time (min)

Temp (deg C)

Sheet1

TimeTemp

040

1040

1590

20140

25190

30240

40240

50240

Sheet1

0

0

0

0

0

0

0

0

Temp

Time (min)

Temp (deg C)

Linear Ramp

Sheet2

Sheet3

Phases

Chromatograms - 551.1Same Organic Mixture Different Capillary Columns

Instrumentation - DetectorsDestructive Mass Spectral (CI/EI) [625] Flame Ionization (FID) [604] Nitrogen-Phosphorus (NPD) [8141A] Flame Photometric (FPD) [8141A] Electrolytic Conductivity (Hall/ELCD) [502.2]Non-Destructive Thermal Conductivity (TCD) Electron Capture (ECD) [551.1] Photo Ionization (PID) [502.2]

How Mass Spectrometry Detectors Works

All Organic Molecules are made up of combinations of atoms containing Carbon and Hydrogen

In addition to Carbon and Hydrogen, other elements are frequently a part of a molecule to provide a variety of chemical and physical properties (e.g. Oxygen, Nitrogen, Chlorine, Fluorine, etc.)

Molecular weights can be calculated knowing the elemental composition of a molecule.

Mass Spectrometry analyzes (identifies) organic molecules according to their molecular and fragment weights.

How Mass Spectrometry (Mass Analysis) Works (Use Table to Calculate Molecular Weights)Isotopes

PRIVATEElement

Symbol

Nominal Mass

Exact Mass

Abundance

Hydrogen

HD or 2H

12

1.007832.01410

99.990.01

Carbon

12C13C

1213

12.000013.0034

98.911.09

Nitrogen

14N15N

1415

14.003115.0001

99.60.37

Oxygen

16O17O18O

161718

15.994916.999117.9992

99.760.0370.20

Fluorine

F

19

18.9984

100

Silicon

28Si29Si30Si

282930

27.976928.976529.9738

92.284.703.02

Phosphorus

P

31

30.9738

100

Sulphur

32S33S34S

323334

31.972132.971533.9679

95.020.744.22

Chlorine

35Cl37Cl

3537

34.968936.9659

75.7724.23

Bromine

79Br81Br

7981

78.918380.9163

50.549.5

Calculating Molecular Weight (Mass)Benzene(C6H6)Pyridine(C5H5N)6 x 12 = 726 x 1 = 6MW = 78 amu5 x 12 = 605 x 1 = 51 x 15 = 15MW = 79 amuElement atomic mass (amu)Carbon(C) 12Hydrogen(H) 1Chlorine(Cl) 35Fluorine (F) 19Oxygen(O) 16Nitrogen(N) 14amu - atomic mass units

Gas Chromatography Mass Spectrometry(Operational Description)Introduction System - Gas Chromatography Ionization Mass Separation Mass DetectionData SystemMass SpectrometerIonization SourceMass AnalyzerParticleDetectorGas ChromatographyDedicatedData SystemVacuum System - approx. 10-6 torr

The Ionization Process(Electron Impact) Neutral molecules are converted into Ions (charged particles)MolecularIone- ++ 2e-(70 Electron Volts)Neutral MoleculeFragment Ion 1Fragment Ion 2, etc.e- ++. Mass Analysis can only work for charged species - not for neutrals.+ 2e-+ .

GC/MS - Mass AnalysisIonsMass Separation(quadrupole)Wavelength SeparationContinuous Light

Principles of Gas Chromatography/Mass Spectrometry(NIST Library Mass Spectra for Benzene)Abundance (Signal)mass/charge (m/z) ------>Benzenem/z 7878 amu

Principles of Gas Chromatography/Mass Spectrometry(NIST Library Mass Spectra for Pyridine)Abundance (Signal)mass/charge (m/z) ------>Pyridinem/z 7979 amu

One More Example for o-Xylene(Fragment Ions contain Useful Information)Xylene(C8H10)8 x 12 = 9610 x 1 = 10MW = 106Element massCarbon(C)12Hydrogen(H)1Chlorine(Cl)35Fluorine (F)19Oxygen(O)16Nitrogen(N)14+.+.+.Molecular Ions can break down into smaller fragmentsm/z 106m/z 91m/z 77

Principles of Gas Chromatography/Mass Spectrometry(NIST Library Mass Spectra for Xylene)o-Xylene(C8H10)8 x 12 = 9610 x 1 = 10MW = 106Element massCarbon(C)12Hydrogen(H)1Chlorine(Cl)35Fluorine (F)19Oxygen(O)16Nitrogen(N)14Abundance (Signal)mass/charge (m/z) ------>

What Does GC/MS Data Look Like?GC/MS Chromatogram of a 4 Component MixtureRetention Time ------>Abundance (Signal)

What Does GC/MS Data Look Like?GC/MS Chromatogram From EPA Method 524.2 AnalysisRetention Time ------>Abundance (Signal)*Courtesy of the NJDHSS Laboratory

What Does GC/MS Data Look Like?Reviewing of Mass Spectra*m/z 78Abundance (Signal)Retention Time ------>mass/charge ------>6.99 min.

What Does GC/MS Data Look Like?Reviewing of Mass Spectra*m/z 78*Abundance (Signal)Retention Time ------>mass/charge ------>6.77 min.1,1-dichloropropene/carbon tetrachloride

Mass Spectrometry does not always provide an easily interpretable compound identification: e.g. MTBEuse of mass spectral libraries for ID determinationuse of manual interpretation techniquesuse of alternate MS and other techniques

Difficult Mass SpectrausuallyMTBE MW= 88

GC/MS Interpretation Procedures

Identify Molecular Ion if presentEvaluate any Isotopic ObservationsUse Isotopes to calculate probable carbon #s for Molecule and/or fragmentsReview all losses observed to determine substructuresReview major fragmentsHypothesize a molecular structure consistent with above observations

Must Confirm Hypothesis with additional data.Typically chemical ionization MSHigh resolution mass spectrometryInfra Red SpectroscopyNuclear Magnetic Resonance SpectrometryObtaining a pure standard and confirming mass spectra with unknownMass Spectral Interpretation Procedures

Powerful analytical tool combining the separation capability of Gas Chromatography and the identification capability of Mass Spectrometry.

Provides for a higher level of confidence in the identification of organics (Both retention time AND the mass spectrum are used).

Capable of analyzing upwards of 80 pollutants in one analysis.

Typical Detection Limits (Aqueous) are in low ppb and high ppt range.

Appropriate calibrations and controls must be performed before any samples can be analyzed.

GC/MS Summary

GC/MS Analysis (Special Topics)

From Raw Data Chromatograms to final report

Proper and Improper Peak Integrations Data Processing

Dealing with Interferences

Mass Spectrometry Libraries and Tentatively Identified Compounds (TICs)

GC/MS Data ProcessingImportant to Review Peak IntegrationChromatogram(maximum information content)Final ReportGC/MS

GC/MS Data ProcessingImportant to Review Peak IntegrationCalibrations and quantitation of organics all rely on correct chromatographic peak integrationsStandards (Ax/Ais)----->Response Factors----->Sample QuantitationAisAx

NJDEPOQA

OCTOBER 2002

Manual IntegrationFOR GC/MS

Definition A Manual Integration is any editing of the area of integration by the chemist. Manual integration is a perfectly acceptable, scientifically valid, analytical technique used to accurately reflect the area of a peak when auto-integration fails.

A Manual Integration is not a way to compensate for an improperly maintained instrument. Manual integrations are not to be used in lieu of establishing appropriate integration events using the analytical system software.

Manual integration may be done in the following cases where the automatic integrator has:

failed to integrate a peak or part of a peakintegrated one peak as two peaksintegrated the wrong peak out of two similar peaksnot integrated from baseline to baselineintegrated a peak due to an elevated baselineintegrated a negative peakintegrated a peak beyond baseline resolution (too much area)Any additional situations in which the auto-integrator fails to perform properly and/or consistently

Manual integrations are NOT to be performed for the sole purpose of making a calibration curve, ICV,CCV, &/ or a QC check sample (LCS, MS, surrogate, etc.) pass acceptance criteria.

History

Most of the software programs used for chromatography are capable of quantitating, using either peak area or peak height and employ mathematical algorithms related to the slope of the response to detect the beginning and end of peaks.

History

Due to the complex nature of some sample matrices, the ability to manually adjust an incorrect integration became necessary. This flexibility is necessary in the production of quality data.

Much of this process is based on analyst judgment. Each peak must be evaluated and adjusted when necessary. However, this flexibility has led to several instances of improper laboratory activities.

IMPROPER INTEGRATIONS

According to the EPA Region 5s SOP on manual integrations, inappropriate integration is any integration, either automated or manual, which excludes area associated with the target peak or includes area not reasonably attributable to the target peak, such as area due to a second peak or excessive peak tailing due to a noisy baseline.

CORRECT INTEGRATIONS

This is an example of proper integrations when several peaks are not completely resolved (i.e., the response does not return to the baseline between peaks). The lowest point between two points, the valley, is selected as the appropriate start and stop points.

CORRECT INTEGRATIONS

Peaks with slight interferences either just prior to or immediately after the target peak. In these cases, part of the automatic integration may include the interfering analyte. The following integration techniques may be employed:

TYPES OF IMPROPER INTEGRATIONS

Peak shaving is the common term for unjustifiably excluding area when integrating a chromatographic peak.

Almost all of us would agree that cutting a peak in half horizontally or vertically is unjustified. But what to do about the in between cases? How can judgment be applied correctly when integrating peaks?


Baseline addition or subtraction

Do not add or subtract from the baseline. Another example of an incorrect manual integration


Poor sensitivity. Signal is not 3 times the background.

WHY IS THIS HAPPENING?

Cost factorsLevel of Expertise factorsUnethical Behavior

Cost Factors

The price paid is often not sufficient to cover the costs of producing the product.

The client should not accept low bids without considering the quality factor.

This is a free market economy - Let the buyer beware or You get what you pay for.

Level of Expertise Factors

Some laboratories have let their most experienced staff go.

Lack of understanding regarding the fundamentals of analytical chemistry at both the laboratory and data user levels.

Thinking that the computer will always give you the correct answer.

Why Unethical Behavior Occurs

Real or perceived pressuresLack of ethics education and awarenessLack of management oversite and reviewLack of knowledge or confidence in appropriate ways to solve problems

Prevention

Efforts should be made during method development to include the best instrument parameters that allow for automatic integration by the data system in most cases.However, regardless of the sophistication of the software, instances occur when the automated software does not integrate a peak correctly.

Prevention

The failure of the software to appropriately integrate a peak is usually obvious from visual inspection of the chromatogram (at an appropriate scale). Electronic review of analytical raw data is essential in detecting improper activities.

The use of proper documentation protocols should be established to allow manual integrations to be reviewed during data validation.

DOCUMENTATION

All data must be integrated consistently in standards, samples and QC samples. Integration parameters, both automated and manual, must adhere to valid scientific chromatographic principles. Manual integration is employed to correct an improper integration performed by the data system and must always include documentation that clearly states the reason manual integration was performed.

Proper documentation is vital when conducting manual integrations. The following is an example documentation requirement:Print the improperly integrated peak. initial, date and provide a reason on the original for the manual integration. Perform the necessary manual integration. Print the manually integrated peak, initial and date. Submit the manual integration data along with the original automatic integration data as part of the final data package.

GENERAL OBSERVATIONS

The fundamental principle of quantitative integration is that samples should be integrated in the same style chosen for integrating calibration standards.

If properly documented and conducted in a scientifically defensible manner, manual integrations are perfectly acceptable.

WHAT CAN LABS DO TO PREVENT IMPROPER ACTIVITIES

Develop a detailed standard operating procedure that includes examples and documentation requirements.

Enforce a zero tolerance policy for any improper activities.

Have all analysts sign an ethics statement.

Electronically review random data files.

Questions?

GC/MS Interferences What are Interferences?Any compound or mixture of compounds that elutes at the same time as the compound of interest. Therefore the compound of interest can not be properly identified or quantified.

EPA Office of Water Says:

Stating that the sample couldnt be analyzed is not sufficient and will not be accepted as justification for a claim of matrix interference.

Interferences in GC/MS AnalysisProblems they cause

quantitation accuracy of targets may be negatively impacted

can make identification of target among interferences difficult or impossible

if dilution is required, may raise detection limits above required regulatory limits.

Interferences - What do they look like(Example 1)interferencesExtract could only be concentrated to 5 mls.

Interferences - What Do They Look Like(Example 2 )a - Napthaleneb - dimethyl phthalatec - diethyl phthalated - di-n-butyl-phthalateabcd- Can only concentrate to 5 mls.- diluted extract 1:5- total dilution factor = 25x

Interferences - What Are They(Example 2)1,3-dichloro-2-propanol(a chlorinated alcohol)1-methyl, 2,4-diisocyanato benzene(a diisocyanate)

Interferences - What Can Be Done(Example 2)Analyze Base/Neutrals and Acid Fractions Separately - may isolate interferences into fraction of less interest

Perform GPC Analysis to remove any potential high molecular weight interferencesmay help for samples that can only be blown down to 5 mls.no guarrantee that GC or GC/MS analysis is seeing all of the sample

Perform Appropriate CleanUpsmethods exist for cleaning up samples so that analytes of interest can be analyzed

GPC and Cleanups can be performed on the same sample.

Identify interferences and clean up waste streampermittee likely has most intimate knowledge of their own waste stream.Additional non-EPA method testing may be appropriate to identify interferences.

Clean-Up Techniques from SW-846(stand alone methods strictly for cleanups)Analytes of Interest Methods

Aniline & aniline derivatives 3620Phenols 3630, 3640, 8041aPhthalate esters 3610, 3620, 3640Nitrosamines 3610, 3620, 3640Organochlorine pesticides & PCBs 3610, 3620, 3630, 3660, 3665Nitroaromatics and cyclic ketones 3620, 3640Polynuclear aromatic hydrocarbons 3611, 3630, 3640Haloethers 3620, 3640Chlorinated hydrocarbons 3620, 3640Organophosphorus pesticides 3620Petroleum waste 3611, 3650All base, neutral, and acid 3640priority pollutants

From the EPA OCPSF (Organic Chemicals, Plastics and Synthetic Fibers) rules Guidance on Evaluation, Resolution, and Documentation of Analytical Problems Associated with Compliance Monitoring

Stating that the sample couldnt be analyzed is not sufficient and will not be accepted as justification for a claim of matrix interference.

EPA provides for flexibility in wastewater methods and allows use of cleanups etc provided method QA/QC are met.As per Fed Reg. 49 FR 43234

Department can require additional work to be performed to get at an accurate number. Not just take the easy way out and say interferences are present.

Alternate methodsuse of clean-up proceduresidentify source of interference

Interferences in GC/MS Analysis

Mass Spectral LibrariesWhat are mass spectral libraries?

A compendium of electron impact mass spectra collected from a variety of sources

Why are they important?

Identifying non-target or tentatively identified compounds (TICs), relies exclusively on these libraries

Mass Spectral LibrariesWhy are they important - cont.?

Site Remediation for example typically requests:VOAs + 10 TICsBNs + 15 TICsfrequently drinking water methods

If TICs are found, proper identification is very importantmay need correct ID for remediationmay need to provide data to County Health Dept and Owners of Potable Well (as in BUST)

Mass Spectral LibrariesWhy are they important - cont?

Waste Water Permitting:some industries indicate that interferences are present which preclude them analyzing the sample to permit detection limitsIdentification of interferences can be used to determine what options for cleanup may exist. Interferences may also be environmentally unfriendly compounds that may need to reviewed.

Bureau of Safe Drinking WaterBSDW reporting form has ability to enter TIC observations from a laboratory.

Mass Spectral LibrariesHow many libraries are there?

NIST/NIH/EPA Mass Spectral LibraryNBS 75KNIST 98NIST 02

Wiley Mass Spectral Library

Combination Wiley/NIST

Custom Librariesindustry specificproprietary

Mass Spectral LibrariesFor NIST75K Library

Approx 50,000 Mass Spectra Approx 25,000 Replicates

This library is very oldNot very well reviewedVariety of Sources not well filtered.

Labs should NOT be using this!

Mass Spectral LibrariesFor NIST98 Library - (a 75% increase over NBS75K library)

107,886 Compounds 107,829 Chemical Structures 129,136 Spectra 21,250 Replicate Spectra 13,205 Compounds with Replicate Spectra 93 Average Peaks per Spectrum 78 Median peaks per Spectrum 75% Increase in coverage from high quality sources

Labs should be using at least this revision!

Mass Spectral LibrariesFor NIST98 Library(where does this 75% increase come from?)

Mass Spectra from other sources were added inChemical Concepts - including Prof Henneberg's industrial chemicals collection Georgia and Virginia Crime Laboratories TNO Flavors and Fragrances AAFS Toxicology Section, Drug Library Association of Official Racing Chemists St. Louis University Urinary AcidsVERIFIN & CBDCOM Chemical Weapons

Mass Spectral LibrariesFor NIST 02 Library - 35% increase in coverage over NIST 98 Library

27,750 Replicate Spectra from high quality sources147,198 Compounds with Spectra18,598 Compounds with Replicate Spectra147,194 Chemical Structures111 Average Peaks/Spectrum174,948 spectra98 Median Peaks/Spectrum

Mass Spectral LibrariesComparison of NIST/NIH/EPA Libraries - different revisions

NBS75KNIST98NIST 02

Total Spectra75, 000129,136 174,948

Total Replicates20,000 21,250 27,750

Mass Spectral LibrariesWiley Registry of Mass Spectral Data - 7th Edition

the world's largest reference database of over 250,000 Electron-Impact mass spectra

Wiley Library may or may not include NIST libraryWiley contains mass spectra that are not as well reviewed.Still very useful, if NIST library comes up short.

Why MS Library Version is Important(example)Air Analysis Example from 1996 (TO-14)

Samples consistently showed large peak in the analysis but compound could not be identified by library.

Library search result was so poor, even a good quality TIC could not be obtained.

At the time the NBS75K library was the only one available.Pre 1998

Requested Chromatogram and Mass Spectrum to evaluate

Mass Spectral Libraries(example of why version of library is important)

Mass Spectral Libraries(example of why version of library is important)Synonyms

1,1-Dichloro-1-fluoroethane Ethane, 1,1-dichloro-1-fluoro- Freon 141

Mass Spectral LibrariesLibrary Search Against NIST 98 Library produced an excellent hit.Best HitNBS75KBest HitNIST98

SummaryMass Spectral Libraries

Identities of non-target compounds (TICs) may be dependent on the version of library being used.

Most laboratories still use NBS75K library.

Be aware that other libraries exist.

Dioxins and PCB Analysis

Using

GC/High Resolution Mass Spectrometry

(actually high resolution gas chromatography/high resolution mass spectrometry- HRGC/HRMS)

OverviewReview of EPA Dioxins and PCB structures & methods

Typical Mass Spectrometry Instrumentation

Why High Resolution Mass Spectrometry?

High Resolution Mass Spectrometry (MS) Overview

Use of Isotopically Labeled Targets

Comparison of PCB congener and Aroclor methods

Toxicity Equivalents (TEQs) and TEFs

Dioxin/Furans/PCBs(Chemical Structures)

Dioxin Analysis Target CompoundsBoth 1613 and 8290 analyze for these 17 Dioxins and Furans

Drinking water regulates only the 2,3,7,8-TCDD

PCB TerminologyPCBs (can mean anything)Aroclors (mixture of PCBs)PCB Congeners (209 individual)Dioxin-Like PCBs Coplanar PCBsWHO PCBs - a list of 12 specific PCBsHomologs (all congeners having same # of chlorines attached)

More PCB TerminologyBZ/IUPAC Congener Number Prefix to ChlorobiphenylPCB-773,3',4,4'-Tetra-Chlorobiphenyl PCB-813,4,4',5-Tetra-PCB-1052,3,3',4,4'-Penta-PCB-1142,3,4,4',5-Penta-PCB-1182,3',4,4',5-Penta-PCB-1232,3',4,4',5'-Penta-PCB-1263,3',4,4',5-Penta-PCB-1562,3,3',4,4',5-Hexa-PCB-1572,3,3',4,4',5'-Hexa-PCB-1672,3',4,4',5,5'-Hexa-PCB-1693,3',4,4',5,5'-Hexa-PCB-1892,3,3',4,4',5,5'-Hepta-

a total of 209 PCB congenerse.g. PCB 1 ---> PCB 209

Still More PCB TerminologyPCBs as Arochlorsno longer referring to individual PCBsArochlors are complex mixtures

Often designated Aroclor XXXXe.g Aroclor 1242on average, this molecule contains 42 % by weight of Chlorine

Dioxin Analyses

EPA Method 1613B1 - For drinking water and waste water useEPA Method 8290 - for SHW samples

PCB Congener Analysis Methods

EPA Method 1668 - Revision Acan be used for all matricesdated December of 1999contains all 209 possible PCB congeners

EPA Method 8082 - usually used for Aroclors - SHW Sampleshas the option to perform limited set of 19 congenerscan be modified to do other congeners

1 as old NPDES permits requiring dioxin analyses by 613, they will be reissued with requirement for 1613BMethod Overview

PCB as Aroclors Analysis Methods

EPA Method 508 and 608 - for Drinking and Wastewater

EPA Method 8082 - solid and hazardous waste samples use

all of these are GC/ECD techniques

Method Overview

Dioxin and PCB Analysis(Why analyze for them?)Dioxins and PCBs have been shown to be toxic at varying levels.

e.g. Drinking Water MCLs (NJ State Standards)

2,3,7,8-T etra C hloro D ibenzo D ioxin (TCDD)

3 x 10-5 ppbor 0.00003 ppb

PCBs

0.5 ppb

Why did EPA choose GC/High Resolution Mass Spectrometry to analyze for Dioxins and PCBs?

MCLs for these compounds are very low

Need sensitive method(s) capable of low detection limits

Provide a high level of confidence in compound identification

Need to minimize effect of interferences

How Does GC/High Resolution Mass Spectrometry Accomplish These Criteria?

1)Need for Very High Identification Certainty/Minimize Interferences

High Resolution Mass AnalysisUse of Chlorine Isotope Massestwo masses for each targetIsotope Ratios must meet theoretical value

2)Need for very low detection limits

Combination of High Voltage Operation and Selected Ion Monitoring Scan (SIM)Concentrate sample to ul range instead of ml.

Nominal and Exact Masses for Common Elements

PRIVATEElement

Symbol

Nominal Mass

Exact Mass

Abundance

Hydrogen

HD or 2H

12

1.007832.01410

99.990.01

Carbon

12C13C

1213

12.000013.0034

98.911.09

Nitrogen

14N15N

1415

14.003115.0001

99.60.37

Oxygen

16O17O18O

161718

15.994916.999117.9992

99.760.0370.20

Fluorine

F

19

18.9984

100

Silicon

28Si29Si30Si

282930

27.976928.976529.9738

92.284.703.02

Phosphorus

P

31

30.9738

100

Sulphur

32S33S34S

323334

31.972132.971533.9679

95.020.744.22

Chlorine

35Cl37Cl

3537

34.968936.9659

75.7724.23

Bromine

79Br81Br

7981

78.918380.9163

50.549.5

What is High Resolution Mass SpectrometryHigh Resolution Mass Spectrometry is capable of obtaining mass spectra and measuring masses to approximately the fourth decimal place. C12H435Cl4O2

MW = 319.896542

C12H437Cl135Cl3O2

MW = 321.8936

320

VG70-250SE High Res. MSHP5973 Low Res. MSSpecialized MS Instrumentation

What is Mass Resolution?Very Simply - The ability to distinguish between different masses.

e.g. Can we distinguish mass 78 from 79? Can we distinguish between mass 78.003 and 78.004?

A quantitative approach to determining how well we can distinguish different masses is called Resolving Power. Different than chromatographic resolution.

Resolving Powerby definition:Resolving Power(R.P.) = m/m

ppm = R.P. / 1 x 106

a resolving power of 10,000 = 100 ppm

***All High Resolution EPA Methods use an R.P. of 10,000***

Calculation of Mass Resolution

Resolution Example

were asked to separate a three component gas mixture containing

carbon monoxide

nitrogen

ethylene

Calculating Molecular Weights

nominal mass accurate masscarbon monoxide - CO 1 x 12 = 12 1 x 12.0000 = 12.0000 + 1 x 16 = 16 + 1 x 15.9949 = 15.9949 28 27.9949

nitrogen - N2 2 x 14 = 28 2 x 14.0031 = 28.0062 28 28.0062

ethylene - C2H4 2 x 12 = 24 2 x 12.0000 = 24.0000 + 4 x 1 = 4 + 4 x 1.0078 = 4.0312 28 28.0312

What Resolution Do We Need to See All 3 Components?

carbon monoxide - CO 1 x 12.0000 = 12.0000 + 1 x 15.9949 = 15.9949 27.9949

nitrogen - N2 2 x 14.0031 = 28.0062 28.0062

ethylene - C2H4 2 x 12.0000 = 24.0000 + 4 x 1.0078 = 4.0312 28.0312

0.01130.025

What Resolution Do We Need to See All 3 Components? (contd) nominal exact mass Resolving Power mass mass Needed (m / m)

CO 28 27.9949 0.0113 2,478N2 28 28.0062 0.0250 1,120C2H4 28 28.0312

- Need at least 2,500 to see all three components.

Low and Medium Resolution Mass Spectra of Ternary Mixture

Applications to Dioxins/PCB Congener Analyses(2,3,7,8-TetrachloroDibenzoDioxin (TCDD))

2,3,7,8-TCDDC12H4Cl4O2Nominal MassExact MassC12 x 12 = 14412 x 12.000000 = 144.000000H 4 x 1 = 4 4 x 1.007825 = 4.031300Cl 4 x 35 = 140 4 x 34.968853 = 139.875412O 2 x 16 = 32 2 x 15.994915 = 31.989830 320 C12H435Cl4O2 319.896542

37Cl = 36.9659 C12H437Cl4O2 321.8936Calculation of Mass for 2,3,7,8-TCDD Analysis

Table From EPA Method 1613Bm/z 320 & 322

Nominal MassExact MassC12 x 12 = 14412 x 12.000000 = 144.000000H 4 x 1 = 4 4 x 1.007825 = 4.031300Cl 6 x 35 = 210 6 x 34.968853 = 209.813118 358 C12H435Cl6 357.844418

37Cl = 36.9659C12H437Cl137Cl3O2 359.8415C12H437Cl237Cl2O2 361.8385

Same Calculation for PCB CongenersHexaChloroBiphenylC12H4Cl6

From Table 7 of EPA Method 1668Am/z 360 362 364

Isotope Ratio QA/QC Requirements(from EPA 8290)(same as 1613B & 1668A) 15 %

Common Chemical Interferences in the GC/MS determination of 2,3,7,8-TCDD

Selected Ion Monitoring - Better DLs(Sector Instruments Use Voltage Scanning for Accuracy)Full Scan

Detect all masses over a given scan range.e.g. m/z 100-500SIM

Look only for masses releventto targets

High Resolution MS AdvantagesEnhanced Identification Capabilities

Ability to analyze exact masses provides for better identification capability over LRMSDetecting multiple isotopes (chlorine) adds yet another level of confidence in compound identificationEliminates or minimizes interferences in dirty samplesCleanups are the rule not the exception

Enhanced Sensitivity

High Resolution Mass Spectrometers operate at High Voltage (8 KV)Voltage Scanning Selected Ion Monitoring (SIM) Concentration of Sample down to ul as opposed to mls.

Approximate Method Detection LimitsDepends on Matrix

Method AqueousOther

Method 1668 5-300 ppq1-25 ppt

Method 1613B 3 ppq1 ppt

Method 8290 10 ppq1 ppt

high capital cost ( approx. $400,000)

higher maintenance maint. contract 8% of purchase price annually.

skilled staff required

analysis costs high $1,000 /analysis

special facility requirements Vibration Footprint is largeTemp/Humidity Control Special Power RequirementsDisadvantages of HRMS

Use of Isotopic LabelingMethods 1613B, 8290 and 1668A all make use of Isotopic Labeling

13C and 37Cl labeled target compounds are used for quantitation (internal standards are also used)

Use of Isotopic LabelingMethods 8290 - Dioxins/Furans9 out of the 17 targets are labeled

Methods 1613B - Dioxins/Furans15 out of 17 targets are labeled

Methods 1668A - PCB Congeners27 out of 209 are labeled.

Benefits of Isotopic LabelingIsotopically labeled target compounds will behave identically to targets of interest.If targets are lost during processing, labeled standards will also be lost. Corrects for recovery 100%Provides for more accurate quantitation of targets.

Isotopically labeled target compounds elute seconds prior to target of interestEnable analyst to readily identify the targetIf interferences are present, this is extremely helpfulThis too increases ID accuracy

Sample Chromatogram (showing Isotopically Labeled Standards)

Sample Chromatogram (Typical Raw Data Page Dioxins - HxCDD)TargetHxCDDLabeledHxCDDInterferingCompoundsLock MassCheck Channel

PCB Congener Analysis vs. Aroclor Analysis(pros)The toxicity of PCBs is very congener specificmeasurement on an Aroclor basis may not accurately reflect toxicity.

Identification of a PCB is more definitive. Interferences are more easily detected.

Quantitation of individual congeners is more accurate than estimating Aroclors

Composition of weathered, degraded and metabolized PCB mixtures can be measured and interpreted easier using congener vs. Aroclor analysis

Aroclor concentrations can be estimated using congener concentrations (depending on the list of congeners being analyzed for)

PCB Congener Analysis vs. Aroclor Analysis(cons)Very high cost (typically greater than $1,000

TEFs are not available for all congenersWorld Health Organization (WHO) has a list of 12 TEFs

Comparability among laboratorieslabs vary in how they perform PCB congener analysisdifferent labs may use different columns (different coelutions)PCB congener ID comparabilityno good PE samples are available (some SRMs)there is a NIST Intercomparison Exercise for Organic Contaminants in the Marine Environmentcost $2500 for two matrices

Comparison of EPA Method 8082 and 1668A8082DLsCost $75 - $300Aroclor Using GC/ECDMay not meet DQOs.Aroclor analysis may over or underestimated PCB concentrations.Does not measure individual congeners but rather relies not a pattern recognitionAroclor analysis may severely underestimate toxicity.1668APCB Congeners using GC/HRMSDetection LimitsCost > $1,000

EPA Method 1668A MisnomersAll 209 congeners are analyzed for, BUT

Does not provide quantitative values for each of the 209 individually

Not all 209 are quantitated in the same manner.Multipoint vs. single point calibration

Not all 209 congeners are chromatographically resolvedabout 130 congeners are fully resolved

everything else is reported as coelutions

Analyzed under low voltage conditions

not at 70 eV (Typically 30 - 40 eV)

Proficiency Evaluation (PE) Samples for Dioxin and PCB AnalysisPE samples for 2,3,7,8-TCDD - AvailablePE Samples for Aroclors - AvailablePE samples for PCB congeners - NOT AVAILABLE

For congenersSRMs are available from NISTSome standards available from CIL, Wellington, others?Still a problemnone of the above contain all of the WHO PCBs - presumably the most important ones.Need to go with a reliable lab

Dioxins and PCB AnalysisHold TimesFrom 1613B Dioxins8.4.1 There are no demonstrated maximum holding times associated with CDDs/CDFs in aqueous, solid, semi-solid, tissues, or other sample matrices. If stored in the dark at 0-4C and preserved as given above (if required), aqueous samples may be stored for up to one year. Similarly, if stored in the dark at

Reporting Dioxin and PCB Data ResultsTwo Approaches

1) Provide a quantitative value for each target compound

2) Report a single number a Toxicity Equivalent

This approach used frequently for Risk Assessment purposes, Dioxins/Furans/PCBs are often combined together as a Toxic Equivalent Quantity (TEQ)

To calculate TEQ, need to use Toxic Equivalency Factors(TEFs)

TEFs and TEQsToxic Equivalent Quantity (TEQ)Over the years, researchers have determined the relative toxicities for a variety of different compounds with the most toxic -

2,3,7,8-Tetrachlorodibenzodioxin

- being assigned a toxic equivalency factor (TEF) of One (1)

TEFs and TEQs

TEF for 2,3,7,8-TCDD = 1

TEF = Toxic Equivalency Factors. A method of weighting the toxicity of individual dioxin/furan/coplanar PCB compounds, as compared to 2,3,7,8-TCDD.

TEFs and TEQs1994 WHO TEFs(1)1997 WHO TEFs(2)Humans/MammalsFishBirdsPCB-770.00050.00010.00010.05PCB-81--0.00010.00050.1PCB-1050.00010.0001

Calculating TEFs and TEQs

Sheet1

Conc.MDLTEFTEQ

DIOXINSng/kgng/kgng/kg

OCDD5500100.00010.550

1234678-HpCDD4105.00.014.100

123478-HxCDDnd2.50.10.125

123678-HxCDD105.00.11.000

123789-HxCDD8.85.00.10.880

12378-PeCDDnd4.812.400

2378-TCDDnd0.7510.375

FURANS

OCDF130100.00010.013

1234678-HpCDF395.00.010.390

1234789-HpCDFnd3.10.010.016

123478-HxCDFnd4.10.10.205

123678-HxCDFnd1.80.10.090

234678-HxCDFnd1.00.10.050

123789-HxCDFnd0.940.10.047

12378-PeCDFnd0.610.050.015

23478-PeCDFnd2.70.50.675

2378-TCDFnd2.900.10.145

COPLANAR PCB'S

3,4,4',5-TCB (#81)1704.00.00010.017

3,3',4,4'-TCB (#77)124.00.00010.001

3,3',4,4',5-PeCB (#126)194.00.11.900

3,3',4,4',5,5'-HxCB (#169)nd4.00.010.020

2,3,3',4,4'-PeCB (#105)10004.00.00010.100

2,3,4,4',5-PeCB (#114)714.00.00050.036

2,3',4,4',5-PeCB (#118)23004.00.00010.230

2',3,4,4',5-PeCB (#123)934.00.00010.009

2,3,3',4,4',5-HxCB (#156)2804.00.00050.140

2,3,3',4,4',5'-HxCB (#157)714.00.00050.036

2,3',4,4',5,5'-HxCB (#167)5004.00.000010.005

2,3,3',4,4',5,5'-HpCB (#189)474.00.00010.005

TOTAL TEQ13.574

Sheet2

Sheet3

Calculating TEFs and TEQs

Sheet1

Conc.MDLTEFTEQ

ng/kgng/kgng/kg

COPLANAR PCB'S

13,4,4',5-TCB (#81)1704.00.00010.017

23,3',4,4'-TCB (#77)124.00.00010.001

33,3',4,4',5-PeCB (#126)194.00.11.900

43,3',4,4',5,5'-HxCB (#169)nd4.00.010.020

52,3,3',4,4'-PeCB (#105)10004.00.00010.100

62,3,4,4',5-PeCB (#114)714.00.00050.036

72,3',4,4',5-PeCB (#118)23004.00.00010.230

82',3,4,4',5-PeCB (#123)934.00.00010.009

92,3,3',4,4',5-HxCB (#156)2804.00.00050.140

102,3,3',4,4',5'-HxCB (#157)714.00.00050.036

112,3',4,4',5,5'-HxCB (#167)5004.00.000010.005

122,3,3',4,4',5,5'-HpCB (#189)474.00.00010.005

TOTAL TEQ13.574

Sheet3

SummaryPCB Congener Data can be obtained by two methods: EPA Method 8082 and 1668A.

GC/High Resolution Mass Spectrometry provides for the analysis of compounds with excellent identification capability and sensitivity.

PPQ detection levels can only be achieved using GC/High Res Mass Spectrometry

Packed - As suggested by the term, it is filled with a coated inert solid support such as fire brick, alumina, and graphite with a specific mesh size. The coatings are called phases and for best results are chemically bonded to the support. Chemical bonding provides for longer column life and less bleeding (major source of background noise) contributing to lower sensitivity. Column dimensions 1/8 - 1/4 ID x up to about 6 using glass or stainless steel. Advantages - higher capacity (higher conc). Disadvantages: low resolution and low S/N.

Capillary - Here the phase (film) is coated on the inside diameter of the capillary wall with film thickness range of 0.1 to 5 where the ticker film provides for better resolution but also allows for more bleed. Typical dimensions .25mm - .53mm ID x up to 60m made of fused silica coated with polyamide. Advantages: high resolution and better S/N. Disadvantages: low capacity and cost.Chromatography - Greek for color writing - from 1902 Mikhail Tswett trying to separate plant pigments using crushed chalk (CaCO3) and petroleum ether. GC- first instrumental chromatographic technique invented by AJP Martin and RLM Synge in 1941 to improve amino-acid sepns. Earned them the 1952 Nobel Prize in chem for discovering partitioning.Isothermal - Keep oven at one temp thru run. Not very useful. Possibly useful for series of very similar compounds differing by boiling points such as alcohols ( MeOH, EtOH, n-PrOH, i-PrOH, BuOH, i-BuOH). BP 64.6 78.3 97.2 82.4 117.6 99.5

Gradient - temp profile: 40 deg hold for 10 min then 10deg/min to 240 deg and hold there for 20 min. Advantages: 1- resolution and 2- analysis time.Here are some of the commonly used phases. They range in polarity from non-polar such as low polarity DMS to the higher polarity of mixtures with DPS with any combination available. There are specialty phases with very high polarity such as cyanopropylphenyl siloxane or trifluoropropyl methyl siloxane for fluorinated compounds. Some of these columns are used for separation confirmation such as the cyanopropylphenyl siloxane column for method 551.1.Note peaks 15, 16 17 & 18 on the DB-5 column and note the same peaks on the DB-1701 column. This shows the need for confirmatory columns (columns with different phases) so that separation of the compounds can be verified.MS - Mass Spectral - EI Electron impact - used for absolute confirmation. Molecules are ionized and their mass to charge ratio is plotted against its abundance. The resulting spectrum is unique to each molecule and can be looked up in a standard library and the % fit is noted. CI- chemical ionization is used for research and for structure elucidation.FID - Air/Hyd. Flame combusts the compound and the conductivity due to the ionization of the resulting carbon is determined and is presented as a signal. Very large range.NPD - Similar to FID except the combusted compound is passed over a heated bead of rubidium which provides for specificity in determining N and P where P is 500x more sensitive than N. NPD-P limited range, NPD-N broader range.FPD - Similar to FID but detector is light-tight where a PMT/filter assembly collects signal from emission for P at 393nm and S at 529nm.Haal/ELCD - furnace at about 900C produces ionized acidic gases such as HCl or HF which is dissolved into a deionized solvent to produce conductivity proportional to the mass of the halogen in the org. compound. Recently, the term manual integration has become associated with improper laboratory activities.

Manual integrations are not only allowable, but quite necessary..

Every effort should be made to educate the analysts that well maintained systems will help reduce the number of MIsWhen evaluating manual integrations, extra attention should be given to QC data that goes from out to in. This can be accomplished by reviewing the audit trail and comparing re-generated quantation reports.

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