leco gcxgc-(hr)ms 2013-06-20€¦ · nitrogen-substances 118 130 65 000 terpenes 81 220 79 000...
TRANSCRIPT
Peter Haglund, Conny Danielsson, Erik Spinnel, Ulrika OlofssonUmeå University, Sweden
David Alonso, Joe Binkley, Liz Humston‐Fulmer, Kevin SiekLeco Corp.
2
Presentation Outline
ChallengesChromatography
SolutionsChromatography TOF-MS
GCxGC-HRMS
Comprehensive Identification
ConclusionComprehensive characterization
Isomer/enantiomerseparations
3
Trace Environmental Analysis: ChallengesChallenges Low concentrations Low concentrations
Complex matricesp
Numerous classes of organic compounds
Many isomeric target compounds
4
You cannot get it all…Selectivity
Simplicity,$$$
Speed$$$
Sensitivity 5
Trace Mixture AnalysisSelectivity
Simplicity,$$$
Speed$$$
Sensitivity 6
GC SolutionsSelectivity
Front end: GCxGCBack end: ECDBack end: ECD
ToF-MS
Simplicity,$$$
Speed$$$
Sensitivity 7
GC SolutionsSelectivity
Front end: GCxGCBack end: ECD
Screening/IdentificationBack end: ECD
ToF-MSIdentification
TargetTargetQuantification
Simplicity,$$$
Speed$$$
Sensitivity 8
Screening/ Identification
GCxGC‐ToF‐MS GCxGC ToF MS
Non‐target Screening of House Dust
Non‐target Screening of City Dump Leachate
Di t d S i f H l t t d C d i Bi t Directed Screening for Halogentated Compounds in Biota
”Omics”‐Identification of Compounds Poorly Removed in p yMunicipal Sewage Treatment Plants (STPs)
9
GCxGCDet.Inj.
10 seconds modulation period
Cryo-trapGC 1 GC 2
6 min 10 s
6
43
5
0
2
0 10 s1
3
3 6 min
0
0 s 10 s3 min
10
GC×GC
Primary separation based on volatility
Secondary separation based on polarity, polarizability, H‐bonding ability, shape, etc.
High peak capacity
High sensitivityHigh sensitivity
GC×GC‐ToF‐MS 3D data 3D data Deconvolution of peaks and spectra Exceptional separation power Exceptional separation power
11
Non-Target screening ofpollutants in house dustpollutants in house dust
Examples: House dust from Umeå City Hall
Full‐scan GC×GC‐ToF‐MS
Total Ion Chromatograms – OverviewLibrary search / Selected ion traces – Identification
12
City Hall dust
Kommun
13
City Hall dustPhthalates
Kommun
14
City Hall dustPhthalates
Kommun
15
Organosphosphatescreening/Identifiction (m/screening/Identifiction (m/z =
99)99)
Name Similarity Reverse Probability CASEthanol, 2-butoxy-, phosphate (3:1) 857 860 8680 78-51-3Ethanol, 2-butoxy-, phosphate (3:1) 839 862 8520 78-51-37-Methyl-Z-tetradecen-1-ol acetate 524 537 85 0-00-0
16
City Hall dustOrganophosphates
Kommun
17
City Hall dustPBDEs
Kommun
18
City Hall dust
SterolsSterols
Kommun
19
Summary of major components
• Petroleum hydrocarbons
• Phthalate plasticizers
• Organophosphate plasticizers/flame retardants
B i t d fl t d t• Brominated flame retardants
• Bacteria sterolsBacteria sterols
20
Non-Target Screening of City Dump LeachateCity Dump Leachate
Liquid‐liquid extraction with DCM
Base‐neutral fraction
Acidic fraction (methylated with diazomethane)
Total Ion Chromatograms Overview Total Ion Chromatograms – Overview
Library search / Selected ion traces – Identificationy /
21
Leachate, Base/Neutral
2-Piperidin-Sulfamide,
N,N-dimethyl-
2(3H)-Benzo-thiazolone
Bi h l A
Caffeine
Column bleedCyclohexan-
dione
2 Piperidinone 1(3H)-Isobenzo-
furanone
N'-phenyl- Bisphenol A
2 C lTriethyl
phosphate
2-methyl-phenol
2H-Indol-2-one, 1,3-dihydro-
Phenol
N-(2-Cyano-ethyl)-benzenesulfonamide
2-Cylco-hexanol
2-Cylco-hexanone
phosphate
Camphor
Phenol, 2,6-dimethyl-4-nitro-
Diisobutyl-phthalate
Tris(1,3-dichloro-isopropyl)phosphate
”Sterols”
1,3-Oxa-thiolane
2-Piperi-dinol DEHP
DINP2,6-Di-tert-butyl-4-nitrophenolPhenol
”Terpenes”
Sterols
CycloalkanesP 1 th 2 th l
C18
C25
C17C16C19 C20 C21 C22 C23 C24 C26 C27 C28 C29 C30 C31
C15C14C
Linear and branchedalkanesPropane, 1,2-dimethoxy-
Propane, 1-ethoxy-2-methyl-
Aliphatic ethers Sulphur (S8)
1,4-DioxaneToluene
C14C13C12
C11
Sulphur (S8)
22
Leachate, Acidic
3H-1,2-Dithiole-3-thione, 4-methyl-
Dimethylf
Carbonodithioic acid, O S
Dimethyl, pentasulfide Terpenoid background
Caffeine3-Methyl-3H-benzothiazol-
2-one
tetrasulfidO,S-dimethyl ester
Dimethyltrisulfide
Methane sulfonic
acid, methyl ester
Sulfuric acid, dimethylester
Benzoic acid, 2,6-dichloro-, methyl ester
Benzeneaceticacid 4 chloro Mecoprop
Acetic acid, (4-chloro-2-methylphenoxy)-,
methyl ester
Dimethyl, disulfide
acid, 4-chloro-, methyl ester
Benzeneacetic acid, à-methyl-
Mecopropmethyl ester
Saturated FAMEs
Monounsaturated FAMEs
4-(2-methylpropyl)-, methyl ester
**
Fatty acid methyl esters (FAMEs)
Saturated FAMEs
Hexanoic acid, 2-ethyl-
Low molecular weight acidsSulfur
2-ethyl-, methyl ester
Hexathiepane
23
Identified compoundsChemical group Substances Concentration in ng/L
Number Median Maximum
Nitrogen-substances 118 130 65 000
Terpenes 81 220 79 000
Ketones 52 92 51 000
Sulfur-substances incl. aromatic sulphonates 52 180 740 000
Aliphatic acids 39 380 200 000
P t d th t 37 270 32 000P-esters and other esters 37 270 32 000
Aromatic and polyaromatic hydrocarbons 34 54 48 000
Phenols (incl. bisphenol A) 33 860 480 000
Aliphatics 28 150 92 000
Other aromatic acids 27 950 170 000Other aromatic acids 27 950 170 000
Other alcohols 32 180 170 000
Ethers 22 105 14 000
Phthalates and adipates incl. metabolites 16 570 190 000
Other halogenated hydrocarbons 14 340 46 000Other halogenated hydrocarbons 14 340 46 000
Phenoxy acids 13 1200 180 000
Aldehydes 12 42 1 700
Chlorophenols 5 2 450 15 000
Cyclic hydrocarbons 5 94 20 000
Siloxanes 4 44 19 000
Sum: 611
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Hazard Assessment (PBT)Hazard Assessment (PBT)Substances
Detected 600Assessed (2/6; >100 ng/L) 140PropertiesHighly persistent 3Persistent 47
Highly bioaccum. 1g yBioaccumulating 3Highly toxic 26g y to c 6Toxic 50
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Directed Screening of Br-Compounds in BiotaCompounds in Biota Identification of persistent compoundsde t cat o o pe s ste t co pou ds
Identification of metabolites
Example: Diloma snail from New Zealand
26
Sea Snail extract (non-polar)
27
Sea Snail extract (non-polar)
28
Isotope Cluster Search
100
Br
100
Br2
100
Br3
100
Br4
60
80
100
60
80
100
60
80
100
60
80
100
20
40
20
40
20
40
20
40
0
M +2 +4 +6 +8 +10
0
M +2 +4 +6 +8 +10
0
M +2 +4 +6 +8 +10
0
M +2 +4 +6 +8 +10
29
Script Proceedure1. Search High toHigh toLow mass
3.Relative
2.
Relative abundance
30
Intensity
Script Proceedure
44.Evaluation of IsotopeClusters
31
2 Br-ScriptRules:
Intensity > 100 auFunction Bromine2()
Bromine2 = FalseMass = EndMass() Finish = StartMass()
Abundance > 20% Ion ratios M+/(M+2)+ 48‐58%
Finish = StartMass()Trappmass = 0Do While Mass>Finish
If intensity(Mass) > 100 thenIf Trappmass = 0 and /( )
(M+4) +/(M+2) + 43‐53% (M+3) +/(M+2) + < 25%
If Trappmass 0 and _intensity(Mass) > 100 and _abundance(Mass) > 20 thenTrapmass = Mass
End ifR = Ratio(Mass–2 , Mass)R1 = Ratio(Mass+1, Mass)R2 = Ratio(Mass+2, Mass)If R>0.48 and R<0.58 and _
R2<0 53 and R2>0 43 and 80
100
Br2
Donald C. Hilton, LecoCurrent Trends in Mass SpectrometryJuly 2007
R2<0.53 and R2>0.43 and _R1<0.25 then Bromine2 = TrueExit do
End ifEnd if20
40
60
80
End ifMass = Mass – 1 Loop
End Function0
20
M +2 +4 +6 +8 +1032
Sea Snail extract (non-polar)
22 Br
3 Br
4 Br
33
Tribromoanisole (Cork Smell)
900
1000 62
Peak True - sample "3241_14 Diloma :1", peak 3379, at 1274 , 0.300 sec , sec
800
900
600
700 74
400
500 329 141
200
300 344
301
155 250
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
100250
222 118 90 197
171 266
34
MeO-TeBDE
900
1000 75
Peak True - sample "3241_14 Diloma :1", peak 9574, at 2414 , 0.770 sec , sec
800
900
600
700 62
356 420
400
500
516 204 178 154 313 131
200
300
341 235
102
50 100 150 200 250 300 350 400 450 500 550
100102 260 90
277 297 404
35
Sea Snail extract (non-polar)
2
MeO-TeBDEMeO-TrBDE
454-Br4
TeBDE
2 Br
3 Br
4 Br
MeO-TeBDDs ?446-Br4
TrBADiBA
Br4
TrBDDTeBDD
DiBA
36
MeO-TeBDD ?
900
1000 74
Peak True - sample "3241_14 Diloma :1", peak 10649, at 2750 , 0.770 sec , sec
800
900
600
700
400
500 530
281 61
93
200
300
155 124 193
265 487 327 434
50 100 150 200 250 300 350 400 450 500 550
1003
249 515 218 408 299 355 378
37
MeO-TeBDD: GC-HRT confirmation
Exp: 509.7096Theory: 509.7103Diff 1 3 ppmDiff. - 1.3 ppm
38
Natural Products in Marine Sponge
Utkina, N.K. et al. J. Nat. Prod., 2001, 64 (2), pp 151–153. DOI: 10.1021/np000354439
GC-HR-ToF-MSFolded Flight Path of up to 40 m yields ultra-high resolution
Vernchikov et.al.US Patent 7385187
Allows ultra-fast capture of high resolution spectra
40
Chlorine-filter Isolates peaks that may contain chlorines Retains spectral peaks spaced by the mass difference p p p yof 35Cl and 37Cl, i.e. 1.997 +/‐ 0.001
Tested on an marine bird egg sample (50 to 60 min)Tested on an marine bird egg sample (50 to 60 min)
41
Low intensity HxCB
TIC
*
XIC, low resolution
* XIC, high resolution*
42
Low intensity HxCB
43
STP efficiency evaluation
Focus on POPs and Semi‐POPs
GC×GC analysis of, e.g. raw and treated water
All peaks (compounds) are detected and integrated All peaks (compounds) are detected and integrated
Treated water used as template to which the samples are dcompared
Area ratio = 1, treatment failed Area ratio << 1 treatment successful Area ratio << 1, treatment successful Area ratio > 1, transformation product
44
Expected STP Removal vs. RT
2 0
1.5
2.0 s
y
0 5
1.0
Polarity
0
0.5
0 1000 2000 3000 4000 s(67 min)(67 min)
Volatility
45
Expected STP Removal vs. RT
2 0
1.5
2.0 s
y
Polar, Non volatile
Volatile, Polar
0 5
1.0
Polarity Non‐volatile
Non‐Polar
Polar
Volatile
0
0.5
0 1000 2000 3000 4000 s(67 min)
Non Polar, Non‐volatile
Volatile, non‐polar
(67 min)
Volatility
46
Bubble Plot of % STP Removal
2 0
1.5
2.0 s
y
0 5
1.0
Polarity
0
0.5
0 1000 2000 3000 4000 s(67 min)(67 min)
Volatility
47
STP Breakthrough %
1.5
1.0
2 0
0.0
0.5
1.5
2.0
seconds
)
> 35%
0.5
1.0
Polarity
2D (
35%
0.0 0 500 1000 1500 2000 2500 3000 3500 4000
Volatility 1D (seconds)48
Library IdentificationTentative name
Effluent conc.
(ng/L)
Functional group(s) Retention time Break‐through (%)
% in water Origin
Acid Am
ine/amArom
atic HalogenateKetone/estNitro
OH
PhosphateS/O
‐ether S,N
,O‐hete
Other
1D (s) 2D (s)
Influent
Effluent
ide
ed ter
erocyclic
2,4,7,9‐Tetramethyl‐5‐decyn‐4,7‐diol 12000 X X 1042 0.46 73 85 97 Defoamer in paintBenzenesulfonamide, N‐butyl‐ 5500 X X 1770 1.53 100 38 89 PlasticizerTris(butoxyethyl) phosphate 3600 X X 2586 0.75 100 94 98 Floor polishTris(butoxyethyl) phosphate 3600 X X 2586 0.75 100 94 98 Floor polishBenzothiazole, 2‐(methylthio)‐ 2200 X S NS 1454 1.70 100 88 99 Rubber industryTricyclo[5.2.1.0(2,6)]dec‐3‐en‐10‐one 1500 X 842 1.17 77 100 100 NaturalTris(3‐chloropropyl) phosphate (TCPP 1) 1500 X X 1758 1.04 67 85 98 Flame retardantBenzophenone 930 X X 1494 1.53 100 87 94 UV initiatorBenzothiazole, 2‐(methylthio), methyl 650 X S NS 1820 0.23 56 100 100 Rubber industryEthyl citrate 520 X X 1506 0 97 71 100 100 Plasticizer food additiveEthyl citrate 520 X X 1506 0.97 71 100 100 Plasticizer, food additive Caffeine 460 X N 1916 1.82 100 87 100 Coffee, soft drinksTris(3‐chloropropyl) phosphate (TCPP 2) 390 X X 1780 1.05 63 81 98 Flame retardantTris(2‐chloroethyl) phosphate 360 X X 1726 1.44 100 97 98 Flame retardant2,2,2‐Trichloro‐1‐phenylethanol 320 X X X 1268 1.34 100 100 100 Flavor and fragranceOxybenzone 230 X X O 2144 1.54 100 68 97 Cosmetics, sunscreen4‐tert‐butyl‐cyclohexanone 220 X 750 0.71 100 100 100 Cosmetics, fragranceEthosuximide 150 X N 812 1.08 98 79 98 PharmaceuticalTris(1,3‐dichloroisopropyl) phosphate (TCDPP) 150 X X X 2534 1.39 72 76 98 Flame retardantIsoquinoline 140 X N 770 1.18 100 97 100 Traffic4‐tert‐octyl‐phenol 140 X X 1410 0.96 69 37 86 Surfactants; resinsHexadecenoic acid, Z‐11‐* 120 X 1944 0.53 37 0 40 Natural Diethyltoluamide (DEET) 110 X X 1386 1.20 100 100 100 Insect repellentBenzenesulfonamide, N‐ethyl‐2‐methyl‐ 100 X X 1554 1.70 100 99 100 Plasticizer2,3,6,7‐Tetramethylquinoxaline 100 X N 1586 1.16 56 73 100 Traffic
Tentative structures – HR‐ToF‐MS verification in progress ! 49
GC-HRT: Modes of Operation
High ResolutionNominal
Mirr
Ultra‐High Resolution
AA LensAAD
AD
Lens
Mirr
D
Mirr
L = 2mR = 1 800
L = 20mR = 25 000
L = 40mR = 50 000R = 1,800 R = 25,000 R = 50,000
50
GCxGC-HRMS validation
One false positive detected
Remaning compounds were in excellent agreement!
%
40%
10%
20%
30%
0%
10%
‐2 to ‐3 ‐1 to ‐2 0 to ‐1 0 to 1 1 to 2 2 to 3
ppm deviation51
Identification of unknown unknown
800
1000
ance
)
181.00167
151.
0091
014
8.02
210
0041
3
860
69 595
Caliper – Effluent sample
200
400
600
800
Area
(Ab
unda
108.
0
153.
978
134.
0066
196.
9965
90.0
3545
68.9
8131
63.0
2519
122.
0068
8
81.9
8893
60 80 100 120 140 160 180 2000
M/Z
1000
nce)
181Library: NIST- Benzothiazole, 2-(methylthio)-
Similarity 680
200
400
600
800
Area
(Ab
unda
n
148
108
69 134
63 122
165
60 80 100 120 140 160 180 2000
M/Z
52
Identification of unknown unknown
800
1000
ance
)
181.00167
151.
0091
014
8.02
210
0041
3
860
69 595
Caliper – Effluent sample+ 0.8 ppm
15.99492 amu(oxygen 15 99491 amu)
200
400
600
800
Area
(Ab
unda
108.
0
153.
978
134.
0066
196.
9965
90.0
3545
68.9
8131
63.0
2519
122.
0068
8
81.9
8893
(oxygen 15.99491 amu)
60 80 100 120 140 160 180 2000
M/Z
1000
nce)
181Library: NIST- Benzothiazole, 2-(methylthio)-
Similarity 680OH
200
400
600
800
Area
(Ab
unda
n
148
108
69 134
63 122
165
60 80 100 120 140 160 180 2000
M/Z
53
b l d d hMBT bacterial degradation pathway
54
Alternative ”Dioxin” Analysis HRMS – The Golden Standard 2 GC injections (non‐polar + polar column)j ( p p )
Alternatives/ GCxGC‐ECD/LRMS
GC‐MS‐MS GC‐HR‐ToF‐MS
Analysis of 17 2,3,7,8‐PCDD/F and 12 WHO‐PCBs in Food and FeedFood and Feed
Full Congener‐Specific Analysis of PCBs and PCDD/Fs for u Co ge e Spec c a ys s o C s a d C / s oSource Tracking
55
Selection of GCxGC columns
The non‐polar J&W DB‐XLB gives the best 1D‐resolution !
What 2D‐column to use ?
56
DB-XLBxNN correlationsLiquid crystalCyanopropyl (dipolar)
SolwaxSil88LC50
CP-C18DB210HP-35
O t lA1701DB1HP1301
SPB-20DB5CP-13Octyl
Optima6HT-8PMCDSPB-20
0.94 0.95 0.96 0.97 0.98 0.99 1.00
DBXLBOpt a6
57
209 PCBsec
LC-50 (liquid crystal)TCN
81
77105 126 169
1574
5sec
28 128156
170
194
206
31 47
5666 85
99 110
118 138153
87
74137
180
114123
167
189
2
3
4020 50 60 7030 80
44
49
52101 141
149151
9787 180
1872021
4020 50 60 7030 80
SP-2340 (polar, cyanopropyl) 7781 16912656
4
sec
81
194
189
169
170180
156157128
167
141138
105114118
149110
85
8797
99
56
66
74
31
47
52
44
TCN28
2
3
4020 50 60 70 min30 80
167
202187
137123151101
7449
1
58
Ortho-Effect on Liquid CrystalsR i l b i
Tetra Tri o o’ Di o o Di Mono Non
Rotational energy barriers:
Tetra Tri o,o’-Di o,o-Di Mono Non
60 40 17 - 20 14 - 17 7 - 8 ca. 2
kcal/mol
59
PCB enantiomer separation
P
Permethylated -cyclodextrin (PMCD)
60
PCBs i Seal Blubber
61
All 209 resolved except... DB-XLB / SP2340 (cyanopropyl) 15 PCB pairs 7 Aroclor PCB pairs7 Aroclor PCB pairs
DB-XLB / LC-50 (liquid crystal) 15 PCB pairs15 PCB pairs 5 Aroclor PCB pairs All WHO and indikator PCBs resolved!
Dual second columns 6 PCB pairs
All A l PCB All Aroclor PCBs Chirasil-Dex / LC-50 or 100% cyanopropyl (VF-23MS) 9 chiral PCBs resolved 7 separated from Aroclor PCBs
62
Quantification of PCB/dioxins
Fish Oil
63
Quantification of PCB/dioxins
Compound feed
64
Summary• DBXLB×LC50 provides separation of: WHO‐PCBs and 2,3,7,8‐PCDD/Fs from each other , , , /
and from matrix WHO‐PCBs from other PCBs 16/17 Toxic PCDD/Fs from other PCDD/Fs
• The results agree well with GC‐HRMS data
GC GC h t ti l ti th d f th• GC×GC has potential as a routine method for the determination of TEQs in food and feed samples
• GCxGC may also provide PCB enantiomer signatures65
Conclusions/ Recommendations
ToF‐MS and HR‐ToF‐MS are powerful techniques!p q
Balance front‐end and back‐end selectivity!y(Don’t forget the chromatography part)
Adopt to the problem at hand – Simplify if possible!
GCxGC‐soft ionization‐HRMS would be very useful for identification work involving complex mixtures
66
The End...
Contact information:P t H l d
Thank you for your Peter Haglund
Umeå UniversityD f Ch i
your attention
Department of Chemistry90187 Umeå, Sweden
h l d [email protected]+46‐90‐7866667
67