Gas Chromatography/Mass Spectrometry Analysis (GC/MS) : Fundamentals and Special Topics
Gas Chromatography/Mass Spectrometry Analysis (GC/MS) : Fundamentals and Special Topics
Gas Chromatography/Mass Spectrometry Analysis (GC/MS) : Fundamentals and Special Topics
Spectrometry Analysis
(GC/MS)
Fundamentals and Special Topics
50 min.
Break
10 min.
50 min.
Break
10 min.
50 min.
Introduction
Break
Break
Mass
Spectrometry
1900
1906
Sir J.J. Thompson
Nobel Prize for
discovery of electron
1979
USEPA Publishes
Wastewater Methods
Under Clean Water Act
EPA Born
1942
First Commercial
Mass Spectrometer
1971
USEPA Purchases 6
Finnigan GC/Mass Specs
2000
GC/MS
LC/MS
ICP/MS
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.
Sample Introduction
Ionization
Mass Separator
Quadrupole
Detector
Gas Chromatography
EI (electron impact)
channeltron
Liquid Chromatography
Electrospray
CI (chemical ionization)
Ion Trap
discrete dynode
NCI negative CI
Time-of-Flight(TOF)
photo-optical
FAB(fast atom bombardment) Sector(BE, EB, EBE)
image current
n
API (atmospheric pressure)
FTMS (MS )
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/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?
LC/MS?
approx. 150,000
approx 700
e.g.
Most Organic Analyses:
approx. 10 to 80 compounds in one analysis
Polarity *
Technique
Ionic
high
high
HPLC, HPLC/MS
NonVolatiles
high
high
HPLC, HPLC/MS
SemiVolatiles
medium
low-medium
Volatiles
low
low-medium
GC; GC/MS
CERCLA (Superfund)
OLMO contracts
- 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. $ >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
Sample Site
Contaminated Site
5.6 ppb
Monitoring Well
Benzene
Permittee Effluent
Drinking Water Facility
Laboratory Side
Sample Analysis
Sample
Preparation
Sample
Clean-Up
(optional)
Determinative Step
Gas Chromatography (GC)
Gas Chromatography/Mass Spectrometry
(GC/MS)
High Pressure Liquid Chromatography (HPLC)
Sample
Sample
Preparation Clean-Up
(optional)
2)
Liquid/Liquid Extraction
(Separatory Funnel)
(Aqueous Samples / Semivolatiles Analysis)
Sonication
(Soils, Solids / Semivolatiles)
Sample
Cartridge
Soxhlet Extraction
(Soils, Solids / Semivolatiles)
Analytes of Interest
Methods
3620
3630, 3640, 8041a
3610, 3620, 3640
3610, 3620, 3640
3610, 3620, 3630, 3660, 3665
3620, 3640
3611, 3630, 3640
3620, 3640
3620, 3640
3620
3611, 3650
3640
Gas
Chromatography
Ionization
Source
Mass
Analyzer
Particle
Detector
Dedicated
Data System
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
Gas Chromatography
Columns
Packed
Capillary
Cross section
MOBILE PHASE
Sample
out
Sample
in
STATIONARY PHASE
(solid or heavy liquid coated onto a solid or support system)
240
200
Temp (deg C)
160
120
80
40
0
0
10
20
30
Time (min)
40
50
60
Phases
Chromatograms - 551.1
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]
Symbol
Nomi
nal
Mass
Exact
Mass
Abundanc
e
Hydrogen
H
D or 2H
1
2
1.00783
2.01410
99.99
0.01
C
C
12
13
12.0000
13.0034
98.91
1.09
N
N
14
15
14.0031
15.0001
99.6
0.37
O
O
18
O
16
17
18
15.9949
16.9991
17.9992
99.76
0.037
0.20
19
18.9984
Si
Si
30
Si
28
29
30
27.9769
28.9765
29.9738
92.28
4.70
3.02
31
30.9738
100
S
S
34
S
32
33
34
31.9721
32.9715
33.9679
95.02
0.74
4.22
Cl
Cl
35
37
34.9689
36.9659
75.77
24.23
Br
Br
79
81
78.9183
80.9163
50.5
49.5
Carbon
Nitrogen
12
13
14
15
16
Oxygen
Fluorine
17
28
Silicon
Phosphorus
29
32
Sulphur
Chlorine
Bromine
33
35
37
79
81
100
Isotopes
H
H
H C C C H
H C
C
N
C
C
H
H
Benzene
(C6H6)
6 x 12 = 72
6x1 = 6
MW = 78 amu
amu - atomic mass units
Pyridine
(C5H5N)
5 x 12 = 60
5x1 = 5
1 x 15 = 15
MW = 79 amu
Gas
Chromatography
Ionization
Source
Mass
Analyzer
Particle
Detector
Dedicated
Data System
e +
Neutral Molecule
+.
Molecular
Ion
+ 2e
Fragment Ion 1
H
H
e +
*
H
C
H C C C H
H C C C H
Mass Analysis can only work for charged species - not for neutrals.
+ 2e
Continuous Light
Mass Separation
(quadrupole)
m
4V
z = qr22
Ions
Abundance (Signal)
m/z 78
Benzene
H
H C C C H
H
78 amu
Abundance (Signal)
H
H
Pyridine
H C
m/z 79
C
C
H
H
79 amu
Element
Carbon(C)
Hydrogen(H)
Chlorine(Cl)
Fluorine (F)
Oxygen(O)
Nitrogen(N)
mass
12
1
35
19
16
14
C
C
Xylene
(C8H10)
8 x 12 = 96
10 x 1 = 10
MW = 106
H
C
H
H
H
C
C
H
H
C
H
m/z 106
H
H
.
C
C
H
m/z 91
H
H
C
H
C
C
C
C
H
m/z 77
C
C
H
H
H
C
H
Abundance (Signal)
Element
Carbon(C)
Hydrogen(H)
Chlorine(Cl)
Fluorine (F)
Oxygen(O)
Nitrogen(N)
o-Xylene
(C8H10)
8 x 12 = 96
10 x 1 = 10
MW = 106
Abundance (Signal)
Abundance (Signal)
Abundance (Signal)
6.99 min.
Abundance (Signal)
mass/charge ------>
6.77 min.
Abundance (Signal)
mass/charge ------>
1,1-dichloropropene/carbon tetrachloride
100
73
MTBE MW= 88
50
15
0
41
29
27
39
31
57
45
10
20
30
40
( m a in lib ) P ro p a n e , 2 - m e t h o x y - 2 - m e t h y l-
55
50
79
60
70
80
90
100
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.
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)
Chromatogram
(maximum
information content)
GC/MS
Final Report
Ax
Ais
NJDEP
OQA
OCTOBER 2002
Manual Integration
FOR 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.
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
Cost Factors
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
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.
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.
interferences
a - Napthalene
b - dimethyl phthalate
c - diethyl phthalate
d - di-n-butyl-phthalate
b c
1,3-dichloro-2-propanol
(a chlorinated alcohol)
Analytes of Interest
Methods
3620
3630, 3640, 8041a
3610, 3620, 3640
3610, 3620, 3640
3610, 3620, 3630, 3660, 3665
3620, 3640
3611, 3630, 3640
3620, 3640
3620, 3640
3620
3611, 3650
3640
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
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
NIST98
NIST 02
Total Spectra
75, 000
129,136
174,948
Total Replicates
20,000
21,250
27,750
Best Hit
NBS75K
81
100
Cl
50
61
26
31
0
35
37
10
20
30
40
50
( m a in lib ) 1 , 1 - D ic h lo ro - 1 - fl u o ro e t h a n e
Freon 141
45
Cl
63
60
83
70
80
101
90
100
110
120
130
Best Hit
NIST98
Summary
Mass 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.
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
Dioxin/Furans/PCBs
(Chemical Structures)
PCB Terminology
BZ/IUPAC
Congener
Number
Prefix to Chlorobiphenyl
PCB-77
3,3',4,4'-Tetra-Chlorobiphenyl
PCB-81
3,4,4',5-Tetra-
PCB-105
2,3,3',4,4'-Penta-
PCB-114
2,3,4,4',5-Penta-
PCB-118
2,3',4,4',5-Penta-
PCB-123
2,3',4,4',5'-Penta-
PCB-126
3,3',4,4',5-Penta-
PCB-156
2,3,3',4,4',5-Hexa-
PCB-157
2,3,3',4,4',5'-Hexa-
PCB-167
2,3',4,4',5,5'-Hexa-
PCB-169
3,3',4,4',5,5'-Hexa-
PCB-189
2,3,3',4,4',5,5'-Hepta-
Method Overview
Dioxin Analyses
EPA Method 1613B1 - For drinking water and waste water use
EPA Method 8290 - for SHW samples
as old NPDES permits requiring dioxin analyses by 613, they will be reissued with
requirement for 1613B
Method Overview
PCBs
0.5 ppb
or
0.00003 ppb
Symbol
Nomi
nal
Mass
Exact
Mass
Abundanc
e
Hydrogen
H
D or 2H
1
2
1.00783
2.01410
99.99
0.01
C
C
12
13
12.0000
13.0034
98.91
1.09
N
N
14
15
14.0031
15.0001
99.6
0.37
O
O
18
O
16
17
18
15.9949
16.9991
17.9992
99.76
0.037
0.20
19
18.9984
Si
Si
30
Si
28
29
30
27.9769
28.9765
29.9738
92.28
4.70
3.02
31
30.9738
100
S
S
34
S
32
33
34
31.9721
32.9715
33.9679
95.02
0.74
4.22
Cl
Cl
35
37
34.9689
36.9659
75.77
24.23
Br
Br
79
81
78.9183
80.9163
50.5
49.5
Carbon
Nitrogen
12
13
14
15
16
Oxygen
Fluorine
17
28
Silicon
Phosphorus
29
32
Sulphur
Chlorine
Bromine
33
35
37
79
81
100
100
320
35
C12H4 Cl4O2
MW = 319.896542
Cl
Cl
Cl
Cl
50
C12H437Cl135Cl3O2
257
194
74
MW = 321.8936
50 62
0
160
97
85
113
122
144
50
80
110
140
170
( m a in lib ) 2 , 3 , 7 , 8 - T e t ra c h lo ro d ib e n z o - p - d io x in
187
287
229
200
230
260
290
320
Specialized MS Instrumentation
VG70-250SE High Res. MS
Resolving Power
by 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***
Resolution Example
were asked to separate a three component gas mixture
containing
carbon monoxide
nitrogen
ethylene
nitrogen - N2
ethylene - C2H4
accurate mass
1 x 12 = 12
+ 1 x 16 = 16
28
1 x 12.0000 = 12.0000
+ 1 x 15.9949 = 15.9949
27.9949
2 x 14 = 28
28
2 x 14.0031 = 28.0062
28.0062
2 x 12 = 24
+ 4x 1= 4
28
2 x 12.0000 = 24.0000
+ 4 x 1.0078 = 4.0312
28.0312
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
28
28
28
exact
mass
mass
Resolving Power
Needed (m / m)
27.9949
0.0113
2,478
0.0250
1,120
28.0062
28.0312
2,3,7,8-TCDD
C
H
Cl
O
37
Nominal Mass
12 x 12 = 144
4x 1 =
4
4 x 35 = 140
2 x 16 = 32
320
Cl = 36.9659
C12H4Cl4O2
Exact Mass
12 x 12.000000 = 144.000000
4 x 1.007825 =
4.031300
4 x 34.968853 = 139.875412
2 x 15.994915 = 31.989830
C12H435Cl4O2
319.896542
C12H437Cl4O2
321.8936
100
HexaChloroBiphenyl
C12H4Cl6
50
290
145
109
37
49
218
127
74
98
61
180
163
0
30
50
70
1,1'-Biphenyl, 3,3',4,4',5,5'-hexachloro-
C
H
Cl
37
90
110
Nominal Mass
12 x 12 = 144
4x 1 =
4
6 x 35 = 210
358
Cl = 36.9659
130
150
254
204
170
190
324
240
210
230
250
270
290
310
330
350
Exact Mass
12 x 12.000000 = 144.000000
4 x 1.007825 =
4.031300
6 x 34.968853 = 209.813118
C12H435Cl6
357.844418
C12H437Cl137Cl3O2 359.8415
C12H437Cl237Cl2O2 361.8385
m/z 360
362
364
15 %
100
Full Scan
Detect all masses
over a given scan
range.
e.g. m/z 100-500
Cl
Cl
Cl
Cl
50
50 62
74
85
97
113
160
194
187
143
50
80
110
140
170
( m a in lib ) 2 , 3 , 7 , 8 - T e t ra c h lo ro d ib e n z o - p - d io x in
257
287
229
200
230
260
290
322
100
SIM
Look only for
masses relevent
to targets
320
Cl
Cl
Cl
Cl
50
50 62
0
74
85
97
113
160
143
50
80
110
140
170
( m a in lib ) 2 , 3 , 7 , 8 - T e t ra c h lo ro d ib e n z o - p - d io x in
194
187
257
287
229
200
230
260
290
320
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.
Aqueous
Other
Method 1668
5-300 ppq
1-25 ppt
Method 1613B
Method 8290
3 ppq
1 ppt
10 ppq
1 ppt
Disadvantages of HRMS
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 large
Temp/Humidity Control
Special Power Requirements
Sample Chromatogram
(showing Isotopically Labeled Standards)
Sample Chromatogram
(Typical Raw Data Page Dioxins - HxCDD)
Target
HxCDD
Labeled
HxCDD
Interfering
Compounds
Lock Mass
Check Channel
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 > $1,000
(TEFs)
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
<0.000005
0.0001
PCB-114
0.0005
0.0005
<0.000005
0.0001
PCB-118
0.0001
0.0001
<0.000005
0.00001
PCB-123
0.0001
0.0001
<0.000005
0.00001
PCB-126
0.1
0.1
0.005
0.1
PCB-156
0.0005
0.0005
<0.000005
0.0001
PCB-157
0.0005
0.0005
<0.000005
0.0001
PCB-167
0.00001
0.00001
<0.000005
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
<0.000005
0.00001
DIOXINS
OCDD
1234678-HpCDD
123478-HxCDD
123678-HxCDD
123789-HxCDD
12378-PeCDD
2378-TCDD
FURANS
OCDF
1234678-HpCDF
1234789-HpCDF
123478-HxCDF
123678-HxCDF
234678-HxCDF
123789-HxCDF
12378-PeCDF
23478-PeCDF
2378-TCDF
TEF
TEQ
ng/kg
ng/kg
ng/kg
5500
410
nd
10
8.8
nd
nd
10
5.0
2.5
5.0
5.0
4.8
0.75
1E-04
0.01
0.1
0.1
0.1
1
1
0.550
4.100
0.125
1.000
0.880
2.400
0.375
130
39
nd
nd
nd
nd
nd
nd
nd
nd
10
5.0
3.1
4.1
1.8
1.0
0.94
0.61
2.7
2.90
1E-04
0.01
0.01
0.1
0.1
0.1
0.1
0.05
0.5
0.1
0.013
0.390
0.016
0.205
0.090
0.050
0.047
0.015
0.675
0.145
1
2
3
4
5
6
7
8
9
10
11
12
COPLANAR PCB'S
3,4,4',5-TCB (#81)
170
3,3',4,4'-TCB (#77)
12
3,3',4,4',5-PeCB (#126)
19
3,3',4,4',5,5'-HxCB (#169)
nd
2,3,3',4,4'-PeCB (#105)
1000
2,3,4,4',5-PeCB (#114)
71
2,3',4,4',5-PeCB (#118)
2300
2',3,4,4',5-PeCB (#123)
93
2,3,3',4,4',5-HxCB (#156) 280
2,3,3',4,4',5'-HxCB (#157) 71
2,3',4,4',5,5'-HxCB (#167) 500
2,3,3',4,4',5,5'-HpCB (#189) 47
TEF
ng/kg
4.0
0.0001
4.0
0.0001
4.0
0.1
4.0
0.01
4.0
0.0001
4.0
0.0005
4.0
0.0001
4.0
0.0001
4.0
0.0005
4.0
0.0005
4.0
1E-05
4.0
0.0001
TOTAL TEQ
TEQ
ng/kg
0.017
0.001
1.900
0.020
0.100
0.036
0.230
0.009
0.140
0.036
0.005
0.005
13.574
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