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.

Presentation Outline

ChallengesChromatography

SolutionsChromatography TOF-MS

GCxGC-HRMS

Comprehensive Identification

ConclusionComprehensive characterization

Isomer/enantiomerseparations

Trace Environmental Analysis: ChallengesChallenges Low concentrations Low concentrations

Complex matricesp

Numerous classes of organic compounds

Many isomeric target compounds

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)

GCxGCDet.Inj.

10 seconds modulation period

Cryo-trapGC 1 GC 2

6 min 10 s

0 10 s1

3 6 min

0 s 10 s3 min

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

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

City Hall dust

Kommun

City Hall dustPhthalates

Kommun

City Hall dustPhthalates

Kommun

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

City Hall dustOrganophosphates

Kommun

City Hall dustPBDEs

Kommun

City Hall dust

SterolsSterols

Kommun

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

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 /

Leachate, Base/Neutral

2-Piperidin-Sulfamide,

N,N-dimethyl-

2(3H)-Benzo-thiazolone

Bi h l A

Caffeine

Column bleedCyclohexan-

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

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

Sulphur (S8)

Leachate, Acidic

3H-1,2-Dithiole-3-thione, 4-methyl-

Dimethylf

Carbonodithioic acid, O S

Dimethyl, pentasulfide Terpenoid background

Caffeine3-Methyl-3H-benzothiazol-

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

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

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

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

Sea Snail extract (non-polar)

Isotope Cluster Search

M +2 +4 +6 +8 +10

Script Proceedure1. Search High toHigh toLow mass

3.Relative

Relative abundance

Intensity

Script Proceedure

44.Evaluation of IsotopeClusters

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

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

End ifMass = Mass – 1 Loop

End Function0

M +2 +4 +6 +8 +1032

Tribromoanisole (Cork Smell)

1000 62

Peak True - sample "3241_14 Diloma :1", peak 3379, at 1274 , 0.300 sec , sec

700 74

500 329 141

300 344

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

MeO-TeBDE

1000 75

700 62

356 420

516 204 178 154 313 131

341 235

50 100 150 200 250 300 350 400 450 500 550

100102 260 90

277 297 404

MeO-TeBDEMeO-TrBDE

454-Br4

MeO-TeBDDs ?446-Br4

TrBADiBA

TrBDDTeBDD

MeO-TeBDD ?

1000 74

500 530

281 61

155 124 193

265 487 327 434

50 100 150 200 250 300 350 400 450 500 550

249 515 218 408 299 355 378

MeO-TeBDD: GC-HRT confirmation

Exp: 509.7096Theory: 509.7103Diff 1 3 ppmDiff. - 1.3 ppm

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

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)

Low intensity HxCB

XIC, low resolution

* XIC, high resolution*

Low intensity HxCB

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

Expected STP Removal vs. RT

Polarity

0 1000 2000 3000 4000 s(67 min)(67 min)

Volatility

Expected STP Removal vs. RT

Polar, Non volatile

Volatile, Polar

Polarity Non‐volatile

Non‐Polar

Volatile

0 1000 2000 3000 4000 s(67 min)

Non Polar, Non‐volatile

Volatile, non‐polar

(67 min)

Volatility

Bubble Plot of % STP Removal

Polarity

0 1000 2000 3000 4000 s(67 min)(67 min)

Volatility

STP Breakthrough %

seconds

Polarity

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

PhosphateS/O

‐ether S,N

,O‐hete

1D (s) 2D (s)

Influent

Effluent

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

Ultra‐High Resolution

AA LensAAD

L = 2mR = 1 800

L = 20mR = 25 000

L = 40mR = 50 000R = 1,800 R = 25,000 R = 50,000

GCxGC-HRMS validation

One false positive detected

Remaning compounds were in excellent agreement!

‐2 to ‐3 ‐1 to ‐2 0 to ‐1 0 to 1 1 to 2 2 to 3

ppm deviation51

Identification of unknown unknown

181.00167

69 595

Caliper – Effluent sample

60 80 100 120 140 160 180 2000

181Library: NIST- Benzothiazole, 2-(methylthio)-

Similarity 680

69 134

63 122

60 80 100 120 140 160 180 2000

Identification of unknown unknown

181.00167

69 595

Caliper – Effluent sample+ 0.8 ppm

15.99492 amu(oxygen 15 99491 amu)

(oxygen 15.99491 amu)

60 80 100 120 140 160 180 2000

181Library: NIST- Benzothiazole, 2-(methylthio)-

Similarity 680OH

69 134

63 122

60 80 100 120 140 160 180 2000

b l d d hMBT bacterial degradation pathway

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

Selection of GCxGC columns

The non‐polar J&W DB‐XLB gives the best 1D‐resolution !

What 2D‐column to use ?

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

209 PCBsec

LC-50 (liquid crystal)TCN

77105 126 169

28 128156

5666 85

99 110

118 138153

114123

4020 50 60 7030 80

52101 141

149151

9787 180

1872021

4020 50 60 7030 80

SP-2340 (polar, cyanopropyl) 7781 16912656

170180

156157128

141138

105114118

149110

4020 50 60 70 min30 80

202187

137123151101

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

PCB enantiomer separation

Permethylated -cyclodextrin (PMCD)

PCBs i Seal Blubber

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

Quantification of PCB/dioxins

Fish Oil

Quantification of PCB/dioxins

Compound feed

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

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 hpeter.haglund@chem.umu.se+46‐90‐7866667

Leco GCxGC-(HR)MS 2013-06-20€¦ · Nitrogen-substances 118 130 65 000 Terpenes 81 220 79 000...

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