new sample preparation and sample introduction techniques ... tcjjp... · 1 new sample preparation...

1

New Sample Preparation and Sample Introduction Techniques

for GC and HPLC

Ed Pfannkoch

Director, Technology Development

GERSTEL Inc.

OutlineOverview

Sample introduction- Liquid, HS, DHS, SPME

Sample Preparation- Twister SBSE

- Liquid extractions

- Filtration

- Automated SPE

- Benchtop Workstation

Example:Packaging

Example:Coconut water - SBSE

- Multidimensional GC-GC

What Can I Do with

My System?

2

GERSTELTechnique Overview

Cooled Injection (PTV)

CIS

Thermal Desorption

TDS

Twister (SBSE)

Twister

MultiPurpose Sampler

MPS

Dynamic HeadspaceOlfactory Detection

ODP

Automated SPEAutomated DPX

Prep Fraction Collector

PFC

GERSTEL

Company Overview Family-owned business

Founded in 1967

Headquarters: Mülheim an der Ruhr, Germany

Subsidiaries: - GERSTEL Inc., U.S.A.

- GERSTEL AG, Switzerland

- GERSTEL KK, Japan

- GERSTEL LLP, Singapore

- GERSTEL Brasil, Brazil

150+ employees

ISO 9001 Certified since 1997

Represented in more than 70 countries.

3

5

CIS - The Heart of the System

General PTV AdvantagesHeadspace

- Inject cool to avoid oxidation

- Solvent vent mode minimizes pressure/flow perturbation

SPME- SLH eliminates inlet septum coring

- SLH more compatible with SPME needle

- Narrow liners (1.0, 0.8mm) improve peak shape

Liquid- Cool injection minimizes discrimination/artifacts

- Allows LVI

On-column conversion kit available

4

Eliminates septum coring, particles or bleeding

Compatible with standard and metal SPME fibers

Minimum maintenance

Septum Less Sampling Head (SLH)

LVI Considerations

Injection port liner• Injection volume 15 uL or less use an unpacked baffled liner• Injection volume more than 15 uL use glass wool or beads• It is sometimes possible to improve solvent-analyte discrimination in the inlet using adsorbent-packed liners (TenaxTA, PDMS foam).• LVI accelerates build up of non-volatiles in the liner, check frequently

Injection port temperatureBest results are obtained when the BP difference between solvent and analyte is at least 150 ºC.Set initial temperature to 30 ºC below the BP of the solvent

5

QuEChERS Extract of Bell PepperInlet liner contamination after one 5uL injection

6

50000

250000

450000

650000

850000

1050000

1250000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Injection No.

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

Chlorthalonil

Endosulfan-sulfat

Dichlorvos

Automated liner exchange at user selectable intervals

12

ALEX

7

Proven pneumatic sealing mechanism

- Used in GERSTEL TDS and TDU systems

13

ALEX

Liner trays available with 14 or 98 liners

- Uncontaminated liners are always ready ensuring best possible analytical results

14

ALEX

8

Special Headspace Techniques

Multiple Headspace Sample Enrichment (MHSE)

Several withdrawals are taken from the same sample vial and the analytes are cryo-focused in the PTV liner. One start signal signal is sent after the final injection.

Benefit: Increased sensitivity

Software

9

MHSE of an Herbal Based Liqueur

MPS PrepAhead for Headspace AnalysisSequential: 38.4 min/sample

Prep Ahead: 11.2 min/sample

Sample Equilibration: 30 minGC Run Time: 6.0 minGC Cool Down: 2.0 min

384 min

112 min= 3.4 times higher throughput

10

Dynamic Headspace System DHS

CIS

TDU

MPS DHS

Liquid or solid samples in standard 10mL or 20mL vials

Samples can be heated, cooled or agitated

Headspace volatiles purged onto adsorbent tube

Adsorbent tube desorbed in TDU Options: use one adsorbent tube

for all samples, or use a different adsorbent tube for each sample

Dynamic Headspace (DHS) for MPS

11

• 2

DHS

Comparison of gas phase extraction techniquesAnalysis of ground coffee

• 22

DHS

SPME

Headspace

• 5 min, 40 °C, • 20 mL/min, • Adsorbent: • TENAX TA

• 40 °C, • Incubation: 5 min, • Extraction: 30 min, • Phase:

DVB/Carboxen• /PDMS 50/30 μm

• 40 °C,• Incubation: 30 min, • Injection volume: 1000

µL

DHS

12

Compound Name RSD /% Compound Name RSD /%

Acetaldehyde 8.8 Thiophene 2.8

Methyl Formate 15 2,3-pentanedione 7.9

Ethanol 9.3 Pyrazine 5.1

Furan 8.8 Dimethyl Disulfide 6.2

DMS 21 Pyridine 5.7

Methyl Acetate 9.2 Methyl Pyrazine 5.6

2-Methylfuran 7.3 Furfural 5.8

Butenal 5.4 Furfuryl Alcohol 6.0

Methyl Propanoate 6.9 Methyl Furfural 5.2

Repeatability of Coffee Analysis by DHS/GC/MS(n = 6)

GERSTEL Stirred Agitator

Selectable standard agitation or stirring

Stirring eliminates stress on SPME fiber

Expanded stirring speeds: 500-1500 RPM

Temperature range:

30 ˚C – 120 ˚C

13

Immersion SPME- Influence of Agitation Speed on Equilibration

Aldehydes In Wine

Pre-Extraction Derivatization

14

Software

Derivatized Cabernet Savignon

15

Calibration

0

500000

1000000

1500000

2000000

2500000

3000000

0 2 4 6 8 10 12 14

Isovaleraldehyde [ppb]

Are

a

Sample [Isovaleraldehyde] %RSD Winery 1: Cabernet Savignon 5.8 3.5 Winery 2: Cabernet Savignon 5.1 2.8 Winery 3: Cabernet Franc 7.4 2.7

OutlineOverview




- Filtration

- Automated SPE


Example:Packaging



What Can I Do with

My System?

16

Technique is called SBSEStir Bar Sorptive ExtractionSame principle as liquid/liquid extraction

- But with a low amount of immobilized „solvent“ (PDMS)

Extraction is based on sorption- Predictable recovery due to proportionality with log Ko/w

- No displacement effects

PDMS- Very inert- Retains no water- Selectivity eliminates polar matrix interferences

Desorption with TDS or TDU- Fast and mild- Extremely low detection limits (ppq to ppt)

GERSTEL-TwisterDesign

Magnetic core, sealed in glass and coated with PDMS

SPME : max. 0.5 µl

l = 10 mm, df = 0.5 mmdf = 1.0 mm

l = 20 mm, df = 0.5 mmdf = 1.0 mm

24 µl63 µl47 µl

126 µl

17

• Add stir bar to vial

Extremely easy to use


• Stir 1hr to overnight


18



• Remove stir bar with

forceps and rinse briefly

in distilled water




• Remove stir bar with

forceps and rinse briefly

in distilled water

• Dry with lint-free tissue


19

Emulsion formed duringsolvent extraction of liquid hand soap.

Ethyl acetate extract of antibacterial liquid hand soapAbundance

5000000

Time--> 10.00 15.005.00 20.00

1e+07

25.00 30.00

20

Twister extract of antibacterial liquid hand soapAbundance

Time--> 10.00 15.005.00 20.00

1

3

4

2

5

6

7

8 9

104000000

1.2e+07

2e+07

25.00

11

8000000

1.6e+07

30.00

Peak Identity

1-5 Short chain esters

6 Limonene

7 Ethyl vanillan

8 Lauric acid

9 Triclosan

10 C20-C30 hydrocarbons

11 Unidentified

Determination of Pesticide Residues in Food

from Classical to Novel Techniques

Aniko Kende, Tamas Rikker, Kornel Torkos

EKOL – Separation Science Research and Training Laboratory

Budapest, Hungary

21

DFG S19 analogue:- 50 g sample

homogenised with 50 ml acetone

- After filtration LLE with cyclohexane:ethyl-acetate 1:1

- Drying on Na2SO4, concentration to 10 ml

- GPC clean up- Concentration to 10 ml

After 42 sample injections

Strategy 3 – Sample Preparation

- 15 g sample and 30 ml methanol homogenised

- Centrifuged and 1 ml of the supernatant layer diluted to 10 ml with distilled water

- Extracted with stir bar for 2 hours and analysed

Stir Bar Sorptive Extraction

After 42 sample injections

Strategy 3 – Sample Preparation

MPS2 - Twister® Gerstel

22

Stir Bar Sorptive ExtractionRecovery and RSD results of 10 ppb spiked apple sample

Calibration curve of Chlorthiophos

44

Twister storage stability after extraction

0,E+00

5,E-01

1,E+00

2,E+00

2,E+00

3,E+00

3,E+00

Day 1 Day 5 Day 9 Day 13 Day 18 Day 21 Day 24 Day 27 Day 30

Are

a ra

tio

phenol 4-CPhenol 23diCphenol 246triCphenol PCP

8%5%2%1%2%

23

SBSE of Coconut water samples

OutlineOverview




- Filtration

- Automated SPE


Example:Packaging



What Can I Do with

My System?

24

Liquid/Liquid Extractions

MASE- Membrane Assisted Solvent Extractions

Filtration options

25

Automated Solid Phase Extraction

GERSTEL style 3 mL 500 mg cartridge

Original 3 mL 500 mg cartridge

26

www.gerstelus.com

SPE steps

Eva

po

rate

/ C

on

cen

trat

e

Solid Phase Extraction

27

Pesticides in Fruits and Vegetables

28

Pesticides in Orange Oil

Method Performance

Recoveries of 70-90% from Orange Oil and 80-100% from Vegetable Extracts

Total Analysis Time of 35 Minutes

Interlaboratory Comparison Study for 185 Compounds spiked into zucchini; 46 particpating Labs

-1 of 12 labs to identify all 185 compounds

- 128 of 185 Compounds identified by LC/MS/MS

29

ITSP- Instrument Top Sample Preparation

www.gerstelus.com

GERSTEL DPX Automation

Transport adapter

Standard pipette tip

Sorbent materialFrit

DPX

30

•Agilent QuEChERS Dispersive SPE

Weigh 15g food sample into 50mL centrifuge tube

Add IS and water, vortex 1 min

Add 15mL acetonitrile, vortex 1 min

Add QuEChERS salt packet, cap and shake

Centrifuge @4700 RPM 5 min

Transfer 1 mL of ACN layer for cleanup step

dSPE cleanup, centrifuge

Transfer to vial, LC/MS or GC/MS

QuEChERS Extraction Workflow

31

Transfer 1mL ACN to dSPE vial

Mix for 60s

Centrifuge

Vortex mix

Transfer 100 µL to vial

QuEChERS ExtractiondSPE Cleanup Procedure

Add 400uL buffer

Filter 0.45µ Transfer to tray and inject

Automated QuEChERS Pesticide Extraction

32

CF-100 Compact Centrifuge

Agitate and Centrifuge

2mL/10mL vials

3000 RPM

Agilent QuEChERS Automation

Sample preparation (homogenize, extract with ACN, centrifuge) performed manually. Transfer acetonitrile extract to vial.

Dispersive SPE cleanup, centrifugation, filtration and injection are automated.

Matrix matched standard prep can be automated.

SPE cleanup (10 min) is performed during LCQQQ analysis.

Agilent LCQQQ database contains > 750 pesticides.

Current work will screen >220 pesticides in one run.

Collaborated with the USFDA and Center for Food Safety and Technology on automation methodology.

Data presented at AOAC meeting in September

33

Prep Sequence for Automated QuEChERS-LC/MS/MS

QuEChERS

QuEChERS

Total of 200 pesticides in Hops

34

www.gerstelus.com

Res

pons

es

QuEChERS

www.gerstelus.com

Poster Presentation – 2011 AOAC meeting -“Automated Dispersive Solid Phase Extraction of Pesticide Residues in Botanicals using Triple Quadrupole LC/MS/MS” Yang Chen, Fadwa Al-Taher, Rima Juskelis and Jack Cappozzo, Institute for Food Safety and Health, Illinois Institutes of Technology, IL

“Results of the current study demonstrate that the automated dSPE clean-up system coupled with LC-MS/MS injection and analysis can be used for quantitation of multiple pesticide residues in botanical matrices with good sensitivity and recovery.”

QuEChERS

35

www.gerstelus.com

Spike 25 ppb Spike 100 ppb Spike 250 ppb Spike 5000 ppb

Analytes Recovery (%) SD (%) Recovery (%) SD (%) Recovery (%) SD (%) Recovery (%) SD (%)

3‐Hydroxycarbofuran 83 4 80 1 80 1 88 6

Acephate 106 4 113 4 110 4 114 4

Acetamiprid 91 6 100 2 104 2 109 2

Aldicarb 91 4 100 2 103 1 107 1

Aldicarb sulfone 107 6 86 1 110 2 93 2

Aldicarb sulfoxid 84 4 93 5 89 3 98 2

Ametryn 91 5 97 2 101 2 107 1

Amicarbazone 96 5 107 3 110 2 114 1

Aminocarb 104 6 94 3 96 2 101 1

Aspon 104 6 110 4 85 3 92 3

Atrazine 99 7 102 4 105 2 109 2

Azoxystrobin 105 9 102 1 114 3 121 1

Benalaxyl 102 4 106 3 111 2 116 1

Bendiocarb 94 7 104 5 104 5 101 3

Benoxacor 105 12 109 8 98 8 112 6

Benthiavalicarb 86 4 98 7 102 2 108 2

Bifenazate 75 9 103 4 102 4 103 3

Excerpt from Table of Recovery Data for ginseng validation standards

Total of 200 pesticides analyzed

QuEChERS

ATEX Option

Transfer adapter includes septum allowing addition of liquids in a microvial within the TDU tube

Volatiles can be thermally desorbed into the inlet leaving the non-volatile materials behind

36

(TIC)

Enhanced view of Phosalone

TDU and ATEX for Liquid InjectionOverlay of Replicates of Spiked Carrot Extract

(SIM)

www.gerstelus.com

AutomatedDisposable Pipette Extraction DPX

37

www.gerstelus.com

Rapid dispersive solid phase extraction

Small sample volumes

Low solvent consumption and waste

Readily automated, including injection into GC or LC

Compatible with most GC or LC systems

Efficiency based on equilibration time not on flows: Simple and accurate control

Formats: WAX, RP, CX, Q

DPX

www.gerstelus.com

DPX

38

DPX Quechers Extracts

AB SCIEX 3200 Q-Trap equipped with single rail MPS-3 with DPX option

QuEChERS

39

Extract 0.25 mL ACN solution in and out of QuEChERS tip 3 timesDispense into vialAdd 1 mL waterInject into LC/MS system—start LC/MS run

QuEChERS

500 ppb in different matrices (diluted to 100ppb)

broccoli

carrots

greenbeans

tomatoes

QuEChERS

40

QuEChERS

Pesticide Carrot Tomato

%Recovery (%RSD) %Recovery (%RSD)

Aldicarb 88 (11) 92 (10)

Aldicarb sulfone 93 (10) 97 (10)

Aldicarb sulfoxide 96 (12) 88 (7)

Carbaryl 87 (6) 77 (10)

Carbofuran 85 (12) 80 (8)

Oxamyl 94 (3) 93 (10)

Methomyl 77 (9) 76 (12)

Methiocarb 92 (13) 86 (11)

Hydroxycarbofuran 86 (14) 91 (10)

Propoxur 81 (14) 76 (9)

Acephate 90 (12) 79 (11)

Diazinon 79 (7) 76 (8)

Demeton‐S 67 (6) 91 (11)

Azinphos‐methyl 86 (13) 79 (6)

Dichlorvos 68 (6) 70 (5)

Mevinphos 68 (14) 75 (10)

Ethoprophos 79 (14) 78 (9)

Coumaphos 89 (13) 90 (12)

Poster Presentation (P-99) – 2011 FPRW - “Automated LC/MS Analyses of Pesticides and Mycotoxins Using QuEChERS and DPX Tips”

William E. Brewer et al., University of South Carolina, Columbia, SC

OutlineOverview




- Filtration

- Automated SPE


Example:Packaging



What Can I Do with

My System?

41

Residual Flavor Compounds in PET Bottles

• PET bottle from soft drink rinsed 3x with DI water/air dried

• 25mg sample cut from side walls of rinsed and unused PET bottles and thermally extracted

• Residual flavor components in PET easily detected

min6 8 10 12 14

pA

-20

0

20

40

60

80

100

Rinsed PET Bottle

Unused PET Bottle (Control)

Lim

onen

e

0

20

40

60

80

100

120

140

0 50 100 150 200 250

Limonene Thermal Extraction from PET

Are

a

Desorption Temperature

First Desorption

Second Desorption (200C)

42

y = 717,82x - 4,16

R2 = 1

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

0 2 4 6 8 10 12

Limonene Calibration Curve- Tenax Tube

1ng-10ug

Are

a

Mass (ug)

Residual Flavor Compounds in PET bottle

Conditions• Thermal extract at 200°C for

5 minutes (45ml/min)

• Cold trap on glass wool at -70°C

• Heat inlet to 280°C (30:1 split)

• Analyze on HP5MS 30m x 0.25mm x 0.25um

• Detection: FID

ResultsSample Conc. (ug/g)

1 7.08

2 6.993 6.804 7.125 7.176 7.097 6.818 7.149 7.2010 7.00Mean 7.04 ppm%RSD 2.00

43

Two Approaches to Multidimensional GC

Comprehensive GCxGC Heartcutting GC-GC

GC x GC: 3D Map

44

Gerstel “Selectable” 1D2D System- Manual

Single dimension system uses both detectors after first column

Second column gas flow provided by crosspiece sweep gas

Gerstel “Selectable” 1D2D System- Manual

Multidimensional system uses both detectors after second column

Crosspiece used to heartcut fractions from first column to second column

45

B

AHeart Cut19-21 min

Hydrocarbons

Abundance

2000000

Time--> 16.00 18.00

4000000

8000000

6000000

1e+07

20.00 22.00 24.00 26.00 28.00 30.00

Time--> 26.00 30.00 34.00 38.00 42.00 46.00 50.00 54.00 58.00

Abundance

1000000

2000000

4000000

3000000

5000000

Vinyl Shower Curtain Odor Components

Scan 12204 (46.273 min)

#112637: 1-Hexanol, 2-ethyl-

B

A"Shower Curtain"

Abundance

2000

31

57

41

6000

8000

m/z--> 20 40 8060 100

4000

70 83

11298

120

2000

m/z-->

6000

4000

8000

Abundance

20 40 8060 100 120

31

57

41 70 83

11298

2-ethyl-1-hexanol

Primary Shower Curtain Odor Component

46

Average of 42.503 to 42.507 min.

#111722: 2-Hexenal, 2-ethyl-

B

A"Musty"

Abundance

2000

55

416000

8000

m/z-->

400069

83 11193

2000

m/z-->

6000

4000

8000

Abundance

20 40 8060 100 120

29

97126

55

41

69 83111

93

97126

20 40 8060 100 120

2-ethyl-2-hexenal

Trace “Musty” component

47

Flavored Coconut H2O-1D

Flavored Coconut H2O-1D

Backflush

48

Flavored Coconut H2O-2D1Twister vs. 3 Twisters

MAESTRO software allows the autosampler to go from this…

49

…to this!

Thank you very much for your attention!

new sample preparation and sample introduction techniques ... tcjjp... · 1 new sample preparation...

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