the chemistry of solid phase extraction - stfxpeople.stfx.ca/tsmithpa/chem361/2011 handouts/d... ·...

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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5

Glass Syringe Barrels

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7

8

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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12

13

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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

O

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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