The Chemistry ofSolid Phase Extraction
The Leader in Solid Phase Extraction Technology
For Over 22 years
The force known as
solid phase
extraction is
unusually strong.
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Solid Phase Extraction (SPE) is a broad term to
describe the digital separation technique where liquids
contact sorbents, and organic compounds or ions in the
liquid adsorb to the functional group(s) of the sorbent.
Sorbents are chosen to either retain the components of
interest, or selectively release them into a strong liquid
phase.
SPE manufacturers commonly pack these sorbents into
cartridges, well plates, dispersive tubes or other convenient
devices.
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What is Solid Phase Extraction
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Glass Syringe Barrels
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Adapters
Adapter cap has a tapered fit for 1, 3 and 6ml size
reservoirs with a standard luer fitting on top. These
adapters are ideal when a sample volume exceeds the
capacity of the SPE column or when sequential
extractions are desired.
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48 Well Plates
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96 Well Plates
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15
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Universal Vacuum Manifold
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Positive Pressure Manifold
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All 48 positions are individually
regulated to provide even
pressure to each column
Air flow to each row can be
individually controlled
Capacity 1 to 48 columns of
either 1mL, 3mL, 6mL, 10mL or
15mL
Positive Pressure Manifold
Components
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SPE & Sorbents
SPE is “digital”
Theoretical plates aren’t used
SPE is used for extraction, concentration and
purification
Sorbents are available in many forms: Silica based,
Polymeric, Alumina, and Carbon(s)
For all fields of application, silica based sorbents are
the most common sorbents used
Silica based sorbents give the greatest number of
choices for extraction
Silica based sorbents are very rugged (Stable over a
wide pH and solvent range)
Silica based sorbents are the most cost effective
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Silica Gel Structures
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Core: PorousPore: ContinuousShape: Irregular
MicroparticulateCore: Porous
Pore: ContinuousShape: Irregular
Terminal vs. Continuous Pores
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cross section of packing showing TERMINALpores
A
cross section of packingshowing CONTINUOUS pores
A
A = analyte
Continuous Pores
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Pore Diameter60A
0
OH
HO
OH
HO
OH
HO SiOSi
SiOSi
Irregular 5 - 20 60
40 - 60 60
60 - 90 60
90 - 125 60
60 - 200 60
120 - 200 60
Silica
Shape
Particle Size
Range (µm)
Pore Diameter
(in Å)
Particle Size and Pore Diameter
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Surface Area
Surface area = sum of internal + external surfaces
External area = geometric surface of particles per gram of silica
Internal area = surface area of open pores
Surface area of bonded silica is approximately 500 m2/g
97% of surface area is due to internal porosity
Surface area depends mostly on pore size & pore volume
Particle size primarily affects flow characteristics and not
surface area
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Solid Phase Extraction (SPE)
Can be very selective or generic
Wide range of sorbent chemistries
Parallel sample processing
Keys to success are:
pH manipulation
Choice of sorbent
Choice of solvents
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Choosing the Sorbent
Identify the functional groups present in the analyte Look up the structure (Merck Index or Internet)
Understand how an analyte behaves in response to changing extraction conditions (sample pH, % organic, etc.)
Determine pKa if possible
Manipulate the conditions to meet defined method objectives Polar or nonpolar
Ionic or nonionic
Analyte solubility Rule of thumb: Aqueous solubility >200 mg/L difficult to extract using
reversed phases
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A = Sorbent Active Sites
B = Analyte
C = Matrix Molecules
Select sorbent chemistry that encourages B to interact strongly with A, weakly with C.
1. Analyte must adsorb to SPE medium
2. Analyte residence time must be sufficient
3. Analyte must be removed from SPE medium
B
A
A
A
C C
CC
C
C
Sorbent
B
B
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Choosing the Sorbent
A simple rule: “Like attracts like”
Non-polar
analytes
Polar
analytes
Ionic
analytes
Alumina, Silica, Florisil®, Diol,
Carbon
Ion Exchange
C8, C18, C30, DVB, CYH, PHY
Increasing solubility
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SPE as a Filter
ex. Florisil Clean-up
apply sample
wash solvent
A
M
M
M
A
A
A
M
M
AA
M
A
A A
M
MM
M
A
A
A = Analyte
M = Matrix
Collect
Reverse Phase• Nonpolar Interactions• Van der Waals Forces• / Interactions• Secondary Interactions
Normal Phase• Polar Interactions• Hydrogen Bonding• Dipole-Dipole Interactions
Ion Exchange• Cationic Interactions• Anionic Interactions
Mixed Mode• Combine Ionic, Nonpolar
and Polar Interactions
Types of Solid Phase Extraction
Binding Energies
1-5 kcal/mole
3-10 kcal/mole
50 to >100kcal/mole
3-100 kcal/mole
-Si-O-C
-Si-O-C
(CH2-CH-CH2)n
Si-O-C
Si-O-Si-C SO3-
-Si-OH
-Si-CH2-CH2-CH2-CN
-(NR )+3
NH2
-COOC
-SO3-
C
CC
Strong
Weak
Sorbent Code Structure
Epoxy EPX -Si-(CH2)3_ O _ CH2
_ CH _ CH2
□□
Aldehydic ALD -Si-(CH2)4CHO
Isocyanate ICN -Si-(CH2)3NCO
Thiopropyl THX -Si-(CH2)3SH
Covalent Phases
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Forms Schiff Base with amines
Used to filter out primary amines, hydrazines, reducing agents, and other nucleophiles
Covalent bonding for proteins, enzymes and other bioactive molecules
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Retention Mechanisms
Reversed Phase Hydrophobic (nonpolar)
Load un-ionized
Elute un-ionized non-polar solvent
Secondary interactions of amines with unreactedsilanols on bonded silicas Elute with polar solvent
Ion Exchange Strong and weak ionic
attraction Load as ionized
Elute in acid or base
Mixed Phase Both reversed phase and
ionic attraction Load as ionized
Elute as un-ionized non-polar solvent
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Non-Polar or Hydrophobic
Sorbent Code Structure
C8 octyl C08 -Si-(CH2)7CH3
C18 octadecyl C18 -Si-(CH2)17CH3
C30 tricontyl C30 -Si-(CH2)29CH3
Cyclohexyl CYH1 -Si
Phenyl PHY1 -Si
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Non-Polar Extractions
Also called hydrophobic or reverse phase
Interactions between sorbent C-H bonds and
analyte C-H bonds
Involves van der Waals / dispersion forces
Applications - PCBs, flame retardants, pesticides,
PAHs, petroleum products
Analytes - protonated / neutral state, aromatics &
alkyl chains
Matrix - biologicals, water, aqueous buffers
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For best hydrophobic retention, the analyte and sorbent
should be uncharged. When using a C18, this is
accomplished by adjusting the sample pH to the
analyte’s pKa.
Elution solvents - typically non-polar to moderately polar
pH Accuracy enhances results.
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Hydrophilic or Polar Phases
Sorbent Code Structure
Silica SIL1 -SiOH
Diol DOL1 -Si-(CH2)3OCH2CHOHCH2OH
Cyanopropyl CNP1 -Si-(CH2)3CN
Florisil® FLS
Alumina, Acidic ALA
Alumina, Neutral ALN
Alumina, Basic ALB
Carbon CARB
Florisil is a registered trade mark of US Silica
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Polar Extractions
Also called hydrophilic or normal phase
Unequal distribution of electrons
Involves hydrogen bonding, pi-pi and dipole/ dipole
interactions
Sorbents - silica, diol, diethylamino,
cyanopropyl, carbon, Florisil
Applications - oil additives, carbohydrates,
phenols, oil soluble vitamins
Analytes - amines, hydroxyls, carbonyls,
aromatic rings, heteroatoms (O, S, N, P)
Matrix - non-polar, organic
Elution solvents - medium to high polarity
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“It’s time we face reality, my friends…We’re not exactly rocket scientists.”
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Ion Exchange Phases
Anion Sorbent Code Structure pKa
Aminopropyl (1° amine) NAX1 -Si-(CH2)3NH2 9.8
N-2 Aminoethyl (1° & 2° amine) PSA1 -Si-(CH2)3NH(CH2)2NH2 10.1, 10.9
Diethylamino (3° amine) DAX1 -Si-(CH2)3N(CH2CH3)2 10.6
Quaternary Amine Chloride QAX1 -Si-(CH2)3N+(CH3)3 Cl- always charged
Quaternary Amine Hydroxide CHQAX1 -Si-(CH2)3N+(CH3)3 CH3CO2- always charged
Quaternary Amine Acetate CAQAX1 -Si-(CH2)3N+(CH3)3 OH- always charged
Quaternary Amine Formate CFQAX1 -Si-(CH2)3N+(CH3)3 CHO2- always charged
Polyimine PAX -Si-(CH2)3-R-[NHCH2CH2]x
CationCarboxylic Acid CCX1 -Si-CH2COOH 4.8
Propylsulfonic Acid PCX1 -Si-(CH2)3SO3H <1
Benzenesulfonic Acid BCX1 -Si-(CH2)2 SO3H always charged
Benzenesulfonic Acid High Load BCXHL1 -Si-(CH2)2 SO3H always charged
Triacetic Acid TAX -Si-(CH2)3NH-(CH2)2N(CH2COOH)2
CH2COOH
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Copolymeric (Multifunctional Phases)
Sorbent Code Structure
Aminopropyl + C8 NAX2 -Si-(CH2)3NH2 & -Si-(CH2)7CH3
Quaternary Amine + C8 QAX2 -Si-(CH2)3N+(CH3)3 & -Si-(CH2)7CH3
Carboxylic Acid + C8 CCX2 -Si-CH2COOH & -Si-(CH2)7CH3
Propylsulfonic Acid + C8 CX2 -Si-(CH2)3SO3H & -Si-(CH2)7CH3
Benzenesulfonic Acid + C8 BCX2 -Si-(CH2)2 SO3H & -Si-(CH2)7CH3
Cyanopropyl + C8 CNP2 -Si-(CH2)3CN & -Si-(CH2)7CH3
Cyclohexyl + C8 CYH2 -Si & -Si-(CH2)7CH3
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“You’re fired, Jack,
The lab results just
came back, and you
tested positive for
Coke.”
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Ion Exchange Mechanisms
Ionic interactions occur between charged
sorbent & analyte of opposite charge
pH is manipulated to ionize analytes functional
group
Ionic bonds are strong & retain analyte
Hydrophobic interferences washed away with
organic solvents
Polar interferences removed with aqueous or
weak aqueous / organic washes
Elute by changing pH
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SPE as a Selective Adsorption Tool
elution solvent
apply sample
wash solvent
A
M
M
AA
M
M
M M
A
AA
M
M
MM
A
A
AA
A
M
M
M
A
A
A = Analyte
M = Matrix
M
The Key to Ion Exchange
pKa - (dissociation constant) the pH at which a compound is 50% ionized
Acids: pH > pKa promotes ionizationpH < pKa suppresses ionization
Bases: pH < pKa promotes ionizationpH > pKa suppresses ionization
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THE MAGIC NUMBER IS 2
ANALYTE SORBENT 2< 1< at pKa 1> 2>
Acid Anion (-) 1 9 50 91 99
Base Cation (+) 99 91 50 9 1
% of Compound in Ionic State
Functionality Ionization State pH units away from pKa
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Anion Exchange Extractions
Anion exchange sorbents positively charged
Acidic analytes manipulated to carry negative charge
Opposites attract forming strong bonds
Sorbents
1, 2 amine pka 10.1, 10.9
Aminopropyl (weak) pka 9.8
Quaternary amine (strong)
Diethylamino (weak) pka 10.6
Applications include phosphates, acidic drugs, organic acids, fatty acids,
vitamins
Analytes
Phosphates
Carboxylic acids
Sulfonic acids (cations)
Matrix - aqueous
Acidic elution solvents to neutralize analyte
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Cation Exchange Extractions
Cation exchange sorbents negatively charged
Basic analytes manipulated to carry positive charge
Opposites attract forming strong bonds
Sorbents
Benzenesulfonic acid (strong)
Propylsulfonic acid (strong) pka <1
Carboxylic acid (weak) pka 4.8
Applications include basic drugs, catecholamines, pharmaceuticals
Analytes Amines Pyrimidines (cations)
Matrix - aqueous
Basic elution solvents to neutralize analyte
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Mixed Mode/Copolymeric
Extractions
Hydrophobic & ionic retention mechanisms
Reverse phase sorbent with cation or anion exchange
Acidic, basic & neutral analyte applications
Matrix - aqueous
Selective washes
Elution solvents mixture of organics with acid or base
Superior sample clean up
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Mixed Mode/Copolymeric Interactions
applysample wash
solventelution
1elution
2
elution3
H2O,
hexane
or
buffer
MeCl2 or
EtAc/Hex
MeOH pH,Ionic
StrengthOrg. Solv.
A-
M
B+
B+
S
N
M
M
A-
M
MM
M
MM
S
M
A
-
B+
NA
-
A
-
A
-
S
S
S
M
M
M
B
B+
B+
N
N
N
wash
MeOH/EtAc
S
S
S
B
B
B+
B
M
M
M
B+
B+M
M
M = MatrixA = Acidic
S = SteroidB = BasicN = Neutral
SPE Steps
Prepare sample
Condition sorbents
Apply sample
Wash interferences
Dry sorbent
Elute analyte
Concentrate
Derivatize
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TROUBLESHOOTING
Common SPE Errors:
Testing pH of deionized water with paper
Absorption of analyte on glass, plastic, or
filters
Over drying sorbent prior to elution
Aggressive extract concentration
Weak or incorrectly prepared elution solvent
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Troubleshooting Continued
Reverse Phase
Needs a neutral charge
Sorbent must be dried
Extract must be dried
Sample loaded too quickly
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Ion Exchange
Sample loaded and/or eluted too quickly
Analysis Errors
Calibrate in same solvent as extract
Normal Phase
Sample loaded too quickly
Sorbent compromised by environment
Extract eluted at wrong polarity
“Do or do not... there is no try.”
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