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GUIDE
TO SAMPLE
PREPARATION

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 GUIDE
TO SAMPLE
PREPARATION

TA B L E O F C ONTENTS

CLICK CHART NAMES TO VIEW CHARTS

Main Chart���������������������������������������������������������������� 5
Supercritical Fluid Extraction �����������������������������������7
Modern Soxhlet Extraction�������������������������������������� 9
Solid-Liquid Extraction������������������������������������������ 10
Solid-Phase Extraction�������������������������������������������11
Classical Liquid–Liquid Extraction ������������������������ 12
QuEChERS Extraction�������������������������������������������� 13

“Guide to Sample Preparation”

is a special project
supplement published
by LCGC Europe. Editorial
contents copyright © UBM.

PROJECT EDITOR:
Ronald E. Majors, LCGC
“Sample Prep Perspectives”
(ChromPrep LLC, West
Chester, Pennsylvania)

Background Image:
Medioimages/Photodisc/
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 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 the sample hard, Semisoft, soft • Macerate
gross sample homogeneous? particle size for extraction • Blend
or dissolution? reduction semisoft, or
soft? • Cut
• Mince
Yes • Homogenize
• Press

Take a representative sample

from the gross homogenate

Consider:
Do you Do you • Headspace sampling
No want to analyze Yes
want to analyze the the volatile portion of • Purge-and-trap sampling
entire sample? the sample? • Thermal extraction
• 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

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 SUPERCRITICAL FLUID EXTRACTION
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

4 Graph absolute recoveries

to evaluate conditions

Is
the recovery No Repeat Steps 1– 4 to
acceptable? fine-tune SFE conditions

Yes
Is
Increase or decrease the the recovery No Add a modifier
supercritical fluid flow rate acceptable? (or different modifier)
to the supercritical
Yes fluid and rerun
Graph relative cumulative steps 1-4
recoveries

Are the
recovery and time No
acceptable?

Yes

Optimize trapping and

reconstitution

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
main flow chart
Yes the recovery
acceptable?
No

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 MODERN SOXHLET EXTRACTION
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

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 SOLID–LIQUID EXTRACTION

or near
boiling
and pressure
(PLE, MASE)

Solid–liquid extraction method:

Modern Soxhlet extraction

liquid (PLE)
Microwave-assisted solvent
extraction (MASE)

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 SOLID-PHASE EXTRACTION
Liquid sample
Remove excess conditioning Select a solid-phase extraction (SPE)
Condition the SPE device with an
solvent: silica based - don’t allow the device — consider:
appropriate solvent:
adsorbent to dry out; Polymer • Mechanism and phase
Does • Reversed phase—methanol or acetonitrile
Per form Does the based - adsorbent can dry out slightly (see table)
Yes Yes the sample contain No • Normal bonded phase—methanol or
liquid–liquid extraction sample contain oils, matrices that may nonpolar solvent • Weight and volume (sorbent: 5–10
fats, or lipids? interfere with SPE mg of sample per gram; ion
to remove experiment? Load the sample onto • Silica gel—nonpolar solvent
an SPE device • Ion exchange—buffer exchange: 0.5–1.5 mequiv/g)
No
Remove salts:
• Perform ion exchange Choose conditions to Choose conditions to retain the matrix
Does retain analytes
• Use a desalting column Yes the sample contain Allow the analytes to pass through Solid-Phase Extraction Mechanisms and Phases
• Dialyze
Dialyze inorganic salts?
Wash away interferences the sorbent unretained
• Pass through a nonpolar Mechanism Sorbent Analyte Type Matrix Type Analyte Eluent
sorbent with an intermediate-
No strength solvent Wash any remaining analytes from
Nonpolar C18, C8, C2, Nonpolar Polar solutions Polar solvents such
the sorbent with a small amount of extraction phenyl, functional (aqueous as methanol,
Remove proteins: the sample solvent Solid-Phase
(reversed Extraction Mechanisms
phase) cyclohexyl, groups such as and Phases acetonitrile,
buffers)
cyanopropyl, alkyl and and water,
• Modify the pH Elute the analytes with a Mechanism Sorbent polymericAnalytearomatic
Type Matrix Type pH adjustment
Analyte Eluent
• Denature with chaotropic stronger elution solvent May have to evaporate some of the
Does volume to concentrate PolarC18,
Nonpolar extraction C8, C2, Silica, diol,
extraction NonpolarPolar Nonpolar
functional Polar
functional solutions Polar solvents
Nonpolar such
solvents
agents or organic solvents Yes phenyl,
(reversed phase) (normal cyclohexyl,
bonded cyano, groups such as solvents, oils assuch as hexane
methanol, ace­
• Precipitate with acid or the sample contain phasecyanopropyl,
or amino, groups such
amineasandalkyl (aqueous and methylene
tonitrile,
chlorideand water,
proteins? adsorption)
polymeric
diamino and aromatic
hydroxyl buffers)
pH adjustment
acetonitrile
SPE devices
• Add a compound that Collect the analyte in the Polar extraction CationSilica,
exchange Polar functional
Strong (sulfonic
diol, cyano,
Nonpolar
Positively charged Buffers such
Aqueous, low Nonpolar as
solvents
No (normal bonded groups such
acid) or weak as groups­solvents,
functional oils
ionic strength acetate,
such citrate, and
as hexane
competes for binding sites No smallest possible volume phase or adsorption)
amino, diamino (carboxylic such
amine and as amines
hydroxyl and phosphate
SPE devices
• Cartridge methylene chloride
• Use restricted-access media Is
acid)

the recovery • Cartridge • Disk Cation exchange AnionStrong (sulfonic

exchange Strong Positively charged
Negatively Aqueous,
Aqueous,lowlow Buffers
Bufferssuch
suchasas
Determine the analyte recovery acid) or weak(tetraalkyl-
functional groups ionic strength
acceptable? • Disk • 96-well plate ammonium),
(carboxylic acid)
charged
such as groups
functional
aminessuch as
ionic strength acetate,
phosphate
acetate
and and
citrate,
phosphate
Is • Pipette tip weak (DEAE, organic acids
Dilute with a Yes the sample No Yes • 96-well plate Anion exchange
amino)
Strong (tetraalkyl­ Negatively charged Aqueous, low Buffers such as
compatible solvent viscous? • Pipette tip
ammonium), weak functional groups ionic strength phosphate and

• Return to the
main flow chart
(DEAE, amino) such as organic
acids
acetate

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 CLASSICAL LIQUID–LIQUID EXTRACTION
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 shake
a separatory funnel to separate
vigorously (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

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 QuEChERS EXTRACTION (Quick, Easy, Cheap, Effective, Rugged, and Safe)
Step 1: Salting out extraction

Sample fruit, vegetable,

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

TCD = trisodium citrate dihydrate

Add acetonitrile (10 mL) DHS = disodium hydrogen citrate
and select extraction sesquihydrate
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

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