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R e s t e k www.restekcorp.com 1
Becky Wittrig, Ph.D.RESTEK CORPORATION
Food & Flavor Analysis:Sample Extraction and Introduction
R e s t e k www.restekcorp.com 2
Food & Flavor Analysis:Sample Extraction and Introduction
I. Extraction TechniquesA. Solvent ExtractionB. Solid Phase Extraction (SPE)C. SPMED. SBSE
II. Headspace SamplingA. Static HeadspaceB. Dynamic HeadspaceC. Thermal Desorption
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Sampling Challenges
• Very Low Concentrations– ppm to ppt
• Matrix Issues– Volatiles can be intracellular– Disruption might be necessary
• Very Complex Volatile Profiles– e.g. coffee has ~800 components
• Wide Range of Volatilities• Air + Heat Instability
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Solvent Extraction Techniques• Solid-Liquid
– Soxhlet extraction
• Liquid-Liquid– Separatory funnel– Liquid-liquid extractors
• Solvents– Methylene chloride – good all-purpose– Diethyl ether, pentane, hydrocarbons, Freons
• Concentration Techniques– Dry over sodium or magnesium sulfate– Concentrate on steam bath
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Distillation Techniques
• Prior to liquid-liquid extraction– Distillation can remove nonvolatile
compounds– Can be done under vacuum – less heat
required
• Supercritical CO2 extractions are gaining in popularity – “cleaner” final sample
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Solid Phase Extraction (SPE)•• “Trap” specific compounds“Trap” specific compounds
–– Compound of interestCompound of interest–– Interferences Interferences
•• Wide range of materials availableWide range of materials available–– NonNon--polar polar –– C18C18–– Polar Polar –– carbon materials, carbon materials, fluorisilfluorisil
•• Standardized proceduresStandardized procedures–– i.e. EPA test methodsi.e. EPA test methods
•• Validation is very importantValidation is very important–– Recoveries, breakthrough levelsRecoveries, breakthrough levels
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1. Conditioninga. Apply 3 mL methanolb. Apply 3 mL deionized water
2. Sample Applicationa. Apply 4 mL sample to moist SPE tube, gravity feed
3. Washa. Pull remaining sample through tube, using vacuumb. Apply 3 mL waterc. Remove excess water from bed under vacuum
4. Elutiona. Apply 2 mL elution solvent, gravity feed, dilute to
volume
SPE Clean-up Procedure
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Solid Phase Microextraction (SPME)
� Solvent-less Extraction Technique� Partitioning of organic components between an
aqueous or a vapor phase and a thin polymeric film
� Independent of the matrix
� Applicable to liquids, solids, and gases
� Factors Affecting the Extraction� Organic/water partition coefficient
� Polarity & volatility
� Volume of sample/headspace, pH, temperature, etc.
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Principle of SPME
Plunger
SPME holder
Fused-silica fiber-expose fiber to liquid, headspace-thermally desorb or solvent extract
Fiber types:PDMSCarboxenCarbowaxDivinylbenzeneCombinations
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SBSE: The Gerstel Twister™
• 1.5cm long magnetic stir bar sealed in glass
• High capacity PDMS phase on glass
• Extremely rugged• Preconditioned for low
background• Stirs and extracts in one step• Splitless desorption of stir bar
gives low detection limits
Slide courtesy of Gerstel Inc.
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• Construction:– Magnetic core material, sealed in glass (1.5 cm)– Glass deactivated and persilylated– PDMS phase (0.5 mm thick)– Solvent washed and thermally conditioned
• Amount of PDMS on stir bar:– Minimum, 1 cm bar: 24 µl– Maximum, 2 cm bar: 126 µl
Stir Bar Sorptive Extraction (SBSE)PDMSGlassMagnet
Slide courtesy of Gerstel Inc.
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SBSE:Lower Detection Limits due to Higher Analyte Recovery
• Typical SPME phase: <0.5µl• Typical Twister phase:
24-126µl• Greater SBSE capacity gives
higher recovery of analytes relative to SPME as polarity increases
Slide courtesy of Gerstel Inc.
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SBSE: How to use• Add stir bar to vial• Stir 1hr-overnight• Remove stir bar with
forceps• Rinse briefly in
distilled water• Dry with lint-free tissue• Transfer bar into thermal
desorption tube• Thermally extract• Refocus analytes in cold
inlet
Slide courtesy of Gerstel Inc.
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Stir Bar Sorptive ExtractionFeatures/Benefits
∇ Immobilized liquid PDMS phase retains negligible water∇ Linear sorption isotherms with minimal displacement effects∇ PDMS phase selectivity enhances extraction of nonpolar
analytes∇ Selectivity eliminates polar matrix interference∇ Extractions from lipophilic matrices (e.g. dairy, soaps)
possible∇ High recovery and splitless sample introduction provide
extremely low detection limits∇ Predictable analyte recovery∇ Sample extraction done in parallel with minimal labor
Slide courtesy of Gerstel Inc.
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3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
340000
360000
380000
400000
420000
440000
460000
480000
500000
520000
540000
560000
Time
Response_
�Polar matrix components (water, acetic acid, ethanol, PG, glycerol) do not partition into PDMS.
�Volatile components (ethanol, methanol, acetone, etc.) can be further reduced by allowing brief evaporation time.
Balsamic Vinegar
Slide courtesy of Gerstel Inc.
Stir Bar Sorptive ExtractionEliminates Matrix Interferences
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1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 20
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
5500000
6000000
Time-->
Abundance TIC: 0620002.DTIC: 0621003.D (*)TIC: 0621004.D (*)
Overlays of 1 yr old Bourbon, Triplicate SBSE
Slide courtesy of Gerstel Inc.
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18.58 18.60 18.62 18.64 18.66 18.68 18.70 18.72 18.74 18.76 18.78 18.80 18.82 18.84 18.86 18.88 18.90 18.92 18.94 18.96 18.98 19.00 19.02 19.04 19.06 19 .08 190
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
260000
280000
300000
320000
Time-->
Abundance TIC: 0621003.DTIC: 0621005.D (*)TIC: 0621009.D (*)TIC: 0621011.D (*)TIC: 0621013.D (*)TIC: 0621015.D (*)
(E,E
)-fa
rnes
ylac
eton
e
Pen
tade
cano
ic a
cid
ethy
l est
er
Overlays of 1yr, 2yr, 4yr, 6yr, 8yr and 10yr Bourbon
Slide courtesy of Gerstel Inc.
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2 .0 0 2 .5 0 3 .0 0 3 .5 0 4 .0 0 4 .5 0 5 .0 0 5 .5 0 6 .0 0 6 .5 0 7 .0 0 7 .5 0 8 .0 0 8 .5 0 9 .0 0 9 .5 0 1 0 .0 0 1 0 .5 0 11 .0 0 11 .5 0
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
1 2 0 0 0 0
1 4 0 0 0 0
1 6 0 0 0 0
1 8 0 0 0 0
Tim e -->
A b u n d a n ce
2 .0 0 2 .5 0 3 .0 0 3 .5 0 4 .0 0 4 .5 0 5 .0 0 5 .5 0 6 .0 0 6 .5 0 7 .0 0 7 .5 0 8 .0 0 8 .5 0 9 .0 0 9 .5 0 1 0 .0 0 1 0 .5 0 11 .0 0 11 .5 0
5 0 0 0 0 0
1 0 0 0 0 0 0
1 5 0 0 0 0 0
2 0 0 0 0 0 0
2 5 0 0 0 0 0
3 0 0 0 0 0 0
3 5 0 0 0 0 0
4 0 0 0 0 0 0
4 5 0 0 0 0 0
5 0 0 0 0 0 0
5 5 0 0 0 0 0
Tim e -->
A b u n d a n ce
Thi
ophe
nede
riva
tives
Sten
ch
Fain
tR
ubbe
ry
Pain
ty Pain
t
Fain
t Stin
k
Fain
t
Fain
t
SBSE of Baking Soda Impurities
Slide courtesy of Gerstel Inc.
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Flavor ProfilingHeadspace Sampling Techniques
•• AdvantagesAdvantages–– “Cleaner” samples“Cleaner” samples
–– Minimal sample preparationMinimal sample preparation
•• TypesTypes–– Static headspaceStatic headspace
–– Dynamic headspaceDynamic headspace
–– Thermal Thermal desorptiondesorption
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Flavor ProfilingStatic Gas Extraction
•• AdvantagesAdvantages–– Excellent screening toolExcellent screening tool
–– InexpensiveInexpensive
–– Minimal sample carryoverMinimal sample carryover
–– Easy to performEasy to perform
•• DisadvantagesDisadvantages–– Less sensitiveLess sensitive
–– Involves preparing calibration in sample matrixInvolves preparing calibration in sample matrix
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Flavor Profiling: Static Gas Extraction
¾¾ Constant ratio between Constant ratio between liquid & gas phases at liquid & gas phases at equilibriumequilibrium
Where K = (Cgas / C liquid)
K = distribution coefficient
� Large K values favor the gas state� Cgas is directly proportional to peak area
Lit. #59895 — Headspace Guide
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Flavor Profiling: Static Gas Extraction
Volatiles from 2 different batches of chewing gum. The headspace was sampled after heating to 60°C.
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Flavor Profiling: Static Gas ExtractionResidual solvents in decaffeinated lemon tea
Minutes0 2 4 6 8 10 12 14 16 18 20
mV
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
TRACE-FIDTea headspace
12
34
5
1. Methanol2. iso-Propanol3. Methylene chloride4. Methyl acetate5. n-Butanol (IS)
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• Concentrates volatiles
• Dynamic extraction of solids and liquids
• Adsorbent trap
• Desorb (10-80 mL/min)
• Narrow bore column (split flow)
• Part of GC system
Flavor Profiling: Dynamic Gas ExtractionPurge & Trap Inlet System
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• Carrier gas bubbled through the matrix• Volatiles in matrix diffuse into carrier gas &
are carried away• Typical flows: 40-50 mL/min. (can heat) • Typical purge time: 10-12 min.
1. Wet Purge
Flavor Profiling: Dynamic Gas ExtractionPurge & Trap Sampling
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•• Trap is dried by purging with gas onlyTrap is dried by purging with gas only
•• Typical time: 1Typical time: 1--4 min.4 min.
2. Dry Purge
Flavor Profiling: Dynamic Gas ExtractionPurge & Trap Sequence
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• Trap is heated without flow• Typical temp: 5o below desorb temperature• Minimizes retention on the trap
3. Desorb Preheat
Flavor Profiling: Dynamic Gas ExtractionPurge & Trap Sequence
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• Trap is backflushed into column• Typical time: 2 - 4 minutes• Typical flow: 10-80 mL/min.
• Typical temp: 180o - 250oC
4. Desorb
Flavor Profiling: Dynamic Gas ExtractionPurge & Trap Sequence
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• Trap is baked clean with flow• Typical time: 8+ minutes• Typical temp: higher than desorb temperature• Avoid overheating adsorbents
5. Trap Bake
Flavor Profiling: Dynamic Gas ExtractionPurge & Trap Sequence
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10cm Carbopack B
6cm Carboxen 1000
1cm Carboxen 1001
High-Boilers
VolatileCompounds
Gases
Vocarb® 3000
Flavor Profiling: Dynamic Gas ExtractionTypical Adsorbents for Purge & Trap
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• Retention of polar & non-polar compounds
• Hydrophobic characteristics
• Reproducible desorption
– Increasing levels of adsorbency
• Able to withstand a broad temperature range
Flavor Profiling: Dynamic Gas ExtractionRequirements of a Trap
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Connecting a Purge & Trap to the GC Column
• Via the injection port
• Directly to the column
– 1/16" union
– Silica transfer line from 6 port valve directly to the column
• Low volume injector
Flavor Profiling: Dynamic Gas Extraction
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� Narrow bore column flows (0.5-1.3 mL/min.) permit a direct interface
� 0.53mm ID columns require:
Open Split Interface
Excess Flow Vacuum
or Jet Separator
Interfacing to a GC/MS System
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GC Parameters¾ Column: Rtx-5MS, 30m x 0.25mm x 1.0um¾ Injector: 250°C, 20:1 split¾ Carrier gas: Helium at 1 mL/min, constant flow¾ Oven: 50 °C to 92 °C at 3 °C/min, to 220°C/min. at
20°C/min. (1 min. hold)
MSD Parameters¾ Temperature: 280°C¾ Scan Range: 35-260, 1 min. solvent delay¾ Ionization: EI @ 70eV
Purge & Trap Parameters¾ Concentrator: Tekmar LSC-3100 with Vocarb 3000 (type K) trap¾ Purge: 10 min. at 40 mL/min, 60°C¾ Dry purge: 3 min. at 40 mL/min.¾ Desorb: 2 min. at 40 mL/min, 245°C
Purge & Trap GC/MS of Volatiles
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• Heat sample to drive volatiles into headspace
• Can trap and concentrate volatiles
– Cryofocusing step
• Minimal sample preparation
• Commercially available units
Thermal Desorption Techniques
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Short-Path Thermal Desorption System
Sample tube
Septum purge vent
Split purge vent
To GC
Valve 1
Valve 2
CarrierGas
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• Sampling techniques for solid and liquid samples
• Food & flavor samples often have extremely challenging matrices
– Need to sample trace level components
– Matrix can vary significantly from sample to sample
• Newer technologies focus on extracting flavor compounds from sample matrix without significant dilution
Summary of Extraction & Introduction Techniques