Automated sample preparation combined with Gas … · Enterprise Plus scheme • Focus ... •...
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Automated sample preparation combined with Gas Chromatography
(GC-MS, GC-MS/MS, GC/Q-TOF and 1D2D GC-ODP) for target and non-target analysis
Dr Kathy Ridgway, Applications ChemistAnatune
www.anatune.co.uk
Purpose of my presentation
“ To show the importance of sample preparation and how to make the right
choices for your applications”
Anatune
• Established almost 20 years• Girton, Cambridge (March 2012)• Member of Royal Society of Chemistry
Enterprise Plus scheme• Focus - Sell and Support Solutions
– Wide number of industries
• Gerstel– MPS – CIS, TDU, DHS, Twister™, ITSP +….
• VAR for Agilent– MSD, QqQ, QTOF
• Expanding Applications team
Outline of Presentation
• Choices and strategies for sample preparation• Overview of a selection of automated sample
preparation techniques– Twister™ (SBSE)– ATEX (Automatic Tube Exchange) – DHS (Dynamic headspace) – +..
• Example applications (MSD, QQQ, QTOF)• The human detector and an extra dimension
• “Known-known’s, known-unknown’s & unknown-unknown’s” (D. Rumsfeld 2002)
• Analytical strategies– Sample type and number, – Number of analytes– selectivity– Sensitivity (action levels/expected concentration)
• Quantitative versus qualitative• Screening
– Laboratory and field analysis• Techniques and TIME available• Method must be ‘fit for purpose’
Analysis Choices
• Why do sample prep?– Removal of matrix interferences
• Increased selectivity• Improved chromatography
– Analyte enrichment• Increase sensitivity – achieve lower limits of detection
– Reduce instrument maintenance
• The ideal sample prep – Selective(?) sensitive, minimum number of steps, environmentally
friendly, robust, – Automated?
Considerations for sample preparation
SPE
Solid samples Liquid samples
SDE
Direct headspace
Solvent extraction (Soxhlet, PLE, MAE, USE )
Liquid-liquid extraction
Dynamic headspace
HS-SPME, HSSE
Sorptive extraction (SPME, SBSE)
Membrane extraction
Extraction + enrichment
Extraction
Concentration
Enrichment Clean-up + enrichment
Extraction
Extraction Extraction + enrichment
Direct Analysis?
Direct headspace
Extraction
GC-MS (MSD, QTOF, MS/MS)
MSPD
Extraction + enrichment
Sample Preparation Options
MultiPurposeSampler MPS for GC-MS
Disposable PipetteExtraction DPX
Solid PhaseExtraction SPE
Twister
ThermalDesorptionSystem TDS
ThermalDesorptionUnit TDU
Automated TDULiner Exchange ATEX
AutomatedLiner EXchangeALEX
MultiFiberEXchangeMFX
DynamicHeadspaceDHS
Cooled InjectionSystem CIS
PreparativeFractionCollector PFC
Olfactory DetectionPort OPDtr
MAESTROPrepAhead
easy LinerExchange eLEX
µFlowManagerTDU PYRO
Selectable1D/2DGC/MS
MultiPositionEvaporationStation mVAP
Balance mVorxFiltration
Automation
MAESTROSoftware
MPS Workstation
Why Automate ?• Reproducibility - precision• Reduces Lab Staff workload
enabling more time to consider data produced and develop methods and themselves
• 24/7 Lab production• Sample preparation
potentially simplified• Reduces exposure to harmful
reagents• Prep ahead – all samples are
treated equally
Outline of Presentation
• Choices and strategies for sample preparation• Overview of a selection of automated sample
preparation techniques– Twister™ (SBSE)– ATEX (Automatic Tube Exchange) – DHS (Dynamic headspace) – +..
• Example applications (MSD, QQQ, QTOF)• The human detector and an extra dimension
TDU liner
CIS liner
No Transferline!
CIS
TDU
CIS
TDU and CIS
Thermal Desorption Unit
Cooled Injection System
TDUUniversal Thermal Desorption Unit
TDU liner with frit for thermal extraction of solid samples
TDU liner packed with adsorbent
TDU liner for Twister desorption
TDU liner for thermal extraction in µ-vials
Twister SBSE– SBSE Stir Bar Sorptive Extraction
- thickness 0.5 mm, 10 mm length (24 µl)- thickness 0.5 mm, 20 mm length (47 µl)- thickness 1.0 mm, 10 mm length (63 µl)- thickness 1.0 mm, 20 mm length (126 µl)
Magnet
Phase Polydimethylsiloxane(PDMS)
Amount of PDMS phase is substantially greater than SPME (0.5 µl)
Can be over 100 fold increase in concentration100ml sample to <100µl extraction phase
SBSE Features
Ø PDMS acts as an absorbent, not adsorbent phaseØ Equilibrium processØ Non-polar partition from water into PDMS phaseØ Use calculated and theoretical Log K o/w (Log P)
Ø Eliminates polar matrix interferenceØ Retains no water
Ø Analytes are stable on stir bar, allowing field samplingØ Parallel extraction of multiple samples possible Ø Analytes recovered by automated thermal desorption
or liquid extraction Ø Extremely low detection limits (ppq to ppt)Ø Excellent bar-to-bar reproducibilityØ Stir bars are reusable
Ø Quantitative, extremely low detection limits possible (ppt-ppq)
• Sorbent phase is a mixture of silicone and ethylene glycol– Efficient concentration of non-polar analytes similar
to the PDMS Twister
– Concentration of polar analytes that form hydrogen bonds acting as proton donors, for example phenols
– Low limits of detection and good recovery due to large phase volume
EG-Silicone
Method• Twisters pre-conditioned
– 280 C for 40 minutes– 220 C for EG twisters– Flow of clean N2 at 40 ml/min per tube
• Take an aliquot of water/sample (10-100 ml)– Twister added and placed onto magnetic stirrer plate– Left to stir for 2 hours (to ensure good recovery)
• After stirring for 2 hours– Remove with magnetic fish – Flush with few ml of deionised water– Wipe with a tissue– Insert into TDU Tube
Twister SBSE Applications• Over 400 publications since 1999• Food, flavor, natural products• Biological fluids, tissues
– Pheromones, drugs, poisons, toxins
• Polymer/packaging- leachables, extractables, • Environmental Applications
– Water odour – Geosmin, MIB, TCA, halophenols– SVOCs – hydrocarbons, PAH, PCB, Pesticides
Anatune examples:• Targeted – low level Cypermethrin in water (PDMS)• Mal odours in water (EG silicone)• HSSE – chocolate flavours
• 2 hour SBSE extraction with PDMS Twister™• Thermal desorption with TDU/CIS followed by analysis by
GC-MS/MS (EI)
Cypermethrin method
(mainlib) Cypermethrin10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430
0
50
100
15 27
39
51
55
65
77
83
91
99
115
127
141
152
163
181
191
209
215 224 235 244 254 265 280 289 310 319 343 353 379 415
O
N O
OCl
Cl
Log Kow 5.3
• Target LOD 0.01 ng/L
Reproducibility:Water @ 0.1 ng/L cypermethrin(n=6,)RSD 9%(without internal standard)
Initial results
R² = 0.9932
0
2000
4000
6000
8000
10000
12000
14000
16000
0 0.1 0.2 0.3 0.4 0.5 0.6
Cypermethrin using Twister
ng/L
Malodours
• Malodours in Water (Enriching analytes: 10 ml water onto EG twisters)
Analyte %RSD R2
2-methylphenol 8.7 0.989
2-isobutyl-3-methoxypyrazine 5.7 0.991
2-chloroanisole 2.7 0.991
2-chlorophenol 3.6 0.993
2,6-dimethylphenol 5.2 0.991
2-chloro-5-methylphenol 4.0 0.991
2-bromophenol 6.1 0.992
2,3,4-trichloroanisole 1.7 0.991
2,4,6-tribromophenol 2.3 0.997
2,5-dimethylphenol 3.1 0.993
Precision achieved for five replicate twister extractions at 0.02 ng/ml, linear regression up to 2 ng/ml (6 point)
Headspace Twister (HSSE)
Analyte %RSD (HSSE)
Methylpyrazine 8.22,5-Dimethylpyrazine 3.92,3-Dimethylpyrazine 9.02-Nonanone 6.1Trimethylpyrazine 2.8
3-Ethyl-2,5dimethylpyrazine 2.4
Tetramethylpyrazine 1.9Butyrolactone 6.2
3-Methylbutanoic acid 8.5
Hexanoic acid 9.0Maltol 9.8
Twicester
• Magnetic positioning of up to three Twisters using Twicester
• Multiple (mSBSE) using two or more Twisters • Simultaneous thermal desorption of the Twisters,
Cryofocusing in the CIS, and GC/MS analysis
Outline of Presentation
• Choices and strategies for sample preparation• Overview of a selection of automated sample
preparation techniques– Twister™ (SBSE)– ATEX (Automatic Tube Exchange) – DHS (Dynamic headspace) – +..
• Example applications (MSD, QQQ, QTOF)• The human detector and an extra dimension
• Uses same hardware as twister• Sample is inserted in Microvial (TDU)• Use transport adaptor with Septa
– automated injection into Microvial in tube
• Volatiles desorbed and trapped in CIS• Non-volatiles (dirty matrix) kept in TDU tube• Extremely useful to keep liner clean
ATEX – Automatic Tube Extraction-look at volatiles in a nonvolatile matrix
CIS liner kept clean
• 132 Spiked Pesticides
• In combination with GC-QTOF Highly selective and sensitive Mass spectrometer
• Complex, dirty matrix, low level target analytes
Pesticides by ATEX
• 5 ppb Pirimphos methyl
Outline of Presentation
• Choices and strategies for sample preparation• Overview of a selection of automated sample
preparation techniques– Twister™ (SBSE)– ATEX (Automatic Tube Exchange) – DHS (Dynamic headspace) – +..
• Example applications (MSD, QQQ, QTOF)• The human detector and an extra dimension
Dynamic Headspace (DHS)
Sample Vial
Purge Gas Exit
Exchangeable Tubewith Adsorbent
Two Needles
Heated Transfer Zone
Multi Purpose SamplerMPS
Dynamic HeadspaceDHS
TDU Tubes
Sample Vials
Thermal Desorption UnitTDU
Dynamic Headspace (DHS)
Dynamic vs Static HeadspaceGin, split 10:1
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.000
500000
1000000
1500000
2000000
2500000
3000000
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.000
500000
1000000
1500000
2000000
2500000
3000000
60°C
10 mL
60°C
1 mL
DHS traps:
TDU Liner with frit packed with:
- Tenax TA (ca. 60 – 70 mg)- Tenax GR- Carbopack B, Carbopack X- Carbopack B, Carbopack X, Carboxen 1000- Carbopack C, Carbopack B, Carbosieve SIII
Dynamic Headspace (DHS)
Dynamic Headspace (DHS) Flavour analysis
Method 1 (100µl, 50ml sampled@ 30)
Method 2 (100µl, 750ml sampled@ 30 + dry)
Method 3 (50µl, 2000ml sampled@60 + dry)
Sequential dynamic headspace sampling
Different trapping conditions for analysis of a wide range of compounds in aqueous samples
New DHS trap for very volatile compounds:Shincar-bon X/Carbotrap B/Carbotrap X (available mid March)
Dynamic Headspace (DHS):Multi-Volatile Method
Dynamic Headspace (DHS):Multi-Volatile Method
Journal of Chromatography AMulti-volatile method for aroma analysis using sequential dynamic headspace sampling with an application to brewed coffeeNobuo Ochiaia,∗, Jun Tsunokawaa, Kikuo Sasamotoa, Andreas Hoffmann
• GC-QTOF chromatogram of whisky (TIC)
• Target analytes - look for a key analyte• Profile?
DHS – whisky
Mass Profiler Professional
• Finding differences between multiple chromatograms can be challenging
• Following deconvolution – export component list • PCA analysis key trends in data
Whisky A
Whisky B
Measured accurate mass: 152.0465Exact mass (based on empirical formula): 152.0473 5ppm
Which instrumentation ?GC-MS• Unknowns in scan and knowns in SIM
• Limited sensitivity and selectivity (unit mass)GC-MS/MS• Target analysis - excellent sensitivity for known analytesGC-QTOF• Increased sensitivity for known knowns/target analysis
• Filter data with v low mass window to obtain good limits of detection (DL approaching MRM QQQ methods)
• Target Screening for known unknowns• Accurate mass acquisition for unknown unknowns
• Structural elucidation• Use of accurate mass fragments (NIST)• Use of ‘Q’ to fragment
- Full data acquisition enables subsequent data analysis
Olfactory detection Port (ODP)
Flow split between MS (and/or other detector) and ODP, so elute at same retention timeDragon voice recognition software and ODP intensity recorder produce annotated Olfactogram
Signals overlaid (MS, NPD, ODP) with annotation
For closely eluting peaks, can be difficult to pick out individual odours and describe in real time. Long run times are not recommended.
Gerstel 1D/2D GC-MS
• Patented configuration of Deans switch and splitter(s)
• Uses LTM II modules for independent control of columns
• Easy switching between 1D and 2D mode– controlled by method parameters in software
• Option for backflush and Optional CTS trap between columns
• All data collected in one chromatogram
Cut 8.8-9.2
Backflush@ 9.7
Peak A
Peak
A
Peak
E
Peak
B Pe
ak C
Peak
D
‘Heartcut’of peaks 1D/2D GC-MS
1D
2D
5x10
1
23
4
56
7
8
9+ TIC Scan 141117 _01.d
6x10
0
0.2
0.4
0.6
0.8
1
1.2
+ TIC Scan 141117 _03.d
Counts vs. Acquisition Time (min)3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13
Cut 4.82-4.96 mins
Creo
sol
Estr
agol
e
Estr
agol
e
Creo
sol
1D
2D1D
backflush from 6.5mins
‘Heartcut’ of odour active compounds
Separation of peaks enables human ‘deconvolution’ of odours
Conclusion
• Not one ideal sample preparation technique for all cases
• In order to determine the best sample preparation and instrumentation for your application:– Define the question – Know your samples
• Variability, sensory data, background information
• More powerful instrumentation can mean less clean up/separation required
• Software improvements to help with data processing• Method needs to be fit for purpose