Gas Chromatography/Mass Spectrometry Analysis (GC/MS) Fundamentals and Special Topics

1
Gas Chromatography/Mass Spectrometry
Analysis(GC/MS) Fundamentals and Special Topics

• Zbigniew Bernie Wilk Ph.D.
• Russell Confer M.S.
• Office of Quality Assurance
• New Jersey Department of Environmental Protection

2
Gas Chromatography/Mass Spectrometry

• GC/MS Overview 50 min.
• the nuts and bolts of how GC/MS works
• Break 10 min.
• GC/MS Analysis Special Topics 50 min.
• Chromatograms Peak Integration
• TICs MS Libraries Interferences
• Break 10 min.
• Dioxins and PCB Analyses 50 min.
• GC/High Resolution Mass Spectrometry

3
Gas Chromatography/Mass Spectrometry

• Introduction
• Organic Analysis Overview
• History
• The wide world of Mass Spectrometry
• How it all works
• Tuning/Calibration
• Break
• Gas Chromatography/Mass Spectrometry (GC/MS)
• From Chromatograms to final report
• Mass Spectrometry Libraries and Compound
  Identification (TICs)
• Proper and Improper Peak Integrations -
  Manipulating Results
• Dealing with Interferences
• Break
• - Dioxin and PCB Analyses Using GC/High
  Resolution Mass Spectrometry
• Review of EPA Methods

4
Gas Chromatography/Mass Spectrometry(GC/MS)
Gas Chromatography - Mass Spectrometry


Gas Chromatography
Mass Spectrometry
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
Identifies (detects) pollutant molecules based on
their molecular weight or mass
5
Gas Chromatography/Mass Spectrometry
6
Historical Timeline of GC/MS
1979 USEPA Publishes Wastewater Methods Under
Clean Water Act
1952 Martin and Synge Nobel Prize Chromatography

1900
2000
EPA Born
GC/MS LC/MS ICP/MS
1942 First Commercial Mass Spectrometer
1971 USEPA Purchases 6 Finnigan GC/Mass Specs
1906 Sir J.J. Thompson Nobel Prize
for discovery of electron
7
Dates of Historical Note

• 1906 - Sir J.J. Thomson (Cambridge) gets Nobel
  Prize for the discovery of the electron.
• 1930 - Aston uses MS to study isotopes
• 1942 - first commercial magnetic mass spectometer
• 1952 - Martin and Synge win Nobel Prize for
  Chromatography
• 1959 - Gas Chromatography interfaced to Mass
  Spectrometer
• 1968 - Finnigan Corp. delivers first Quadrupole
  GC/MS
• 1969 - Finnigan Corp. delivers first Quadrupole
  GC/MS with computer
• 1970 - USEPA is born
• 1971 - USEPA purchases 6 Finnigan GC/MS systems
• 1972 - Federal Water Pollution Control Act (CWA)
  is passed
• 1976 - Hewlett Packard introduces fully
  computerized GC/MS system
• 1976 - RCRA Enacted
• 1979 - USEPA publishes wastewater methods under
  CWA
• 1983 - Development of LC/MS interface by Vestal
  et. al.

8
Various Forms of Mass Spectrometry

• A whole range of possibilities/permutations

Sample Introduction Ionization
Mass Separator
Detector Gas Chromatography EI (electron
impact) Quadrupole
channeltron Liquid Chromatography CI
(chemical ionization) Ion Trap
discrete dynode Electrospray NCI negative CI
Time-of-Flight(TOF)
photo-optical FAB(fast atom bombardment)
Sector(BE EB EBE) image
current API (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.
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GC/MS

• Great For the Analysis of Organics
• Gas Chromatography Analysis Requirement
• Organics 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

10
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. 150000 Federal
Pollutant Database approx 700 e.g. Most
Organic Analyses approx. 10 to 80 compounds
in one analysis
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Classification of Organic Compounds
Boiling Point Polarity Technique Ionic h
igh high HPLC HPLC/MS NonVolatiles high high
HPLC HPLC/MS SemiVolatiles
medium low-medium GC GC/MS HPLC
Volatiles low low-medium GC GC/MS
Increasing polarity Increasing solubility
in water
12
Survey of GC/MS Methods(by Program)

• SDWA
• EPA 500 series e.g. 524.2 525
• Clean Water Act
• EPA 600 and 1600 series e.g. 624 625 1624
  1625 1666
• RCRA (Solid and Hazardous Waste)
• EPA 8000 series e.g. 8260 8270
• CERCLA (Superfund)
• OLMO contracts
• Clean Air Act
• TO (Toxic Organics) series e.g. TO-14 TO-15
  TO-17
• (Some ASTM and Standard Methods are also EPA
  approved)

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Principles of Gas Chromatography Mass
Spectrometry
Advantages - high sensitivity excellent
detection limits. Typically low ppb to high
ppt - high selectivity identification is based
on two parameters not one (retention time and
mass spectrum must match standard) selects
analyte of interest with very high confidence -
Speed typical analysis takes from 1/2 hour to
approx. 1 hour analysis can contain upwards of
80 and more pollutants Disadvantages - higher
capital cost (approx. gt85 K vs. 15 K for
GC) - higher maintenance (time expertise and
money) - for optimum results requires analyst
knowledgeable in both chromatography and mass
spectrometry
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The Analytical Process - GC/MS is Last Step
Data Received by DEP 5.6 ppb Benzene
Sample Site Contaminated Site Monitoring
Well Permittee Effluent Drinking Water Facility
Laboratory Side
Sample Analysis

• Determinative Step
• Gas Chromatography (GC)
• Gas Chromatography/Mass Spectrometry (GC/MS)
• High Pressure Liquid Chromatography (HPLC)

Sample Preparation
Sample Clean-Up (optional)
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The Analytical Process(It all starts with Sample
Preparation)
Sample Analysis

• Determinitive Step
• Gas Chromatography (GC)
• Gas Chromatography/Mass Spectrometry (GC/MS)
• High Pressure Liquid Chromatography (HPLC)

Sample Preparation
Sample Clean-Up (optional)
Purge and Trap Liquid-Liquid Extraction Sonication
Solid Phase Extraction (SPE) Soxhlet
Extraction (not an exhaustive listing)
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Sample Preparation Techniques

• Preparation - 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)

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Purge and Trap(Aqueous and Soils / Volatiles
Preparation)
Courtesy of Environmental Conservation
Laboratories Inc.
18
Liquid/Liquid Extraction(Separatory Funnel)
(Aqueous Samples / Semivolatiles Analysis)
Courtesy of Environmental Conservation
Laboratories Inc.
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Sonication(Soils Solids / Semivolatiles)
Courtesy of Environmental Conservation
Laboratories Inc.
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Solid Phase Extraction(Aqueous / Semivolatiles)
Sample
Cartridge
Courtesy of Stanford Laboratory
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Soxhlet Extraction(Soils Solids / Semivolatiles)
Courtesy of Environmental Conservation
Laboratories Inc.
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Sample Clean-Up Techniques

• Clean-Ups - used if interferences are a problem
• stand alone methods are available
• also procedures written into some methods
• these 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.

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

• Analytes of Interest Methods
• Aniline aniline derivatives 3620
• Phenols 3630 3640 8041a
• Phthalate esters 3610 3620 3640
• Nitrosamines 3610 3620 3640
• Organochlorine pesticides PCBs 3610 3620
  3630 3660 3665
• Nitroaromatics and cyclic ketones 3620 3640
• Polynuclear aromatic hydrocarbons 3611 3630
  3640
• Haloethers 3620 3640
• Chlorinated hydrocarbons 3620 3640
• Organophosphorus pesticides 3620
• Petroleum waste 3611 3650
• All base neutral and acid 3640
• priority pollutants

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Gas Chromatography Mass Spectrometry(Operationa
l Description)
Introduction System - Gas Chromatography
Ionization Mass Separation Mass
Detection Data System
Mass Spectrometer
Dedicated Data System
Gas Chromatography
Ionization Source
Mass Analyzer
Particle Detector
Vacuum System - approx. 10-6 torr
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Gas Chromatography

• Powerful Analytical Chemistry technique used to
  separate and identify organic compounds from
  mixtures.
• One requirement
• organics must be volatile or semivolatile
• any very polar non volatile or ionic compounds
  in sample will not be detected

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Gas Chromatography
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Columns

• Packed

• Capillary

Cross section
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THE CHROMATOGRAPHIC PROCESS - PARTITIONING
(gas or liquid) MOBILE PHASE
Sample out
Sample in
STATIONARY PHASE
(solid or heavy liquid coated onto a solid or
support system)
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Temperature Control Isothermal Gradient
Parameters Affecting Separation
Column Type (Phase) Polar (DB-1701
NonPolar (DB-1)Phase ThicknessColumn Dimensions
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Phases
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Chromatograms - 551.1
Same Organic Mixture Different Capillary Columns
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Instrumentation - Detectors

• Destructive
• 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

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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.
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How Mass Spectrometry (Mass Analysis) Works(Use
Table to Calculate Molecular Weights)
Isotopes
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Calculating Molecular Weight (Mass)
Element atomic mass
(amu) Carbon(C) 12 Hydrogen(H)
1 Chlorine(Cl) 35 Fluorine (F)
19 Oxygen(O) 16 Nitrogen(N)
14
Benzene (C6H6)
Pyridine (C5H5N)
6 x 12 72 6 x 1 6 MW 78 amu
5 x 12 60 5 x 1 5 1 x 15 15 MW 79
amu
amu - atomic mass units
36
Gas Chromatography Mass Spectrometry(Operationa
l Description)
Introduction System - Gas Chromatography
Ionization Mass Separation Mass
Detection Data System
Mass Spectrometer
Dedicated Data System
Gas Chromatography
Ionization Source
Mass Analyzer
Particle Detector
Vacuum System - approx. 10-6 torr
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The Ionization Process(Electron Impact) Neutral
molecules are converted into Ions (charged
particles)
.
e-
2e-
Molecular Ion
Neutral Molecule
(70 Electron Volts)
Fragment Ion 1
Fragment Ion 2 etc.
.
e-
2e-

• Mass Analysis can only work for charged
  species - not for neutrals.

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GC/MS - Mass Analysis
Wavelength Separation

Continuous Light
Mass Separation (quadrupole)
Ions
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Principles of Gas Chromatography/Mass
Spectrometry(NIST Library Mass Spectra for
Benzene)
m/z 78
Benzene
Abundance (Signal)
78 amu
mass/charge (m/z) ------gt
40
Principles of Gas Chromatography/Mass
Spectrometry(NIST Library Mass Spectra for
Pyridine)
m/z 79
Pyridine
Abundance (Signal)
79 amu
mass/charge (m/z) ------gt
41
One More Example for o-Xylene(Fragment Ions
contain Useful Information)
Xylene (C8H10)
Element
mass Carbon(C) 12 Hydrogen(H) 1 Chlorine(Cl) 3
5 Fluorine (F) 19 Oxygen(O) 16 Nitrogen(N) 14
8 x 12 96 10 x 1 10 MW 106
Molecular Ions can break down into smaller
fragments
.
.
.
m/z 106
m/z 91
m/z 77
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Principles of Gas Chromatography/Mass
Spectrometry(NIST Library Mass Spectra for
Xylene)
o-Xylene (C8H10)
Element
mass Carbon(C) 12 Hydrogen(H) 1 Chlorine(Cl) 3
5 Fluorine (F) 19 Oxygen(O) 16 Nitrogen(N) 14
8 x 12 96 10 x 1 10 MW 106
Abundance (Signal)
mass/charge (m/z) ------gt
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What Does GC/MS Data Look LikeGC/MS
Chromatogram of a 4 Component Mixture
Abundance (Signal)
Retention Time ------gt
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What Does GC/MS Data Look LikeGC/MS
Chromatogram of a 4 Component Mixture
Abundance (Signal)
Retention Time ------gt
45
What Does GC/MS Data Look LikeGC/MS
Chromatogram From EPA Method 524.2 Analysis

Abundance (Signal)
Retention Time ------gt
Courtesy of the NJDHSS Laboratory
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What Does GC/MS Data Look LikeReviewing of Mass
Spectra
6.99 min.

Abundance (Signal)
Retention Time ------gt
m/z 78
mass/charge ------gt
47
What Does GC/MS Data Look LikeReviewing of Mass
Spectra
6.77 min.


Abundance (Signal)
Retention Time ------gt
m/z 78
mass/charge ------gt
11-dichloropropene/carbon tetrachloride
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Difficult Mass Spectra
usually

• Mass Spectrometry does not always provide an
  easily interpretable compound identification
  e.g. MTBE
• use of mass spectral libraries for ID
  determination
• use of manual interpretation techniques
• use of alternate MS and other techniques

MTBE MW 88
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Mass Spectral Interpretation Procedures

• GC/MS Interpretation Procedures
• Identify Molecular Ion if present
• Evaluate any Isotopic Observations
• Use Isotopes to calculate probable carbon s for
  Molecule and/or fragments
• Review all losses observed to determine
  substructures
• Review major fragments
• Hypothesize a molecular structure consistent with
  above observations
• Must Confirm Hypothesis with additional data.
• Typically chemical ionization MS
• High resolution mass spectrometry
• Infra Red Spectroscopy
• Nuclear Magnetic Resonance Spectrometry
• Obtaining a pure standard and confirming mass
  spectra with unknown

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GC/MS Summary

• 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.

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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)

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GC/MS Data ProcessingImportant to Review Peak
Integration
Chromatogram (maximum information content)
GC/MS
Final Report
53
GC/MS Data ProcessingImportant to Review Peak
Integration

• Calibrations and quantitation of organics all
  rely on correct chromatographic peak integrations

Standards (Ax/Ais)-----gtResponse
Factors-----gtSample Quantitation
Ais
Ax
54

NJDEP OQA OCTOBER 2002 Manual Integration FOR
GC/MS
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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.

56

• 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.

57

• Manual integration may be done in the
  following cases where the automatic integrator
  has
• failed to integrate a peak or part of a peak
• integrated one peak as two peaks
• integrated the wrong peak out of two similar
  peaks
• not integrated from baseline to baseline
• integrated a peak due to an elevated baseline
• integrated a negative peak
• integrated a peak beyond baseline resolution (too
  much area)
• Any additional situations in which the
  auto-integrator fails to perform properly and/or
  consistently

58

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

59

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.
60

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.
61

• 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.

62

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.
63

• 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

64

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
65

• TYPES OF IMPROPER INTEGRATIONS
• Baseline addition or subtraction
• Do not add or subtract from the baseline. Another
  example of an incorrect manual integration

66

TYPES OF IMPROPER INTEGRATIONS Poor
sensitivity. Signal is not 3 times the background.
67

• WHY IS THIS HAPPENING
• Cost factors
• Level of Expertise factors
• Unethical Behavior

68

• 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.

69

• 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.

70

• Why Unethical Behavior Occurs
• Real or perceived pressures
• Lack of ethics education and awareness
• Lack of management oversite and review
• Lack of knowledge or confidence in appropriate
  ways to solve problems

71

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.
72

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.
73

DOCUMENTATION
74

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.
75

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.
76

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.
77

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.
78

Questions
79
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.

80
Interferences in GC/MS Analysis

• Problems 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.

81
Interferences - What do they look like(Example 1)
interferences
Extract could only be concentrated to 5 mls.
82
Interferences - What Do They Look Like(Example 2
)
- Can only concentrate to 5 mls. - diluted
extract 15 - total dilution factor 25x
a - Napthalene b - dimethyl phthalate c - diethyl
phthalate d - di-n-butyl-phthalate
a
c
b
d
83
Interferences - What Are They(Example 2)
13-dichloro-2-propanol (a chlorinated alcohol)
1-methyl 24-diisocyanato benzene (a
diisocyanate)
84
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 interferences
• may 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 CleanUps
• methods 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 stream
• permittee likely has most intimate knowledge of
  their own waste stream.
• Additional non-EPA method testing may be
  appropriate to identify interferences.

85
Clean-Up Techniques from SW-846(stand alone
methods strictly for cleanups)

• Analytes of Interest Methods
• Aniline aniline derivatives 3620
• Phenols 3630 3640 8041a
• Phthalate esters 3610 3620 3640
• Nitrosamines 3610 3620 3640
• Organochlorine pesticides PCBs 3610 3620
  3630 3660 3665
• Nitroaromatics and cyclic ketones 3620 3640
• Polynuclear aromatic hydrocarbons 3611 3630
  3640
• Haloethers 3620 3640
• Chlorinated hydrocarbons 3620 3640
• Organophosphorus pesticides 3620
• Petroleum waste 3611 3650
• All base neutral and acid 3640
• priority pollutants

86
Interferences in GC/MS Analysis

• 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 methods
• use of clean-up procedures
• identify source of interference

87
Mass Spectral Libraries

• What 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

88
Mass Spectral Libraries

• Why are they important - cont.
• Site Remediation for example typically requests
• VOAs 10 TICs
• BNs 15 TICs
• frequently drinking water methods
• If TICs are found proper identification is very
  important
• may need correct ID for remediation
• may need to provide data to County Health Dept
  and Owners of Potable Well (as in BUST)

89
Mass Spectral Libraries

• Why are they important - cont
• Waste Water Permitting
• some industries indicate that interferences are
  present which preclude them analyzing the sample
  to permit detection limits
• Identification 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 Water
• BSDW reporting form has ability to enter TIC
  observations from a laboratory.

90
Mass Spectral Libraries

• How many libraries are there
• NIST/NIH/EPA Mass Spectral Library
• NBS 75K
• NIST 98
• NIST 02
• Wiley Mass Spectral Library
• Combination Wiley/NIST
• Custom Libraries
• industry specific
• proprietary

91
Mass Spectral Libraries

• For NIST75K Library
• Approx 50000 Mass Spectra
• Approx 25000 Replicates
• This library is very old
• Not very well reviewed
• Variety of Sources not well filtered.
• Labs should NOT be using this!

92
Mass Spectral Libraries

• For NIST98 Library - (a 75 increase over NBS75K
  library)
• 107886 Compounds
• 107829 Chemical Structures
• 129136 Spectra
• 21250 Replicate Spectra
• 13205 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!

93
Mass Spectral Libraries

• For NIST98 Library
• (where does this 75 increase come from)
• Mass Spectra from other sources were added in
• Chemical Concepts - including Prof Hennebergs
  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 Acids
• VERIFIN CBDCOM Chemical Weapons

94
Mass Spectral Libraries

• For NIST 02 Library - 35 increase in coverage
  over NIST 98 Library
• 27750 Replicate Spectrafrom high quality
  sources
• 147198 Compounds with Spectra
• 18598 Compounds with Replicate Spectra
• 147194 Chemical Structures
• 111 Average Peaks/Spectrum
• 174948 spectra
• 98 Median Peaks/Spectrum

95
Mass Spectral Libraries

• Comparison of NIST/NIH/EPA Libraries - different
  revisions
• NBS75K NIST98 NIST 02
• Total Spectra 75 000 129136 174948
• Total Replicates 20000 21250 27750

96
Mass Spectral Libraries

• Wiley Registry of Mass Spectral Data - 7th
  Edition
• the worlds largest reference database of over
  250000 Electron-Impact mass spectra
• Wiley Library may or may not include NIST library
• Wiley contains mass spectra that are not as well
  reviewed.
• Still very useful if NIST library comes up
  short.

97
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

98
Mass Spectral Libraries(example of why version
of library is important)
99
Mass Spectral Libraries(example of why version
of library is important)
100
Mass Spectral Libraries(example of why version
of library is important)
101
Mass Spectral Libraries(example of why version
of library is important)

• Synonyms
• 11-Dichloro-1-fluoroethane
• Ethane 11-dichloro-1-fluoro-
• Freon 141

102
Mass Spectral Libraries

• Library Search Against NIST 98 Library produced
  an excellent hit.

Best Hit NBS75K
Best Hit NIST98
103
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.

104
Dioxins and PCB AnalysisUsingGC/High
Resolution Mass Spectrometry(actually high
resolution gas chromatography/high resolution
mass spectrometry- HRGC/HRMS)
105
Overview

• Review 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

106
Dioxin/Furans/PCBs(Chemical Structures)
107
Dioxin Analysis Target Compounds

• Both 1613 and 8290 analyze for these 17 Dioxins
  and Furans
• Drinking water regulates only the 2378-TCDD

108
PCB Terminology

• PCBs (can mean anything)
• Aroclors (mixture of PCBs)
• PCB Congeners (209 individual)
• Dioxin-Like PCBs
• Coplanar PCBs
• WHO PCBs - a list of 12 specific PCBs
• Homologs (all congeners having same of
  chlorines attached)

109
More PCB Terminology

• BZ/IUPACCongenerNumber Prefix to
  Chlorobiphenyl
• PCB-77 3344-Tetra-Chlorobiphenyl
• PCB-81 3445-Tetra-
• PCB-105 23344-Penta-
• PCB-114 23445-Penta-
• PCB-118 23445-Penta-
• PCB-123 23445-Penta-
• PCB-126 33445-Penta-
• PCB-156 233445-Hexa-
• PCB-157 233445-Hexa-
• PCB-167 234455-Hexa-
• PCB-169 334455-Hexa-
• PCB-189 2334455-Hepta-
• a total of 209 PCB congeners e.g. PCB 1 ---gt PCB
  209

110
Still More PCB Terminology

• PCBs as Arochlors
• no longer referring to individual PCBs
• Arochlors are complex mixtures
• Often designated Aroclor XXXX
• e.g Aroclor 1242
• on average this molecule contains 42 by weight
  of Chlorine

111
Method Overview

• Dioxin Analyses
• EPA Method 1613B1 - For drinking water and waste
  water use
• EPA Method 8290 - for SHW samples
• PCB Congener Analysis Methods
• EPA Method 1668 - Revision A
• can be used for all matrices
• dated December of 1999
• contains all 209 possible PCB congeners
• EPA Method 8082 - usually used for Aroclors - SHW
  Samples
• has the option to perform limited set of 19
  congeners
• can be modified to do other congeners
• 1 as old NPDES permits requiring dioxin analyses
  by 613 they will be reissued with requirement
  for 1613B

112
Method 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

113
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)
• 2378-T etra C hloro D ibenzo D ioxin (TCDD)
• 3 x 10-5 ppb or 0.00003 ppb
• PCBs
• 0.5 ppb

114

• 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

115

• How Does GC/High Resolution Mass Spectrometry
  Accomplish These Criteria
• 1) Need for Very High Identification
  Certainty/Minimize Interferences
• High Resolution Mass Analysis
• Use of Chlorine Isotope Masses
• two masses for each target
• Isotope 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.

116
Nominal and Exact Masses for Common Elements
117
What is High Resolution Mass Spectrometry

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

320
C12H435Cl4O2 MW 319.896542
C12H437Cl135Cl3O2 MW 321.8936
118
Specialized MS Instrumentation
VG70-250SE High Res. MS
HP5973 Low Res. MS
119
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.
120
Resolving Power
by definition Resolving Power(R.P.) m/m ppm
R.P. / 1 x 106 a resolving power of 10000
100 ppm All High Resolution EPA Methods use
an R.P. of 10000
121
Calculation of Mass Resolution
122
Resolution Example
were asked to separate a three component gas
mixture containing carbon monoxide nitrogen eth
ylene
123
Calculating Molecular Weights
nominal
mass accurate mass carbon
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
124
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
0.0113
0.025
125
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 2478 N2 28
28.0062
0.0250 1120 C2H4 28
28.0312 - Need at least 2500 to see all three
components.
126
Low and Medium Resolution Mass Spectra of
Ternary Mixture
127
Applications to Dioxins/PCB Congener
Analyses (2378-TetrachloroDibenzoDioxin (TCDD))
128
Calculation of Mass for 2378-TCDD Analysis
2378-TCDD C12H4Cl4O2
Nominal Mass Exact Mass C 12 x 12 144 12
x 12.000000 144.000000 H 4 x 1
4 4 x 1.007825 4.031300 Cl 4 x 35
140 4 x 34.968853 139.875412 O
2 x 16 32 2 x 15.994915
31.989830 320 C12H435Cl4O2
319.896542 37Cl 36.9659 C12H437Cl4O2
321.8936
129
Table From EPA Method 1613B
m/z 320 322
130
Same Calculation for PCB Congeners
HexaChloroBiphenyl C12H4Cl6
Nominal Mass Exact Mass C 12 x 12 144 12
x 12.000000 144.000000 H 4 x 1
4 4 x 1.007825 4.031300 Cl 6 x 35
210 6 x 34.968853 209.813118
358 C12H435Cl6 357.844418 37Cl
36.9659 C12H437Cl137Cl3O2 359.8415 C12H437
Cl237Cl2O2 361.8385
131
From Table 7 of EPA Method 1668A
m/z 360 362 364
132
Isotope Ratio QA/QC Requirements(from EPA
8290)(same as 1613B 1668A)
15
133
Common Chemical Interferences in the GC/MS
determination of 2378-TCDD
134
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 100-500
SIM Look only for masses relevent to targets
135
High Resolution MS Advantages

• Enhanced Identification Capabilities
• Ability to analyze exact masses provides for
  better identification capability over LRMS
• Detecting multiple isotopes (chlorine) adds yet
  another level of confidence in compound
  identification
• Eliminates or minimizes interferences in dirty
  samples
• Cleanups 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.

136
Approximate Method Detection Limits

• Depends on Matrix
• Method Aqueous Other
• Method 1668 5-300 ppq 1-25 ppt
• Method 1613B 3 ppq 1 ppt
• Method 8290 10 ppq 1 ppt

137
Disadvantages of HRMS

• high capital cost ( approx. 400000)
• higher maintenance
• maint. contract 8 of purchase price
  annually.
• skilled staff required
• analysis costs high
• 1000 /analysis
• special facility requirements
• Vibration
• Footprint is large
• Temp/Humidity Control
• Special Power Requirements

138
Use of Isotopic Labeling

• Methods 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)

139
Use of Isotopic Labeling

• Methods 8290 - Dioxins/Furans
• 9 out of the 17 targets are labeled
• Methods 1613B - Dioxins/Furans
• 15 out of 17 targets are labeled
• Methods 1668A - PCB Congeners
• 27 out of 209 are labeled.

140
Benefits of Isotopic Labeling

• Isotopically 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 interest
• Enable analyst to readily identify the target
• If interferences are present this is extremely
  helpful
• This too increases ID accuracy

141
Sample Chromatogram(showing Isotopically Labeled
Standards)
142
Sample Chromatogram(Typical Raw Data Page
Dioxins - HxCDD)
Target HxCDD
Labeled HxCDD
Interfering Compounds
Lock Mass Check Channel
143
PCB Congener Analysis vs. Aroclor Analysis(pros)

• The toxicity of PCBs is very congener specific
• measurement 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)

144
PCB Congener Analysis vs. Aroclor Analysis(cons)

• Very high cost (typically greater than 1000
• TEFs are not available for all congeners
• World Health Organization (WHO) has a list of 12
  TEFs
• Comparability among laboratories
• labs vary in how they perform PCB congener
  analysis
• different labs may use different columns
  (different coelutions)
• PCB congener ID comparability
• no good PE samples are available (some SRMs)
• there is a NIST Intercomparison Exercise for
  Organic Contaminants in the Marine Environment
• cost 2500 for two matrices

145
Comparison of EPA Method 8082 and 1668A

• 8082
• DLs
• Cost 75 - 300
• Aroclor Using GC/ECD
• May not meet DQOs.
• Aroclor analysis may over or underestimated PCB
  concentrations.
• Does not measure individual congeners but rather
  relies not a pattern recognition
• Aroclor analysis may severely underestimate
  toxicity.
• 1668A
• PCB Congeners using GC/HRMS
• Detection Limits
• Cost gt 1000

146
EPA Method 1668A Misnomers

• All 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
  resolved
• about 130 congeners are fully resolved
• everything else is reported as coelutions
• Analyzed under low voltage conditions
• not at 70 eV (Typically 30 - 40 eV)

147
Proficiency Evaluation (PE) Samples for Dioxin
and PCB Analysis

• PE samples for 2378-TCDD - Available
• PE Samples for Aroclors - Available
• PE samples for PCB congeners - NOT AVAILABLE
• For congeners
• SRMs are available from NIST
• Some standards available from CIL Wellington
  others
• Still a problem
• none of the above contain all of the WHO PCBs -
  presumably the most important ones.
• Need to go with a reliable lab

148
Dioxins and PCB AnalysisHold Times

• From 1613B Dioxins
• 8.4.1 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 lt-10C
  solid semi-solid multi-phase and tissue
  samples may be stored for up to one year.
• 8.4.2 Store sample extracts in the dark at lt-10C
  until analyzed. If stored in the dark at lt-10C
  sample extracts may be stored for up to one year.
• From 1668A PCB Congeners
• 8.5.1 There are no demonstrated maximum holding
  times associated with the CBs in aqueous solid
  semi-solid tissues or other sample matrices. If
  stored in the dark at 0-4 EC and preserved as
  given above (if required) aqueous samples may be
  stored for up to one year. Similarly if stored
  in the dark at lt-10 EC solid semisolid
  multi-phase and tissue samples may be stored for
  up to one year.

149
Reporting Dioxin and PCB Data Results

• Two 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)

150
TEFs and TEQs

• Toxic Equivalent Quantity (TEQ)
• Over the years researchers have determined the
  relative toxicities for a variety of different
  compounds with the most toxic -
• 2378-Tetrachlorodibenzodioxin
• - being assigned a toxic equivalency factor
  (TEF) of One (1)

151
TEFs and TEQs

• TEF for 2378-TCDD 1
• TEF Toxic Equivalency Factors. A method of
  weighting the toxicity of individual
  dioxin/furan/coplanar PCB compounds as compared
  to 2378-TCDD.

152
TEFs and TEQs

• 1994 WHO TEFs(1) 1997 WHO TEFs(2) Humans
  /Mammals Fish Birds
• PCB-77 0.0005 0.0001 0.0001 0.05
• PCB-81 -- 0.0001 0.0005 0.1
• PCB-105 0.0001 0.0001 lt0.000005 0.0001
• PCB-114 0.0005 0.0005 lt0.000005 0.0001
• PCB-118 0.0001 0.0001 lt0.000005 0.00001
• PCB-123 0.0001 0.0001 lt0.000005 0.00001
• PCB-126 0.1 0.1 0.005 0.1
• PCB-156 0.0005 0.0005 lt0.000005 0.0001
• PCB-157 0.0005 0.0005 lt0.000005 0.0001
• PCB-167 0.00001 0.00001 lt0.000005 0.00001
• PCB-169 0.01 0.01 0.00005 0.001
• PCB-170 0.0001 -- -- --
• PCB-180 0.00001 -- -- --
• PCB-189 0.0001 0.0001 lt0.000005 0.00001

153
Calculating TEFs and TEQs

154
Calculating TEFs and TEQs

155
Summary

• PCB 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