SOLID

PHASE
EXTRACTIO
N (SPE)
Lecture notes by:
Mr. Retšepile Matamane
(Bsc. CTECH, Msc. Analytical Chem
Candidate)
Course Outline
• Theoretical background
• Counterparts of SPE
• Advantages of SPE over its counterparts
• Disadvantages of SPE
• Applications of SPE
• Recent development/advanced SPE based
techniques
- SOLID PHASE MICROEXTRACTION [SPME]
- STIR BAR SORBENT EXTRACTION [SBSE]
Theoretical Background
• SPE is a separation process by which compounds that are dissolved or suspended in a
liquid mixture are separated from other compounds in the mixture according to their
physical and chemical properties.
• It is usually used to pre-concentrate and purify samples for chromatographic analysis.
• SPE drives liquid chromatographic mechanisms to their extreme, such that K D
approaches infinity, representing total accumulation of the analyte during retention,
and KD approaches zero during subsequent elution or release of the analyte.
• SPE uses the affinity of solutes dissolved or suspended in a liquid (known as the
mobile phase) for a solid through which the sample is passed (known as the
stationary phase) to separate a mixture into desired and undesired components.
• The stationary phase comes in the form of a packed syringe-shaped cartridge, a 96
well plate or a 47- or 90-mm flat disk, each of which can be mounted on its specific
type of extraction manifold.
Continuation
• The manifold allows multiple samples to be processed by holding
several SPE media in place and allowing for an equal number of
samples to pass through them simultaneously.
Continuation
• Most SPE manifolds are equipped with a vacuum port to speed up the
extraction process by pulling the liquid sample through the stationary
phase.
• The majority of stationary phases are based on silica that has been
bonded to a specific functional group. Some of these functional
groups include hydrocarbon chains of variable length (for reversed
phase SPE), quaternary ammonium or amino groups (for anion
exchange), and sulfonic acid or carboxyl groups (for cation exchange).
Visual display of the interactions between various
sorbents and analytes
a. Cyanopropyl-modified silica sorbent (Polar interactions)
Continuation…
b. Silica sorbent (dipole-dipole interactions)
Continuation….
c. diol-modified silica sorbent (Hydrogen bonding interactions)
Types of SPE
1. Normal phase SPE procedure
• First, the cartridge is equilibrated with a non-polar solvent or slightly polar, which
wets the surface and penetrates the bonded phase.
• Water or buffer of the same composition as the sample, is typically washed
through the column to wet the silica surface.
• The sample is then added to the cartridge. As the sample passes through the
stationary phase, the analytes in the sample will interact and retain on the
sorbent while the solvent, salts, and other impurities pass through the cartridge.
After the sample is loaded, the cartridge is washed with buffer or solvent to
remove further impurities.
• Lastly, the analyte is eluted with a non-polar solvent or a buffer of the
appropriate pH.
Typical normal phase SPE procedure
Purpose
1. Conditioning - To activate the stationary phase in order to allow for consistent
interactions between analyte and sorbent.
2. Equilibrating – To introduce a solvent with similar characteristics (such as
solvent strength, pH, etc.) to the sample. This will ensure maximum retention.
3. Loading – To introduce the sample to the sorbent in a manner that allows for
maximum analyte retention. [Sample solvent should not have any eluting
strength]
4. Washing – To remove (wash away) matrix interferences from the sorbent. [An
appropriate wash solvent should be chosen that will not elutes the analytes
during this step]
5. Eluting – To apply a solvent that will allow for complete elution of analytes.
[Sample can be directly injected or dried down and reconstituted in a more
appropriate solvent].
2. Reverse Phase SPE [RP-SPE] procedure
RP-SPE performs separation on the basis of polarity.
• The stationary phase of a reversed phase SPE cartridge is derivatized
with hydrocarbon chains, which retain compounds of mid to low
polarity due to the hydrophobic effect.
• The analyte can be eluted by washing the cartridge with a non-polar
solvent, which disrupts the interaction of the analyte and the
stationary phase.
3. Ion exchange SPE
This technique separate analytes based on electrostatic interactions between the analyte of interest and the
positively charged groups on the stationary phase. For ion exchange to occur, both the stationary phase and
sample must be at a pH where both are charged.
a) Anion exchange
• Anion exchange sorbents are derivatized with positively charged functional groups that interact and retain
negatively charged anions, such as acids.
• Strong anion exchange sorbents contain quaternary ammonium groups that have a permanent positive
charge in aqueous solutions, and weak anion exchange sorbents use amine groups which are charged when
the pH is below about 9.
• Strong anion exchange sorbents are useful because any strongly acidic impurities in the sample will bind to
the sorbent and usually will not be eluted with the analyte of interest; to recover a strong acid a weak anion
exchange cartridge should be used.
• To elute the analyte from either the strong or weak sorbent, the stationary phase is washed with a solvent
that neutralizes the charge of either the analyte, the stationary phase, or both. Once the charge is
neutralized, the electrostatic interaction between the analyte and the stationary phase no longer exists and
the analyte will elute from the cartridge
b) Cation exchange
Cation exchange sorbents are derivatized with functional groups that interact and
retain positively charged cations, such as bases.
• Strong cation exchange sorbents contain aliphatic sulfonic acid groups that are
always negatively charged in aqueous solution, and weak cation exchange sorbents
contain aliphatic carboxylic acids, which are charged when the pH is above about 5.
• Strong cation exchange sorbents are useful because any strongly basic impurities in
the sample will bind to the sorbent and usually will not be eluted with the analyte
of interest; to recover a strong base a weak cation exchange cartridge should be
used.
• To elute the analyte from either the strong or weak sorbent, the stationary phase is
washed with a solvent that neutralizes ionic interaction between the analyte and
the stationary phase.
Counterparts of SPE
The leading counterpart of SPE is
• Liquid-liquid extraction [LLE] and LLE based techniques…….
Advantages of SPE over its counterparts
SPE vs LLE
• More complete extraction of the analyte
• More efficient separation of interferences from analytes
• Increased separation selectivity
• Reduced organic solvent consumption
• Easier collection of the total analyte fraction
• A trace-enrichment process offering inherent concentration potential
• More convenient manual procedures
• Removal of particulates
• More easily automated
Disadvantages of SPE over its counterparts
SPE vs LLE
• Mixed mechanisms in SPE can occur
• Irreversible adsorption of some analytes on SPE cartridges
• More complex method development is required (3 or more steps
involved)
Applications of SPE
SPE is used for six main purposes in sample preparation:
1. Removal of interferences
2. Concentration or trace enrichment of the analyte
3. Desalting
4. Phase exchange
5. In situ derivatization
6. Sample storage and transport
1. Removal of interferences
a) SPE can remove interferences from the matrix or undesired
compounds in a sample that overlap analyte bands in HPLC or GC
separation which can complicate method development and adversely
affect assay results.
b) SPE can eliminate or reduce interferences in complex samples such as
natural products, food, protein digests etc. which may make it
impossible to separate these from one or more analyte bands with
only one HPLC or GC separation.
c) RP-SPE can be used to remove interfering compounds from some
samples that contain components such as proteins, polymeric
materials, hydrophobic substances or particulates that can either plug
or deactivate HPLC column.
2. Analyte concentration/trace enrichment
SPE can often be used to increase the concentration of a trace
component. If an SPE cartridge can be selected so that the
equilibrium constant (k >>1) for the analyte becomes very large,
then a relatively large volume of a sample can be applied before
the analyte overloads the stationary phase and begins to elute
from the cartridge. The net result is a considerable increase in the
concentration of analyte when eluted with a strong solvent (k <1),
which implies an increase in detection sensitivity (called trace
enrichment).
3. Desalting
RP-SPE is often used to desalt biological and other high-salt samples,
especially prior to ion exchange HPLC. Conditions of pH and %-organic
are selected to retain the analyte initially, which allows the inorganic
salts to be washed from the cartridge with water. The analyte can then
be eluted (salt-free) with a suitable organic solvent.
4. Phase exchange
Phase exchange can be performed by isolating the analyte of interest
on the SPE cartridge, then by flowing air or dry nitrogen through the
cartridge to remove the initial liquid. Next, a second non-compatible
solvent can be used to elute the analyte into a collection device for
further sample preparation or injection into a chromatograph.
5. In situ derivatization
• In one of the procedures of in situ derivatization, an active derivatizing reagent
is sorbed onto an SPE cartridge and a sample containing an active analyte is
passed through the cartridge. Depending on the reaction kinetics, the analyte
can be simultaneously derivatized and concentrated. A strong solvent can then
be used to elute the derivatized compound which can then be handled
chromatographically. E.g. The in situ derivatization of carbonyl-containing
compounds with 2,4-dinitrophenylhydrazine
• Another approach to in situ derivatization involves the in-solution
derivatization in the presence of a SPE sorbent that isolates the derivatized
analyte in solution. Similarly, the derivatized compound is eluted for analysis.
E.g. The thermal hydrolysis and direct methylation of bacterial fatty acids with
tetramethylammonium hydroxide from a sample of bacterial lipids. The
derivatized fatty acid methyl esters were then subjected to GC-ion mobility
mass spectrometry for analysis
6. Sample storage and transport
Oftentimes, analytes adsorbed to a solid support are stabilized relative
to their stability in solution. This observation allows samples to be
stored on a cartridge sorbent allowing for storage and for transport
(through the mail, for example). Before using this approach, individual
set of analytes should be tested in terms of long-term stability when
sorbed to a solid support.
Recent developments/Advanced SPE based techniques

Advancements in SPE have led to the development of more

sensitive techniques which employs the use of coated fibres [Solid
phase microextraction, SPME] and coated stir bar [Stir bar sorbent
extraction, SBSE]

The two slides below depicts the evolution of SPE over the years
since the year of its discovery.
Solid Phase Microextraction [SPME]
What is SPME?
A solvent-less sample preparation method that uses a fused silica fiber which is coated with a
stationary phase.
It is used for sample cleaning before using other analytical methods
It consists of coated fibers that are used to isolate and concentrate analytes into a range of coating
materials
SPME consists of two discrete steps; solute adsorption from the sample matrix into a thick layer of
silicone or related adsorptive material, followed by transfer of the adsorbed analytes into a
chromatography inlet system by thermal or liquid desorption
This technique is an adaptation of SPE in which the solid-phase column has been replaced by a
microliter syringe-like device with a hollow needle and a plunger to which is attached a fused silica
fiber coated with a suitable stationary polymer.
Various SPME fibers and coatings are commercially available and can be used to target different
analytes.
SPME has been applied to both GC and LC.
Instrument of SPME
Modified syringe-like instrument [refer to the next slide]
A typical SPME fiber is made up of fused silica coated with a thin layer (7
µm to 100 µm thick) of immobilized polymer or a solid adsorbent, or a
combination.
The fused silica fiber, having a small size and cylindrical shape, is
connected to a stainless-steel tubing that is used to provide additional
mechanical strength to the fiber assembly for repeated sampling.
This stainless-steel tubing is connected to a specially designed syringe-
like instrument.
A small volume of extraction phase (usually less than 1 µL) coated on
fused silica support is mounted in a modified syringe.
The key feature in this device is an extraction
fiber protected inside the needle of the
syringe. Movement of the plunger allows
exposure of the fiber during extraction and
desorption, and it’s protected in the needle
during storage and penetration of the septum.
How does SPME work?
Firstly, you draw the fiber into the needle.
The needle pierces the septum to a sample (sample can be gas, liquid, or
headspace)
You then depress the plunger to expose the fiber to your sample.
Organic analytes are then adsorbed to the coating on the fiber
After adsorption equilibrium is attained, which can be anywhere from 2
minutes to 1.5 hours, the fiber is drawn back into the needle and is
withdrawn from the sample vial.
Finally, the needle is introduced the GC injector or SPME/HPLC interface,
where the adsorbed analytes are thermally desorbed and delivered to
the instrument's column.
Examples
Continuation…..
Continuation….
Continuation…..
Reaching equilibrium
The extraction is considered to be complete when it reaches equilibrium,
and the conditions can be described by the following equation:

This equation shows the relationship between the analyte concentration in the sample
and amount extracted by the coated fiber. Where ne - amount of solute extracted or
adsorbed by a fiber, Kfs – partition coefficient for analyte between coating and sample
matrix, Vf – volume of the fiber coating, Vs – sample volume and C0 – initial
concentration of the analyte in the sample.
Continuation……

If the amount of solute extracted onto the fiber is an insignificant portion of that
present in the sample, the product of Kfs and Vf is essentially negligible compared to
Vs, and the above equation becomes

This equation indicates that the amount of extracted analyte is independent of the
volume of the sample. This means that;
i. there is no need to collect a defined amount of sample prior to analysis,
ii. the fiber can be exposed directly to whatever is being analyzed, and
iii. the amount of extracted analyte will correspond directly to its concentration in
the sample matrix.
Advantages of SPME
• One of the principal advantages of SPME is its simplicity
• In many cases, the detection limits of SPME are also lower than with
other methods.
• All extracted analytes are transferred to the analytical instrument
• Can sample directly into a sample or the headspace above sample
• During desorption of the analyte, the polymeric phase is cleaned and
ready for reuse
• Absence of solvent makes SPME
 Environmentally friendly
 Separation is faster
 Throughput increases and allows for use of simpler instruments
Disadvantages of SPME
• Polymer coating is fragile, easily broken, and have limited time.
• Can get relatively expensive if one is not care with due to the cost
being roughly $108 to $650 per fiber.
• Its main limitation is its reduced concentration capability due to the
small volume of polymer coating on the fiber
Factors affecting SPME
• Fiber coating selection
• Microextraction temperature
• Microextraction time
• Desorption temperature and time
• Sample agitation
• Salting out effect
Applications of SPME
• The recovery of explosives and ignitable liquid residues from forensic
specimens
• Food analysis
• To study the variety of plasticizers and other additives in lamination
films
• Sample preparation in biomedical analysis
• Flavour analysis
Dependency of SPME to other techniques
1. Derivatization
• Derivatization may be necessary and can be used in SPME just as in the case of LLE and SPE for
chemical transformation of the analyte into a form which is more suitable for analysis
• Derivatization can increase the volatility and/or reduce the polarity of some analytes and
therefore can improve extraction efficiency, selectivity and detection.
• In situ derivatization is often preferred in SPME. In this case a derivatization agent is added to the
sample matrix, derivatization taken place and the SPME fibre extracts the derivatized analytes
either from the solution or from the HS.
• On-fibre derivatization (e.g.with diazomethane or with MSTFA) can be employed after the
extraction procedure. Extracted compounds on the fibre are exposed (in a heated and sealed HS
vial) to the derivatizing reagent in the vapour phase for a given time.
• Derivatization can also be carried out on the SPME fibre in a GC injection port.
2. Quantitation
• At the beginning of the history of SPME, the technique was mainly used for
qualitative or semi-quantitative (screening) studies.
• Dutyiu
• Jggcy
• With SPME, the amount of analyte removed by the fibre (or extraction
capillary) is proportioned to the concentration of the compounds in the
sample.
• The ability to use SPME quantitatively before reaching equilibrium allows
much shorter sampling times, producing a fast economical and versatile
technique.
• The selection criteria as to which quantitation approach is to be selected
depends on the sample matrix, its complexity and the extraction method (HS
or DI) being used.