Definition
Definition of
of SPE
SPE
“Separation
“Separation or
or removal
removal of
of an
an analyte
analyte
or
or analytes
analytes from
from aa mixture
mixture of
of
compounds
compounds by
by selective
selective partitioning
partitioning
of
of the
the compounds
compounds between
between aa solid
solid
phase
phase ((sorbent
sorbent)) and
and aa liquid
liquid phase
phase
((solvent
solvent).”
).”
Definition
Definition of
of aa Robust
Robust SPE
SPE
Procedure
Procedure
“A
“A robust
robust SPE
SPE procedure
procedure is
is one
one that
that can
can
provide
provide sample
sample extracts
extracts of
of adequate
adequate
purity
high
purity and
and
high reproducible
reproducible
extraction
efficiencies
when
extraction
efficiencies
when
performed
performed by
by any
any analyst
analyst on
on any
any
sample
sample of
of the
the matrix
matrix type
type for
for which
which
the
the procedure
procedure was
was optimized.”
optimized.”
A
A Typical
Typical SPE
SPE Procedure
Procedure
Involves
Involves Six
Six Steps
Steps
1.
1.
2.
2.
3.
3.
4.
4.
5.
5.
6.
6.
Sample
Sample pre-treatment
pre-treatment
Column
Column solvation
solvation
Column
Column equilibration
equilibration
Sample
Sample application
application
Interference
Interference elution
elution
Analyte
Analyte elution
elution
Solvent
Solvent Strength
Strength
(Chemical
(Chemical Strength
Strength is
is always
always relative)
relative)
SORBENT
SORBENT
INCREASING
INCREASINGSTRENGTH
STRENGTH
Silica
Silica
Hexane
Hexane------ MeOH
MeOH------HH22O
O
Hydrocarbon
Hydrocarbon
HH2O
- - - - - MeOH - - - Hexane
2O - - - - - MeOH - - - Hexane
Bonded
Bondedsilica
silica
HH2O
<<: MeOH - - - H O: >> MeOH
2O <<: MeOH - - - H22O: >> MeOH
Effect
Effect of
of Solvent
Solvent on
on pKa
pKa of
of HOAc
HOAc at
at 25
25 C
C
11
10
9
8
pKa
pKa
Dioxane
Dioxane
MeOH
MeOH
7
6
5
4
0
20
40
60
80
100
Percent
PercentOrganic
OrganicSolvent
Solventin
inWater
Water
Reference:
Reference:Hendrickson,
Hendrickson,Cram
Cramand
andHammond,
Hammond,
“Organic
“OrganicChemistry”,
Chemistry”,3rd
3rdEdition
EditionMcGraw
McGrawHill
Hillpp303
303
SCX
SCX // PRS
PRS Counter-ion
Counter-ion Selectivity
Selectivity
Ba+2
Pb+2
Ca+2
0.00
Mg+2
K+
Mn+2
NH4+
Na+
H+
Li+
2.00
4.00
6.00
8.00
Relative Selectivity
10.00
SAX
SAX CounterCounter- ion
ion Selectivity
Selectivity
R-Ph-SO3CitrateI-
Br-
HSO4ClO3NO3-
CNHSO3BrO3NO2ClHCO3IO3HPO4RCOOFOH-
Relative Selectivity
Steps
Steps to
to be
be Optimized
Optimized During
During
Method
Method Development
Development
••
••
Sorbent
Sorbentselection
selection
Sample
Samplepreparation
preparation
••
••
Column
Columnconditioning
conditioning
Sample
Sampleloading
loadingrate
rate
••
••
Selection
Selectionof
ofinterference
interferenceelution
elutionsolvent
solvent
Interference
Interferenceelution
elutionsolvent
solventflow
flowrate
rate
••
••
Selection
Selectionof
ofIsolate
Isolateelution
elutionsolvent
solvent
Isolate
Isolateelution
elutionsolvent
solventflow
flowrate
rate
SPE
SPE Column
Column Conditioning
Conditioning
••
••
Solvation
Solvation of
of the
the Sorbent
Sorbent
Purification
Purification of
of the
the SPE
SPE Column
Column // Sorbent
Sorbent
Comparison
Comparison of
of 0.1
0.1 ml
ml and
and 0.4
0.4 ml
ml SPE
SPE
Columns
Columns
0.1
ml
0.4
ml
Contaminant
Contaminant Elution
Elution
••
••
••
••
••
Analyte-Insoluble
Analyte-Insoluble Solvents
Solvents
Selective
Selective Mixtures
Mixtures
Maintain
Maintain Retention
Retention Conditions
Conditions
Verify
Verify no
no Analyte
Analyte Bleed
Bleed
Proper
Proper Flow
Flow Rate
Rate
Why
Why use
use Mixed-mode
Mixed-mode
Columns?
Columns?
•• More
More robust
robust procedures
procedures
(Less
(Lessmatrix
matrixdependent
dependentvs.
vs.100%
100%ion
ionexchange)
exchange)
•• Columns
Columns designed
designed to
to provide
provide multiple
multiple
interaction
interaction
••
••
••
(ion
(ionexchange
exchangeand
andpolar
polarpartitioning
partitioningcapacity
capacity
optimized
optimizedfor
forsome
somePTM
PTMbio-applications)
bio-applications)
Cleaner
Cleaner extract
extract possible
possible
Ability
Ability to
to fractionate
fractionate complex
complex mixtures
mixtures
Methods
Methods for
for biomolecules
biomolecules or
or from
from
biomatrices
biomatrices
Mixed-Mode
Mixed-Mode Extraction
Extraction
Apply Sample
Rins
e
Elute (1)
Elute (2)
C18
C18
C18
C18
IX
IX
IX
IX
Neutral Mol’s.
Basic Mol’s.
Influence
Influence of
of pH
pH on
on Retention
Retention
Non-Polar
Non-Polar
C4
pH 3.0
pH 7.0
RCOOH
RCOO
-
C4
Adjust pH to Suppress Ionization
RCOOH pKa = 5.0
Influence
Influence of
of pH
pH on
on Retention
Retention
Ion
Ion Exchange
Exchange
SAX
pH 3.0
pH 7.0
RCOO
H
RCOO-
SAX
Adjust pH to Ensure Ionization
Anion
Anion Exchange
Exchange
Condition
1. Methanol
2. Water or Buffer
SAX
Apply Sample
2 pH Units
Above
Isolate pKa
SAX
Interference
Elution
Water or
Buffer
SAX
Analyte
Elution
2 pH Units
Below pKa
(500mM H2SO4)
SAX
Homovanillic Acid
Rumsby, et. al.
Weak
Weak Anion
Anion Exchange
Exchange (NH2)
(NH2)
pH 3
Analyte: Weak Anion, pKa 5.0
pH 7
pH 11
R-COOH
R-COO-
R-COO-
Sorbent: NH2, pKa 9.8
R-NH3+
R-NH3+
R-NH2
Analyte: Strong Anion
R-SO3-
R-SO3-
R-SO3-
Sorbent: NH2, pKa 9.8
R-NH3+
R-NH3+
R-NH2
Strong
Strong Anion
Anion Exchange
Exchange (SAX)
(SAX)
pH 3
Analyte: Weak Anion, pKa 5.0
pH 7
pH 11
R-COOH
R-COO-
R-COO-
Sorbent: SAX
R-NR3+
R-NR3+
R-NR3+
Analyte: Strong Anion
R-SO3-
R- SO3-
R-SO3-
Sorbent: SAX
R-NR3+
R-NR3+
R-NR3+
L/L
L/L Extraction
Extraction
••
••
••
••
••
••
••
Two
Two Extraction
Extraction Solvents
Solvents per
per Step
Step
Solvents
Solvents must
must be
be Immiscible
Immiscible
Solubility
Solubility Differentiation
Differentiation (Non-Selective)
(Non-Selective)
Emulsions
Emulsions
Large
Large Solvent
Solvent Volumes
Volumes
Extract
Extract Concentration
Concentration Required
Required
Serial
Serial Process
Process
Solid
Solid Phase
Phase Extraction
Extraction
••
••
••
••
••
••
••
Sorbent
Sorbent Phase
Phase and
and Solvent
Solvent Phase
Phase
Inherent
Inherent “Immiscibility”
“Immiscibility”
Functional
Functional Group
Group Differentiation
Differentiation
No
No Emulsions
Emulsions
Small
Small Solvent
Solvent Volumes
Volumes
Inherent
Inherent Concentration
Concentration
Batch
Batch Process
Process
Interference
Interference Elution
Elution Step
Step
•• Analyte-Insoluble
Analyte-Insoluble Solvents
Solvents
•• Selective
Selective Mixtures
Mixtures
•• Maintain
Maintain Retention
Retention Conditions
Conditions
(pH
(pH control
control can
can be
be important)
important)
•• Verify
Verify no
no Analyte
Analyte Bleed
Bleed
•• Optimize
Optimize Flow
Flow Rate
Rate
Extraction
Extraction Steps
Steps
••
••
••
••
••
••
Sample
Sample Pretreatment
Pretreatment
Column
Column Conditioning
Conditioning
Column
Column Equilibration
Equilibration
Sample
Sample Application
Application to
to Column
Column
Interference
Interference Elution
Elution
Analyte
Analyte Elution
Elution
Column
Column
Conditioning
Conditioning
•• Hygienic
Hygienic Cleaning
Cleaning
•• Solvation
Solvation of
of the
the Sorbent
Sorbent
SPE
SPE Column
Column Conditioning
Conditioning
•• Non-Polar
Non-Polar Sorbents
Sorbents
MeOH,
MeOH,MeCN,
MeCN,THF
THF
•• Bonded
Bonded Polar
Polar Sorbents
Sorbents (HILIC)
(HILIC)
MeOH,
MeOH,MeCN,
MeCN,THF
THFthen
then5-10%
5-10%water
water/w
/w
buffer
buffer
•• Unbonded
Unbonded Polar
Polar Sorbents
Sorbents
Non-Polar
Non-PolarSolvents,
Solvents,Hexane,
Hexane,Ethyl
Ethyl
Acetate,
Acetate,
(Same
(Samesolvent
solventas
asthe
thesample
samplematrix)
matrix)
•• Ion-Exchange
Ion-Exchange Sorbents
Sorbents
MeOH,
MeOH,MeCN,
MeCN,THF,
THF,then
thenwater
water/w
/wbuffer
bufferions
ions
Column
Column Equilibration
Equilibration
••
••
••
Remove
Remove Excess
Excess Solvation
Solvation Solvent
Solvent
Normalize
Normalize Sorbent
Sorbent to
to Sample
Sample Condition
Condition
Promote
Promote Optimum
Optimum Retention
Retention
–– Solvent
SolventComposition
Composition
–– Ionic
IonicStrength
Strength
–– pH
pH
•• Ion-Exchange
Ion-Exchange
–– Optimize
OptimizeChoice
Choiceof
ofCounter-ion
Counter-ion
–– Optimize
OptimizepH
pHfor
forionization
ionizationof
ofsorbent
sorbent
Sample
Sample Pretreatment
Pretreatment
Optimize
Optimize Sample
Sample for
for Analyte
Analyte Retention
Retention
••
••
••
••
Proper
Proper Dilution/Ionic
Dilution/Ionic Strength
Strength
Correct
Correct pH
pH
Analytes
Analytes Free
Free in
in Solution
Solution
Remove
Remove Particulates
Particulates
Sample
Sample
Application
Application
Proper
Proper Flow
Flow Rate
Rate
Analyte
Analyte Elution
Elution
•• Elution
Elution solvent
solvent must
must overcome
overcome both
both
PRIMARY
PRIMARY and
and SECONDARY
SECONDARY
interactions
interactions
•• 100%
100% Elution
Elution in
in 2-10
2-10 Bed
Bed Volumes
Volumes
•• Use
Use Selective
Selective Solvents/Mixtures
Solvents/Mixtures
•• Smaller
Smaller elution
elution volume
volume !
! More
More conc.
conc.
extract
extract
•• Optimize
Optimize Flow
Flow Rate
Rate
Flow
Flow Rate
Rate Considerations
Considerations
Optimize and Specify in Protocol for
– Column Equilibration
(Counter-ion Exchange)
– Sample Loading
– Interference Elution
– Analyte Elution
Molecular
Molecular Interactions
Interactions
••
••
••
Covalent
CovalentInteractions
Interactions
Ionic
IonicInteractions
Interactions
Polar
PolarInteractions
Interactions
100-300
100-300kcal/mole
kcal/mole
50-75
50-75kcal/mole
kcal/mole
3-7
3-7kcal/mole
kcal/mole
–– Hydrogen-Bonding
Hydrogen-Bonding
–– Dipole-Dipole
Dipole-Dipole
–– !-!
!-!
•• Non-Polar
Non-PolarInteractions
Interactions
–– Van
Vander
derWaals
Waalsor
ordispersion
dispersion
1-2
1-2kcal/mole
kcal/mole
Extraction
Extraction Components
Components
Matrix (Sample or Solvent)
Analyte(s)
Sorbent
Retention/Elution
Retention/Elution
Retention
Retention
Elution
Elution
Analyte
Analyte
Analyte !"
!" Sorbent
SorbentAnalyte
Matrix
Matrix
#$
#$
Analyte
Analyte
#$
#$
Sorbent
Sorbent
Sorbent
Matrix !"
!" Sorbent
Sorbent
Sorbent Matrix
#$
#$
Matrix
Analyte !"
!" Matrix
Matrix
Matrix Analyte
Non-Polar
Non-Polar Sorbents
Sorbents
(CH2 )17CH3
Octadecyl
(CH2)7CH3
Octyl
CH2CH3
Ethyl
(CH2 )3 CN
Phenyl
Cyclohexyl
Cyanopropyl
Polar
Polar Sorbents
Sorbents
Si OH
(CH2 )3 OCH2CHCH2
HO OH
Silica
Diol
(CH2 )3NH2
(CH2)3CN
Aminopropyl
Cyanopropyl
Ion-Exchange
Ion-Exchange Sorbents
Sorbents
(CH2 )3NH2
(CH2 )3 N+(CH3 )3
Aminopropyl
SAX (quaternary amine)
O
(CH2 )3 COH
CBA (carboxylic acid)
SO3 H
SCX (benzenesulfonic acid)
(CH2 )3 SO3 H
PRS (propylsulfonic acid)
Barriers
Barriers to
to SPE
SPE Use
Use
•
•
•
•
More complex than liquid/liquid extraction
More choices than liquid/liquid extraction
Less user familiarity
Longer method development cycle
HPLC
HPLC vs.
vs. SPE
SPE
•• Elution:
Elution: Column
Column Chromatography:
Chromatography:
VVrr == VVmm ++ KV
KVss
0.2
0.2 <<
<< K
K <<
<< 200
200
•• Extraction:
Extraction: SPE
SPE Chromatography:
Chromatography:
K
K ==
K
K ==
[stat]
[matrix]
[stat]
[eluent]
>> 1000
1000
Retention
Retention
<< 0.001
0.001
Elution
Elution
Six-Step
Six-Step SPE
SPE Procedure
Procedure
11
2/3
2/3
44
55
66
A
A Typical
Typical SPE
SPE Procedure
Procedure
Involves
Involves Six
Six Steps
Steps
1.
1.
2.
2.
3.
3.
4.
4.
5.
5.
6.
6.
Sample
Sample pre-treatment
pre-treatment
Column
Column solvation
solvation
Column
Column equilibration
equilibration
Sample
Sample application
application
Interference
Interference elution
elution
Analyte
Analyte elution
elution
Non-Polar Interactions
Sorbents
Interactions
Si
van der Waals
C8
Si
P
H
C2
van der Waals
Si
van der Waals
Polar Interactions
Interactions
Sorbents
O
C
N
NH2
Si
C
Si
N
H
H
2OH
Dipole/Dipole
N
H
Si
H
HydrogenBonding
O
O
O
O H
H
O
H
HydrogenBonding
Ionic Interactions
Sorbents
PRS
CBA
SAX
Interactions
H3+N
SO3-
Si
O
Si
H3+N
O-
-O S
3
Si
Electrostatic
N+(CH3)3
Electrostatic
Electrostatic
Conditioning
Conditioning
Secondary
Secondary Silanol
Silanol
Interactions
Interactions on
on Non-Polar
Non-Polar
Phases
Phases
Apply
ApplySample
Sample
Rinse
Rinse
Aqueous, pH 5-7
Si
Si
-+NH3
O
Acetonitrile
Si
Si
O +NH3
Elute
Elute
0.5% HCL in Methanol
Si
Si
O
H
Cl +NH3
Steps
Steps to
to be
be Optimized
Optimized During
During Method
Method
Development
Development
••
••
••
••
••
••
••
••
Sorbent
Sorbent selection
selection
Sample
Sample preparation
preparation
Column
Column conditioning
conditioning
Sample
Sample loading
loading rate
rate
Selection
Selection of
of interference
interference elution
elution solvent
solvent
Interference
Interference elution
elution solvent
solvent flow
flow rate
rate
Selection
Selection of
of Isolate
Isolate elution
elution solvent
solvent
Isolate
Isolate elution
elution solvent
solvent flow
flow rate
rate
Flow
Flow Rate
Rate Considerations
Considerations
Optimize
Optimize and
and Specify
Specify in
in Protocol
Protocol for
for
•• Column
Column Equilibration
Equilibration
•• Counter-ion
Counter-ion Exchange
Exchange
•• Sample
Sample Loading
Loading
•• Interference
Interference Elution
Elution
•• Analyte
Analyte Elution
Elution
Sample
Sample Loading
Loading Flow
Flow Rate
Rate vs.
vs. Extraction
Extraction
Efficiency
Efficiency
100
% Recovery
50
Increasing Flow Rate
Elution
Elution Solvent
Solvent Flow
Flow Rate
Rate vs.
vs. Extraction
Extraction
Efficiency
Efficiency
100
% Recovery
50
Increasing Flow Rate
Sorbent
Sorbent Screening
Screening
Sample
SampleApplication
Application
C18
C18
C8
C8
C2
C2
CN
CN
PH
PH
Influence
Influence of
of pH
pH on
on Non-Polar
Non-Polar Retention
Retention
pH 3.0 - RCOOH
C2
pH 7.0 - RCOO-
C2
Adjust pH to Suppress Ionization
RCOOH pKa = 5.0
Influence
Influence of
of pH
pH on
on Ionic
Ionic Retention
Retention
pH
pH3.0
3.0--RCOOH
RCOOH
SAX
SAX
pH
pH7.0
7.0--RCOO
RCOO-
SAX
SAX
Adjust
AdjustpH
pHto
toEnsure
EnsureIonization
Ionization
Analyte
Analyte Characteristics
Characteristics
••
••
••
••
••
Functional
FunctionalGroups
Groups
Solubilities
Solubilities
pK
pKaaValues
Values
Stability
StabilityCharacteristics
Characteristics
Chromatographic
ChromatographicBehavior
Behavior
Hydrophobic
OH
SH
NH
Cationic
NH2
H-Bonding
NH2
Anionic
O
+
+
NH
O
S
NH3
+
O
P
-
O
-
O
-
COO
-
O
Mechanism
Mechanism Selection
Selection
Analyte-Based
Analyte-Based
Functionality
Hydrophobic
H-Bonding
Analyte
Mechanism
NH2
NH2
Non-Polar
Polar
+
NH3
Ionic
Ion-Exchange
Mechanism
Mechanism Selection
Selection -- Summary
Summary
Analyte
Matrix
Hydrophobic
Non-Polar
Aqueous
H-Bonding
SH
Cations
+
NH
NR3
O
S
O
-
NH3
O
C
NH2
+
Anions
O
Aqueous or
Non-Polar
Solvent
NH2
NH
OH
-
+
O
-
O
-
O
C18
C8
PH
CH
C2
CN
Polar
SI NH2
2OH CN
Aqueous (Low
Ionic Strength)
Cation Exchange
(High ionic strength)
(ENV+ C18 C8)
Aqueous (Low
Ionic Strength)
Anion Exchange
+
P
O
Sorbent
(High ionic strength)
CBA PRS SCX
SAX
NH2
(ENV+ C18 C8)
Secondary
Secondary Interactions
Interactions on
on
Ion
Ion Exchange
Exchange Phases
Phases
Rinse
Apply Sample
Aqueous
, pH 5-7
Acetonitrile
Si
Rinse
Elute
0.1M K2HPO4
Acetonitrile,
0.1M K2HPO4
(50:50)
Si
Si
SO3 -
SO3 -
NH3+
NH3+
Si
SO3 -
SO3 -
H3N+
NH3+
Sorbent
Sorbent Screening
Screening
Analyte
AnalyteElution
Elution
C18
C18
C8
C8
PH
PH
Validate
Validate for
for Ruggedness
Ruggedness
••
••
••
••
••
••
Vary
Vary Solvent
Solvent Strength
Strength by
by ±± 5%
5%
Vary
Vary pH
pH of
of Ionic
Ionic Steps
Steps
Change
Change Sample
Sample Volumes
Volumes
Change
Change Flow
Flow Rates
Rates
Verify
Verify Linearity
Linearity
Check
Check Multiple
Multiple Sorbent
Sorbent Lots
Lots
Ionic
Ionic Combinations
Combinations
Cation
Anion
pH = 3.0
Weak
Weak
NH3
Weak
Strong
NH3
Strong
Weak
NR3
Strong
Strong
NR3
+
+
+
+
pH = 7.0
HO2C
NH3
-O S
NH3
HO2C
NR3
-O S
NR3
3
3
pH = 11.0
+
-O C
NH2
-O C
+
-O S
3
NH2
-O S
+
-O C
NR3
-O S
NR3
2
2
+
3
2
3
+
-O C
2
+
-O S
3
Ion-Exchange
Ion-Exchange Elution
Elution
• High Ionic Strength
• pH Change
– Neutralize Sorbent
– Neutralize Analyte
• High Selectivity Counter-ions
Cleanup
Cleanup Techniques
Techniques
Selective
SelectiveRinse
Rinse
Selective
SelectiveElution
Elution
Typical
Typical SPE
SPE Procedure
Procedure
Column
Sample
Column Column
ColumnPrePreSample
Conditioni
Conditioni Equilibration
Equilibration Application
Application
ng
ng
Interferenc
Interferenc
eeElution
Elution
Analyte
Analyte
Elution
Elution
Typical
Typical SPE
SPE Procedure
Procedure
Sample
Application
Wash
Elution
Cleanup
Cleanup Tips
Tips
1.
1. Non-retaining
Non-retaining sorbents
sorbents (from
(from Sorbent
Sorbent
Screening)
Screening)are
aregood
goodcleanup
cleanupsorbents
sorbents
2.
2. Non-eluting
Non-eluting solvents
solvents (from
(from Analyte
Analyte Elution
Elution
Screening)
Screening)are
aregood
goodrinse
rinsesolvents
solvents
3.
3. Solvents
Solvents intermediary
intermediary between
between sample
sample and
and
elution
elutionsolvent
solventare
aregood
goodrinse
rinsesolvents
solvents
4.
4. Solvents
Solvents in
in which
which the
the analyte(s)
analyte(s) are
are
insoluble
insolubleare
aregood
goodrinse
rinsesolvents
solvents
Capacity
Capacity &
& Elution
Elution Volume
Volume
••
••
••
••
Capacity
Capacity is
is ~~ 11- 5%
5% of
of Sorbent
Sorbent Mass
Mass
Minimum
Minimum Elution
Elution Volume
Volume is
is ~~ 125
125 µl
µl per
per
100
100 mg
mg Sorbent
Sorbent Mass
Mass for
for Spin
Spin Columns
Columns
“Effective”
“Effective” Capacity
Capacity is
is increased
increased through
through
Selective
Selective Extractions
Extractions
IfIf large
large sorbent
sorbent mass
mass is
is required
required initially,
initially,
first
first elution
elution may
may be
be reconcentrated
reconcentrated on
on
smaller
smaller bed
bed
COO
H
Benzoic acid, pKa 4.80
COO
H
NH2
p-Aminobenzoic Acid
pKa's 4.65 , 4.80
Automation
Automation of
of SPE
SPE
•• Routine
Routine Analysis
Analysis
•• Method
Method Development
Development
A
A TYPICAL
TYPICAL SPE
SPE PROCEDURE:
PROCEDURE:
Automation
Automation of
of the
the six
six steps
steps
Step
Step One:
One: SAMPLE
SAMPLE PRETREATMENT
PRETREATMENT
••
••
••
••
••
••
pH
pH
Ionic
Ionic strength
strength
Redox
Redox
Wetting
Wetting // protein
protein binding
binding
Filtering
Filtering
Mixing
Mixing
A
A TYPICAL
TYPICAL SPE
SPE PROCEDURE:
PROCEDURE:
Automation
Automation of
of the
the six
six steps
steps
Step
Step Two:
Two: COLUMN
COLUMN SOLVATION
SOLVATION
••
••
••
Variable
Variable volumes
volumes
Variable
Variable flow
flow rates
rates
Multiple
Multiple solvents
solvents
A
A TYPICAL
TYPICAL SPE
SPE PROCEDURE:
PROCEDURE:
Automation
Automation of
of the
the six
six steps
steps
Step
Step Three:
Three: COLUMN
COLUMN EQUILIBRATION
EQUILIBRATION
••
••
••
Multiple
Multiple solvents
solvents
Variable
Variable volumes
volumes
Variable
Variable flows
flows
A
A TYPICAL
TYPICAL SPE
SPE PROCEDURE:
PROCEDURE:
Automation
Automation of
of the
the six
six steps
steps
Step
Step Four:
Four: SAMPLE
SAMPLE APPLICATION
APPLICATION
••
••
••
Variable
Variable volumes
volumes
Variable
Variable flow
flow rate
rate
Multiple
Multiple collection
collection options
options
A
A TYPICAL
TYPICAL SPE
SPE PROCEDURE:
PROCEDURE:
Automation
Automation of
of the
the six
six steps
steps
Step
Step Five:
Five: INTERFERENCE
INTERFERENCE ELUTION
ELUTION
••
••
••
••
••
Multiple
Multiple solvents
solvents
Variable
Variable volumes
volumes
Variable
Variable flow
flow rates
rates
Multiple
Multiple collection
collection options
options
Drying
Drying options
options
––Volume
Volume
––Flow
Flow
A
A TYPICAL
TYPICAL SPE
SPE PROCEDURE:
PROCEDURE:
Automation
Automation of
of the
the six
six steps
steps
Step
Step Six:
Six: ANALYTE
ANALYTE ELUTION
ELUTION
••
••
••
Multiple
Multiple solvents
solvents
Variable
Variable volumes
volumes
Variable
Variable flow
flow rates
rates