food & flavor analysis: sample extraction and introduction · pdf filefood & flavor...

R e s t e k www.restekcorp.com 1

Becky Wittrig, Ph.D.RESTEK CORPORATION

Food & Flavor Analysis:Sample Extraction and Introduction


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


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


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


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


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


HMF in Grape Juice: No SPE Clean-up

HMF + interferences


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


HMF

HMF in Grape JuiceC18 SPE, HMF Eluted with 20% Methanol


HMF

HMF in Grape JuiceC18 SPE, HMF Eluted with 50% Methanol


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.


Principle of SPME

Plunger

SPME holder

Fused-silica fiber-expose fiber to liquid, headspace-thermally desorb or solvent extract

Fiber types:PDMSCarboxenCarbowaxDivinylbenzeneCombinations


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.


• 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



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



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



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



3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

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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


Stir Bar Sorptive ExtractionEliminates Matrix Interferences


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

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Time-->

Abundance TIC: 0620002.DTIC: 0621003.D (*)TIC: 0621004.D (*)

Overlays of 1 yr old Bourbon, Triplicate SBSE



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

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Abundance TIC: 0621003.DTIC: 0621005.D (*)TIC: 0621009.D (*)TIC: 0621011.D (*)TIC: 0621013.D (*)TIC: 0621015.D (*)

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Overlays of 1yr, 2yr, 4yr, 6yr, 8yr and 10yr Bourbon



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 -->

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SBSE of Baking Soda Impurities



Flavor ProfilingHeadspace Sampling Techniques

•• AdvantagesAdvantages–– “Cleaner” samples“Cleaner” samples

–– Minimal sample preparationMinimal sample preparation

•• TypesTypes–– Static headspaceStatic headspace

–– Dynamic headspaceDynamic headspace

–– Thermal Thermal desorptiondesorption


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


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


Flavor Profiling: Static Gas Extraction

Volatiles from 2 different batches of chewing gum. The headspace was sampled after heating to 60°C.


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)


• 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


• 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


•• 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


• Trap is heated without flow• Typical temp: 5o below desorb temperature• Minimizes retention on the trap

3. Desorb Preheat



• Trap is backflushed into column• Typical time: 2 - 4 minutes• Typical flow: 10-80 mL/min.

• Typical temp: 180o - 250oC

4. Desorb



• Trap is baked clean with flow• Typical time: 8+ minutes• Typical temp: higher than desorb temperature• Avoid overheating adsorbents

5. Trap Bake



10cm Carbopack B

6cm Carboxen 1000

1cm Carboxen 1001

High-Boilers

VolatileCompounds

Gases

Vocarb® 3000

Flavor Profiling: Dynamic Gas ExtractionTypical Adsorbents for Purge & Trap


• 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


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


� 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


THF

Benzene

4-Heptanone

Styrene

Purge & Trap GC/MS of Volatiles


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


• Heat sample to drive volatiles into headspace

• Can trap and concentrate volatiles

– Cryofocusing step

• Minimal sample preparation

• Commercially available units

Thermal Desorption Techniques


Short-Path Thermal Desorption System

Sample tube

Septum purge vent

Split purge vent

To GC

Valve 1

Valve 2

CarrierGas


• 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

food & flavor analysis: sample extraction and introduction · pdf filefood & flavor...

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