Automated sample preparation combined with Gas … · Enterprise Plus scheme • Focus ... •...

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

Maestro software fully integrated with Mass Hunter

No Overlap- Prep Ahead disabled

Maestro software – Prep Ahead

Prep Ahead enabled within this loop

Maestro software – Prep Ahead

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™ (Stir Bar Sorptive Extraction)

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)

Theory of Twister SBSE

– Example (Methylisoborneol)

– Log K o/w = 3.31

• 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 Set up

Twisters placed in twister tray (98 positions)

TDU

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

• 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

Pesticide analysis– dirty matrix

Clomazone comparison unit mass to 20ppm window

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 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) 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

TOF vs Single Quad data

Improved selectivity and signal to noise

Mass Profiler Professional

• Finding differences between multiple chromatograms can be challenging

• Following deconvolution – export component list • PCA analysis key trends in data

Whisky B

Whisky A

Whisky B

Whisky A

TIC – Whisky samples

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

Gerstel 1D/2D GC-MS

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

Choices Strategies and Insights…….It’s no CSI!

Multiflex by Anatune

Image from flowtv.org

Acknowledgements

• Dan Carrier• Sean O’Connor• Anais Maury• Jeff Stubbs

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