LCGC Sample Prep WallChart Ebook v2

GUIDE TO
SAMPLE
PREPARATION
MAIN FLOW CHART
CLASSICAL LIQUID–LIQUID EXTRACTION
SOLID–LIQUID EXTRACTION
SOLID-PHASE EXTRACTION
MODERN SOXHLET EXTRACTION
QuEChERS Extraction
(Quick, Easy, Cheap, Effective, Rugged, and Safe)
SUPERCRITICAL FLUID EXTRACTION
“Guide to Sample Preparation” is a special project supplement published by LCGC North America. Editorial contents copyright © MJH Life Sciences.
GUIDE TO SAMPLE PREPARATION
MAIN CLASSICAL LIQUID– SOLID–LIQUID SOLID-PHASE MODERN SOXHLET QuEChERS Extraction SUPERCRITICAL FLUID
FLOW CHART LIQUID EXTRACTION EXTRACTION EXTRACTION EXTRACTION (Quick, Easy, Cheap, Effective, Rugged, and Safe) EXTRACTION
Sample
• Mill
Hard sample • Grind
Solid • Pulverize Liquid
• Crush
• Sieve
Is the Is • Chop
Homogenize No Is the sample Yes sample in a suitable No Particle size Semisoft, soft • Macerate
particle size for extraction
the sample hard,
gross sample homogeneous? reduction semisoft, or • Blend
or dissolution?
soft? • Cut
• Mince
Yes • Homogenize
• Press
Take a representative sample

from the gross homogenate
Do you Consider:
Do you
No want to analyze Yes • Headspace sampling
want to analyze the the volatile portion of • Purge-and-trap sampling
entire sample? the sample? • Solid phase microextraction
• Thermal desorption
Yes No
Do you
want to analyze
soluble portions
of sample?
Consider the nature of the

analyte–matrix pair
Sample dissolution Solid–liquid extraction:
No
• Boiling
Consider: • Soxhlet extraction Is
• Digestion (acid) Find a solvent that Find a solvent that
• Sonication Choose the extraction the recovery
• Organic solvent with will dissolve the entire dissolves the analyte but
• Accelerated (enhanced) solvent temperature, time, and pressure acceptable?
vortexing sample without affecting extraction/pressurized liquid extraction not the matrix
• Aqueous solvent for salts the analytes • Microwave-assisted
solvent extraction Yes
• Supercritical fluid extraction
Remove
• QuEChERS (fruits and vegetables)
particulates:
• Filtration
• Centrifugation
• Sedimentation
Does Does Is the
Does the sample Is the
the sample sample reactive extract or sample Are you
Yes the sample contain No contain desired high No contain unwanted high No No Is the sample No Is the sample too No Yes
or thermally or hydrolyti- suitable for direct interested in all sample
particulates? molecular weight molecular weight
cally unstable? too dilute? concentrated? components?
components? components? injection?
No Yes Yes Yes Yes Yes Yes No
Consider: Separate high molecular • Derivatize the sample to Increase the injection Dilute the sample to the Selectively extract the
• Size-exclusion weight substances: stabilize volume of the analytical appropriate concentration components of interest —
chromatography • Size-exclusion • Keep the sample cool (bio- technique or concentrate range with a compatible consider:
• Dialysis
Dialysis chromatography logical samples at 4°C)
4 7C) the sample: solvent or use a smaller • Solid-phase extraction
• Ultrafiltration • Dialysis
Dialysis • Do not expose the sample • Liquid–liquid extraction initial sample
• Liquid–liquid extraction
• Ultrafiltration to light
• Solid-phase extraction • Flash chromatography
• Precipitation • Do not expose the sample
to air • Evaporation
• Supported-liquid
• Freeze and lyophilize • Lyophilization
membrane extraction
Determine where the
Is the analyte loss occurred
solvent compatible Yes and correct the
with the analytical procedure
method?
Inject sample
No Reconstitute the
Remove solvent by:
analytes in a solvent Is the recovery
Perform solvent exchange • Evaporation No
and at a concentration acceptable?
• Lyophilization
suitable for the analytical
• Distillation
technique
Yes
•
Is an
Yes
internal standard Add the internal standard
required?
No
Inject sample
Liquid sample Typical Extraction Solvent Pairs for

Liquid–Liquid Extraction
Aqueous Organic
Is the Dissolve in a solvent Pure water Aliphatic hydrocarbons
Yes sample already No suitable for later Acidic solution Diethyl ether and other ethers
in a solvent? extraction steps Basic solution Methylene chloride
(see table) High salt (salting Ethyl acetate and other esters
out effect) Aliphatic ketones (C6+)
Complexing agents Aliphatic alcohols (C6+)
(ion-pairing, Xylenes, toluene
If necessary make a chemical chelating, chiral) Combination of two or more
adjustment such as changing the pH Combination of two of the above solvents
or adding a complexation reagent or more of the
above solvents
Add an immiscible
Place the sample in Allow the phases
solvent and swirl
a separatory funnel to separate
(see table)
Try a different
immiscible solvent
Break the emulsion:
Are No • Add salt to the aqueous phase
Measure the solute Draw off Yes the two liquids • Heat or cool the extraction vessel
in each phase each phase clear? • Filter the emulsion through a
glass wool plug
• Filter the emulsion through
Are the phase-separation filter paper
No solutes extracted • Centrifuge
quantitatively? • Add a small amount of a different
•
organic solvent
Yes Return to the
main flow chart
or near
boiling
and pressure
(PLE, MASE)
Solid–liquid extraction method:
Modern Soxhlet extraction
liquid (PLE)
Microwave-assisted solvent
extraction (MASE)
Liquid sample
Does
Perform Does the the sample contain
Yes sample contain oils, Yes matrices that may No
liquid–liquid extraction
fats, or lipids? interfere with SPE
to remove experiment?
No
Remove salts:
• Perform ion exchange
Does
• Use a desalting column Yes the sample contain
• Dialyze
Dialyze inorganic salts?
• Pass through a nonpolar
sorbent
No
Remove proteins:
• Modify the pH
• Denature with chaotropic
agents or organic solvents Does
Yes the sample contain
• Precipitate with acid or
proteins?
acetonitrile
• Add a compound that
No
competes for binding sites No
• Use restricted-access media Is
the recovery
acceptable?
Is
Dilute with a Yes the sample No Yes
compatible solvent viscous?
• Return to the
main flow chart
Remove excess conditioning Condition the SPE device with an Select a solid-phase extraction (SPE)
solvent: silica based - don’t allow the appropriate solvent: device — consider:
adsorbent to dry out; Polymer • Reversed phase—methanol or acetonitrile • Mechanism and phase
based - adsorbent can dry out slightly • Normal bonded phase—methanol or (see table)
nonpolar solvent • Weight and volume (sorbent: 5–10
Load the sample onto • Silica gel—nonpolar solvent mg of sample per gram; ion
an SPE device • Ion exchange—buffer exchange: 0.5–1.5 mequiv/g)
Choose conditions to Choose conditions to retain the matrix

retain analytes
Allow the analytes to pass through Solid-Phase Extraction Mechanisms and Phases
Wash away interferences the sorbent unretained
Mechanism Sorbent Analyte Type Matrix Type Analyte Eluent
with an intermediate-
strength solvent Wash any remaining analytes from C18, C8, C2, Nonpolar Polar solutions Polar solvents such
Nonpolar
the sorbent with a small amount of extraction phenyl, functional (aqueous as methanol,
the sample solvent (reversed phase) cyclohexyl,
cyanopropyl,
groups such as buffers) acetonitrile,
and water,
alkyl and
Elute the analytes with a polymeric aromatic pH adjustment
stronger elution solvent May have to evaporate some of the
Nonpolar Nonpolar solvents
volume to concentrate Polar extraction
(normal bonded
Silica, diol,
cyano,
Polar functional
groups such as solvents, oils such as hexane
phase or amino, amine and and methylene
adsorption) diamino hydroxyl chloride
SPE devices
Collect the analyte in the Cation exchange Strong (sulfonic Positively charged Aqueous, low
functional groups ionic strength
Buffers such as
acetate, citrate,
acid) or weak
smallest possible volume (carboxylic such as amines and phosphate
• Cartridge acid)
• Disk Anion exchange Strong Negatively Aqueous, low Buffers such as
Determine the analyte recovery (tetraalkyl- charged functional ionic strength phosphate and
• 96-well plate ammonium), groups such as acetate
• Pipette tip weak (DEAE,
amino)
organic acids
Determine the Does the

Yes Mix the sample
minimum sample solid require a
dessicant? with dessicant
size needed
No
Estimate the sample Reduce the

volume in milliliters sample size
Is Is Is
the sample No the sample No the sample No
volume less than volume less than volume less than
40 mL? 60 mL? 85 mL?
Yes Yes Yes
Choose a small Choose an intermediate Choose a large

extraction thimble extraction thimble extraction thimble
(25 mm × 3 100 mm) (33 mm× 3 94 mm) (38 mm × 3 102 mm)
Determine the appropriate

solvent amount — a good
approximation is 70 mL + 1.1
times the sample volume
Determine the appropriate

extraction solvent — choose
a solvent that dissolves the
analyte but not the matrix
Determine the boiling

and extraction times
Is the
No sample easy to Yes
Boil for at least 60 min Boil for at least 20 min
permeate (porous)?
Extract (rinse) Extract (rinse)

for at least 60 min for at least 20 min
Is the
No recovery
acceptable?
Yes
• Return to the
main flow chart
Step 1: Salting out extraction
Sample fruit, vegetable,

50-mL centrifuge tube or meat comminuted
10 or 15 g (see below)
Add acetonitrile (10 mL)

and select extraction
method TCD = trisodium citrate dihydrate
DHS = disodium hydrogen citrate
sesquihydrate
Select AOAC, or EN method MgSO4 = anhydrous magnesium sulfate
PSA = primary/secondary amine
GCB = graphitized carbon black
Add 1% HOAc Add 4g MgSO4 +

Add 4 g MgSO4 + 6g MgSO4 + 1g NaCl + 1g
and 1 g NaCl AOAC Method 1.5g NaOAc
Original method TCD + 0.5g DHS EN 15662 Method
2007.1
unbuffered buffered
buffered
10 or 15 g Add internal Add internal 10 g
standard 15 g
standard
Check and adjust pH

to 5.0-5.5
Shake for 1 min

and centrifuge
Step 2: Dispersive SPE
AOAC method EN method

Take 1-, 6-, or 8-mL aliquot
of acetonitrile layer May require sorbent
adjustment in d-SPE step
General fruits and vegetables General fruits and vegetables

2 mL: 50 mg PSA + Transfer to appropriate 2 mL: 25 mg PSA +
150 mg MgSO4 * size centrifuge tube 150 mg MgSO4 *
Fruits and vegetables with Fruits and vegetables with

fats and waxes Perform dispersive SPE fats and waxes
2 mL: 50 mg PSA + 50 mg (d-SPE) 2 mL: 25 mg PSA + 25 mg
C18 + 150 mg MgSO4 * C18 + 150 mg MgSO4 *
Pigmented fruits and Shake for 1 min Pigmented fruits and

vegetables then centrifuge vegetables
2 mL: 50 mg PSA + 50 mg 2 mL: 25 mg PSA + 2.5 mg
GCB + 150 mg MgSO4 * GCB + 150 mg MgSO4 *
Transfer 1-mL aliquot to
vial for analysis
Fruits and vegetables with Highly pigmented fruits
pigments and fats and vegetables
2 mL: 50 mg PSA, 2 mL: 25 mg PSA, 7.5 mg
50 mg GCB + 50 mg C18 Analyze by LC–MS, GCB + 150 mg MgSO4 *
+ 150 mg MgSO4 * GC–MS, or -MS-MS
• Return to main
flow chart
Yes Is recovery acceptable?
No
1 Place the weighed sample

in extraction thimble
2 Choose an initial set of

extraction conditions —
consider the nature of the
analyte–matrix pair
Run initial experiments at

3 different SF-CO2 densities
Is
the recovery No Graph absolute recoveries
acceptable? to evaluate conditions
Yes
If solubility limited, add 5-20% modifier to extracting
Increase or decrease the fluid. If diffusion limited, increase extraction
supercritical fluid flow rate temperature or decrease sample particle size.
Is
the recovery No Optimize trapping
Return to the acceptable? and reconstitution
Main Flow Chart
Yes
Choose liquid Choose solid

trapping trapping
Is Choose
the recovery No Change the rinse solvent to No Change the
acceptable? trap solvent elute analyte rinse solvent
Yes
Is Is Is
the recovery No the recovery the recovery No
acceptable? acceptable? acceptable?
Change the trap
Yes Yes Yes material and temperature
•
Is
Return to the Yes the recovery No
main flow chart acceptable?

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GUIDE TO

CLASSICAL LIQUID–LIQUID EXTRACTION

MODERN SOXHLET EXTRACTION

SUPERCRITICAL FLUID EXTRACTION

Take a representative sample

Consider the nature of the

No Yes Yes Yes Yes Yes Yes No

Liquid sample Typical Extraction Solvent Pairs for

Solid–liquid extraction method:

Modern Soxhlet extraction

Choose conditions to Choose conditions to retain the matrix

Determine the Does the

Estimate the sample Reduce the

Choose a small Choose an intermediate Choose a large

Determine the appropriate

Determine the appropriate

Determine the boiling

Extract (rinse) Extract (rinse)

Step 1: Salting out extraction

Sample fruit, vegetable,

Add acetonitrile (10 mL)

Add 1% HOAc Add 4g MgSO4 +

Check and adjust pH

Shake for 1 min

AOAC method EN method

General fruits and vegetables General fruits and vegetables

Fruits and vegetables with Fruits and vegetables with

Pigmented fruits and Shake for 1 min Pigmented fruits and

1 Place the weighed sample

2 Choose an initial set of

Run initial experiments at

Choose liquid Choose solid

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