IJRPC 2013, 3(2) More et al.

ISSN: 22312781

INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY

Available online at www.ijrpc.com Research Article

MICROEXTRACTION TECHNIQUES IN ANALYSIS OF DRUGS

VN. More* and DG. Mundhe
Department of Quality Assurance, I.B.S.S. College of Pharmacy, Malkapur - 443 101, India.

ABSTRACT
This review will attempt to provide an overview as well as a theoretical and practical
understanding of the use of microextraction technologies for drug analysis. In many cases only
a small fraction of the initial analyte is extracted for analysis. The extraction efficiency is
determined by the partitioning of analyte between the sample matrix and the extraction phase.
The higher the affinity the analyte has for the extraction phase relative to the sample matrix, the
greater the amount of analyte extracted. Microextraction techniques represent an important
contribution to the improvement of sample preparation performance, which especially
addresses the issues of miniaturization, automation, onsite analysis, and time efficiency. Sample
preparation is essential for isolating desired components from complex matrices and greatly
influences their reliable and accurate analysis. Solid-phase microextraction (SPME) is a new and
effective sample preparation technique. Fibers and capillary tubes coated with an appropriate
stationary phase are usually used for SPME, but alternative microextraction techniques are also
used.

Keywords: Analyte, affinity, microextraction, SPME, stationary phase.

INTRODUCTION drugs has to date, found its greatest

Microextraction is defined as an extraction application with the technique of solid-phase
technique where the volume of the extracting microextraction (SPME) and in particular fibre
phase is very small in relation to the volume of SPME. SPME is a relatively new sample
the sample, and extraction of analytes is not preparation method, which has the potential to
exhaustive. In most cases only a small fraction significantly simplify sample preparation, and
of the initial analyte is extracted for analysis. integrate it with sample analysis1.
The extraction efficiency is determined by the Microextraction techniques represent an
partitioning of analyte between the sample important contribution to the improvement of
matrix and the extraction phase. The higher sample preparation performance, which
the affinity the analyte has for the extraction especially addresses the issues of
phase relative to the sample matrix, the miniaturization, automation, onsite analysis,
greater the amount of analyte extracted1. Thus and time efficiency. Actually, different types of
while the goal of microextraction is to extract microextraction techniques were reported in
based on equilibrium partitioning i.e. the goal literature a long time ago3,4, but the field
is to extract asnear as possible to 100% of the gained in significance with the invention of
2
analytes from a sample . solid-phase microextraction (SPME) in
Partitioning is controlled by the 19905,which later became commercially
physicochemical properties of the analyte, the available. In the commercial version of this
sample matrix and the extraction phase. technique, a small diameter fiber coated with a
Where sample matrix and extraction phase small volume of stationary phase is placed
composition are constant, the degree of either in an aqueous or a gaseous sample.
partitioning and hence the percentage of The analytes partition into the stationary phase
analyte extracted will be constant also. and are subsequently thermally desorbed in
Because partitioning is not dependent on the injector of a gas chromatograph (GC).
analyte concentration, quantification of sample During recent years, SPME has gained
concentration may be determined from substantially in popularity, and from this point,
absolute amount extracted. Microextraction of several alternative approaches have been

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introduced, such as in-tube SPME designed and sorbent bed dimensions will depend on
primarily for high-performance liquid the particle size of the solid extractant. Very
chromatography (HPLC)6. In parallel to the small particles (e.g., 10 mm) are more efficient
development of SPME, attention has also than columns packed with larger particles
been directed to the utility of small volumes of (e.g.,50–100 mm). Therefore, a smaller bed of
liquids for analytical extractions, namely liquid- the smaller particles may be used.
phase microextraction (LPME). This field was
basically initiated in 1996 when the use 3. Washing
ofsmall droplets of organic solvents The most common type of SPE is where
suspended from the tip of amicro-syringe was organic analytes are extracted from an
7,8
described for the first time , and thisapproach aqueous sample. The purpose of the washing
was subsequently refined by implementing the step is to remove salts and other non-
use ofporous hollow fibers for protection of the extracted material as completely as possible
extracting liquids9. without eluting any of the desired analytes.
Water alone is often the appropriate wash
Types of microextraction techniques10 solution, but some solutes may be partially
1. Solid phase extraction (SPE) retained by the SPE column and only slowly
2. Solid phase microextraction (SPME) washed off by water alone. In such cases
3. Liquid phase microextraction (LPME) water containing 10–20% of anorganic solvent
4. Molecularly imprinted polymers (MIPs) might be a better wash liquid. Ofcourse, the
5. Turbulent flow chromatography (TFC) wash solution must not contain a percentage
of organic solvent high enough to elute the
1. SOLID PHASE EXTRACTION sample analytes.
Solid phase extraction (SPE) is an extraction
method that uses a solid phase and a liquid 4. Elution
phase to isolate one, or one type, of analyte In the elution step the adsorbed analytes are
from a solution. It is usually used to clean up a removed from the solid extractant and are
sample before using a chromatographic or returned to a liquid phase that is suitable for
other analytical method to quantify the amount analytical measurement. Most commonly, the
of analyte(s) in the sample11. eluting phase is an organic liquid, although it is
often possible to thermally desorb analytes
Principles of solid-phase extraction12 with the aid of a gas stream. It is usually better
It consists of mainly four steps. to select an eluting solvent that is miscible with
1. Conditioning water, otherwise the effluent may contain two
Before extraction of analytes can begin, the liquid phases. It is common practice to remove
sorbent bed must be prepared so that it will as much of the water as possible from the
make intimateand effective surface contact column just before the elution step. This can
with the liquid sample solution. Most be accomplished by applying gentle vacuum
commonly, conditioning is accomplished by for a few minutes or by passing compressed
passing a small volume of methanol or air or nitrogen through the column.
acetonitrile through the SPE extraction tube. Occasionally centrifugation is used to remove
Some of this organic solvent is adsorbed on liquid from the column.
the surface of the sorbent particles, making
the surface more hydrophilic and thus more Solid phase extraction theory13
compatible with a primarily aqueous sample How compounds are retained by the
solution. Without such treatment the surface of sorbent
many common sorbents is hydrophobic and is  Reversed phase
poorly wetted by the hydrophilic sample Reversed phase separations involve a polar
solution. The polar liquid flows in small (usually aqueous) or moderately polar sample
channels through the solid phase without matrix (mobile phase) and a nonpolar
making the necessary close surface contact. stationary phase. The analyte of interest is
The conditioning step also serves to elute any typically mid- to nonpolar. Several SPE
adsorbed organic impurities from the SPE bed. materials, such as the alkyl- or aryl-bonded
silicas (LC-18, ENVI-18, LC-8, ENVI-8,LC-4,
2. Adsorption and LC-Ph) are in the reversed phase
The liquid sample is passed through the category.
packed SPE device with the aid of a gentle (Polar - liquid phase, non-polar modified solid
vacuum (applied to the end of the column), phase)
applied pressure or a pump. The flow rate Hydrophobic interactions
should be reasonably constant. The flow rate  Nonpolar-nonpolar interactions

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 Van-der Waals or dispersion forces. attractive forces are commonly called van der
Waals forces or dispersion forces. The
5. Normal phase nonpolar solvent, which can disrupt the forces
Normal phase SPE procedures typically between the sorbent and compound, is used
involve a polar analyte, a mid- to nonpolar to elute an adsorbed compound from a
matrix (e.g. acetone, chlorinated solvents, and reversed phase SPE tube or disk. Normal
hexane), and a polar stationary phase. Polar- phase involve a polar analyte, a mid- to non-
functionalized bonded silicas (e.g. LC-CN, LC- polar matrix (e.g. acetone, chlorinated solvents
NH2, and LC-Diol), and polar adsorption and hexane) and a polar stationary phase.
media (LC-Si, LC-Florisil, ENVI-Florisil, and Retention of an analyte under normal phase
LC-Alumina) typically are used under normal conditions is primarily due to interactions
phase conditions. Retention of an analyte between polar functional groups of the analyte
under normal phase conditions is primarily due and polar groups on the sorbent surface.
to interactions between polar functional groups These include hydrogen bonding, л- л
of the analyte and polar groups on the sorbent interactions, among others. A compound
surface. These include hydrogen bonding, pi- adsorbed by these mechanisms is eluted by
pi interactions, dipole-dipole interactions, and passing a solvent that disrupts the binding
dipole-induced dipole interactions, among mechanism, usually a solvent that is more
others. A compound adsorbed by these polar than the sample’s matrix.
mechanisms is eluted by passing a solvent
that disrupts the binding mechanism-usually a
solvent that is more polar than the sample’s Solid phase extraction process13,14
original matrix. The SPE process can provide samples that
(Nonpolar- liquid phase, polar modified solid are in solution, free of interfering matrix
phase) components and concentrated enough for
Hydrophilic interactions detection. Solid phase extraction is achieved
 Polar-polar interactions through the interaction of three components:
 Hydrogen bonding the sorbent, the analyte and the solvent. The
 Pi-pi interactions analyte must be attracted more strongly to the
 Dipole-dipole interactions sorbent than to the matrix. The best solid
 Dipole-induced dipole interactions. phase extraction mechanism and procedures
are defined by the characteristics of the
6. Ion exchange analyte in the sample. The steps of the solid
Electrostatic attraction of charged group on phase extraction process are shown in Fig. 1.
compoundto a charged group on the sorbent’s
surface. Step 1: Select the proper SPE tube or Disk
 Selecting SPE tube size
7. Adsorption Criterion for the selection of SPE tube size is
(Interactions of compounds withunmodified mentioned in table no.1
materials), Hydrophobic and hydrophilic  Selecting SPE disk size
interactions may applyDepends on which solid Criterion for the selection of SPE tube size is
phase is used. mentioned in table no.1
 Selecting an SPE tube:Bed weight
Mechanism of solid phase extraction Reversed phase, normal phase, and
process13 adsorption-type procedures
The most common retention mechanisms in The mass of the compounds to be extracted
SPE are based on van der Waals forces (“non- should not be more than 5% of the mass of the
polar interactions”), hydrogen bonding, dipole- packing in the tube. In other words, if you are
dipole forces (“polar” interactions) and cation- using a 100mg/1mL SPE tube, do not load
anion interactions (“ionic” interactions). more than 5mg of analytes.
Reversed phase involves a polar or
moderately polar sample matrix (mobile Step 2: Condition the SPE tube or Disk
phase) and a nonpolar stationary phase. The To condition the SPE tube packing, rinse it
analyte of interest is typically mid- to nonpolar. with up to one tube-full of solventbefore
Retention of organic analytes from polar extracting the sample. For disks, use a volume
solutions (e.g. water) onto these SPE of 5-10 ml.
materials is due primarily to the attractive
forces between the carbon-hydrogen bonds in Step 3: Add the sample
the analyte and the functional groups on the Accurately transfer the sample to the tube or
sorbent surface. These nonpolar – nonpolar reservoir, using a volumetric pipette or

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micropipette. The sample must be in a form  Air and gas

that is compatible with SPE. The analysis of air and gaseous samples can
be subdivided into analysis of volatile
Step 4: Wash the packing compounds (VOCs) and of particulate matter
If compounds of interest are retained on the (PM). The method classical SPE followed by
packing, wash off unwanted, un-retained GC analysis. The examples include the
materials using the same solution in which the analysis of volatile hydro-carbons, volatile
sample was dissolved, or another solution that organic sulfur compounds.
will not remove the desired compounds.
Usually no more than a tube volume of wash 2. SOLID PHASE MICROEXTRACTION
solution is needed, or 5-10 ml for SPE disks. (SPME)
Microextraction techniques have been
Step 5: Elute the compounds of interest regarded as themost attractive for the
Rinse the packing with a small volume pretreatment of complex samplematrices prior
(typically 200μl to 2 ml depending on the tube to chromatographic and
size, or 5-10 ml depending on the disk size) of capillaryelectrophoretic processes because
a solution that removes compounds of interest, they enable rapid analysis at low operating
but leaves behind any impurities not removed costs and with no environmental pollution.The
in the wash step. Collect the eluate and further recent trend in sample preparation processes
prepare as appropriate. focuses onhow to miniaturize the process and
which medium to use forthe extraction and
Applications of SPE12 pre-concentration of sample components.In
 Biological fluids this section, I review in detail fiber SPME, in-
The compatibility of SPE coupled with HPLC tube SPME(or capillary microextraction), solid-
has been reflected in growing popularity of phase dynamic extraction(SPDE),
SPE–HPLC on-line. A number of papers in the microextraction in a packed syringe (MEPS)
area of SPE–HPLC document that it is a well andstir-bar-sorptive extraction (SBSE), all of
established technique. which have beenwidely used for forensic,
Eg. Determination of methylated arsenic clinical and pharmaceuticalanalysis. Fiber
species in human urine. SPME is the most widely used technique.
IntubeSPME was developed primarily to
 Waters extend SPME tohigh-throughput applications
An interesting example of a simple and automated instrumentation.SBSE was
combination of sample trapping with a non- developed to increase the sensitivity of SPME.
separation analytical technique was presented Various new affinity SMPE sorbents,
by Ackerman et al. [189] when they includingimmunosorbents and MIPs, have
determined polycyclic aromatic hydro-carbons been used for the specificpreparation of
(PAHs) isolated from water using SPE samples and are also described in this section.
combined with a direct in-situ measurement by Other new microextraction techniques, such
fluorescence /phosphorescence. Various as liquid-liquidmicroextraction (liquid-phase15-
17
pollutants were determined in aqueous or single-drop18 microextraction), solvent bar
samples after SPE or SPME. microextraction19 and liquidmembrane
20-22
microextraction, are not reviewed
 Food, beverages and agricultural here,since they have been recently
The use of SPE in the determination of various reviewed23,24.
chemicals in food has increased rapidly in the
last decade and SPE methods have replaced Principle of solid phase microextraction
many of the traditional methods of sample SPME is based on a modified syringe which
pretreatment.As an example, the bulk of lipid contains stainless steel microtubing within its
content of fats and oil in foods has syringe needle. This microtubing has an about
complicated the analysis of pesticide residues 1-cm. fused-silica fiber tip which is coated with
as well as other chemical contaminants. The an organic polymer. The coated silica fiber can
recent examples of food analysis for volatile be moved between two positions, inside and
flavor compounds show ongoing use of SPE– outside the needle, with a plunger as in the
GC methods but especially an increase in the case of a normal syringe the diameter of the
use of SPME–GC. Examples of the first type syringe needle housing the microtubing and
of analysis include trace-level determination of coated silica fiber is not much increased in
polar flavor compounds in butter by SPE with comparison with a normal GC syringe. Thus,
polymeric sorbent. by means of this simple equipment several
steps of sample preparation are combined in

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one device. Extraction and enrichment of the cleaned fiber coating is exposed to a sample
analyteis completed by the coating in the matrix for a predetermined, fixed period, which
position outside the syringe needle. results in the adsorption of the analyte on the
Penetration of the septum of a GC injection fiber coating (Figure 2). This extraction can be
port is possible if the fiber was withdrawn into performed in twoways30: 1) headspace SPME
the syringe needle. Desorption of the analyte or HS-SPME where fiber is exposed in the
and transfer to the capillary is performed after vapor phase above a gaseous, liquid, or solid
again moving the fiber to the position outside sample: 2) direct immersion DI-SPME, where
the syringe25,26. the fiber is directly immersedin liquid
31
samples .
Advantages of solid-phase
27-29
microextraction  MATERIALS AND METHODS
The main advantage of SPME is Before the SPME device was used it was
simplicity,rapidity, solvent elimination. necessary to condition the fibres, this was
1.High sensitivity achieved by exposing the fibres to the GC
2.Small sample volume injector port at 250 ◦C for 1h.Several fibre
3.Low cost. blanks were run to ensure the fibres were fully
conditioned and that no interferences from the
Design of SPME device fibres were present in GC chromatograms.
SPME is a modified syringe-like instrument Once this had been completed successfully a
(Figure 2). The fused silica fiber, having a variety of standard solutions containing
small size and cylindrical shape, is connected individual PAHs were made. Eventually, it is
to stainless-steel tubing that is used to provide hoped the device will be used to extract up to
additional mechanical strength to the fiber 16 PAH compounds simultaneously, however,
assembly for repeated sampling. This for the purpose of optimising extractionand
stainless-steel tubing is connected toa desorption conditions it was decided that
specially designed syringe-like instrument. The working with individual compounds would
fused silica fiber is coated with a relatively thin make extractions both easier and quicker.
film of several polymeric stationary phases. When using SPME it is important that the
The fiber assembly is reusable and device is placed into the hottest part of the GC
replaceable. Supelco (www.sigmaaldrich.com) injector while desorption is taking place,
provides seven different types of fibers. The therefore, a series of desorption’s of a
small size and cylindrical geometry of fiber has standard solution containing 9.98 ng μL−1
some advantages such as easy placement of naphthalene were carried out withdesorption
the sorbent fibercoating into a sample or taking place at different depths in the injector,
headspace above the sample to extract the all other factors were kept constant. It was
analytes; also it can be easily placed in found that a depth of 3 cm in the injector port
desorption chamber of GC or interphase of the was optimal so this depth was used in all
HPLC without any modification of GC or further experiments.
HPLC. Plunger movement and timing must be Investigations into the various parameters
controlled carefully to perform adsorption and governing extraction efficiency were then
desorption correctly. It is very important for carried out using standard solutions of the
field sampling to prevent loss of analyte during PAHs naphthalene, chrysene, fluorene and
transport. To do this, the needle opening of BaP and samples of de-ionised water spiked
SPME devise must be sealed by using a with these compounds. These compounds
septum and/or by cooling the needle30. The were chosen because they cover a range of
SPME device is shown in figure no. 2. PAH physiochemical properties, naphthalene
being one of the smallest and most volatile
Working with SPME device with two aromatic rings, and BaP one of the
During the SPME operation, the fiber is first largest and least volatile of the PAHs of
drawn into the syringe needle, then lowered interest with five fused aromatic rings.
into the vial (which is sealed with a septum Generally, at ambient temperature, headspace
type cap) by pressing the plunger. The fused SPME can only be used to extract compounds
silica fiber of suitable coating is used which is with Henry’s law constants above90
dependent upon the nature of the analyte. The atm.cm3mol−1, this includes three-ring PAHs
fiber should be cleaned before analyzing any or more volatile analytes (Zhang and
sample so as to remove contaminants that Pawliszyn, 1993). Therefore, it was decided to
give a high background in the chromatogram. concentrate on the parameters that affect the
Cleaning can be performed in the desorption extraction of the compounds of interest by
32
chamber of HPLC by running solvent. Now the immersion extraction at this stage .

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Applications of SPME in various fields

 Environmental applications: Disadvantages
In the early developmental period the majority  Relatively low recommended
of applications were in environmental operating temperature (generally in
chemistry. Mostly organic compounds33-40 the range 240–2800C);
have been studied, and pesticides, herbicides  Their instability and swelling in organic
and other biologically active compounds in solvents (greatly restricting their use
aqueous samples41-43. with HPLC);
 Fiber breakage;
 Applications in food chemistry  Stripping of coatings; and,
Food analysis is important for the evaluation of  The bending of needles and their
nutritional value, for quality control of fresh and expense49.
processed products and the monitoring of food
additives and other toxic contaminants. In Types of liquid phase microextraction
general, flavor is sensitive to compositional It can be divided into three main categories
alterations in the case of food (fruit, wine, (1) Single-drop microextraction (SDME)
etc.)44,45. (2) Dispersive liquid–liquid microextraction
(DLLME)
 Applications to biological fluids (3) Hollow-fiber microextraction (HF-LPME).
Sample preparation is one of the most critical
steps in the analysis of biological fluids and 1. Single-drop microextraction (SDME)
46
compounds in biological matrices . SDME, using typically 1–3 lL of an organic
solvent at the tip of a micro syringe, has
3. LIQUID PHASE MICROEXTRACTION evolved from LPME. Afterextraction, the micro
(LPME) drop is retracted back into the syringe and
Sample preparation can include cleanup transferred for further analysis
procedures for very complex (dirty) samples. In practice, two main approaches can be used
This step must also bring the analytes to a to perform SDME:
suitable concentration level47. LPME is a a) Direct immersion (DI)-SDME
solvent-minimized samplepretreatment b) Headspace (HS)-SDME
procedure of LLE, in which only several lLit of
solvent are required to concentrate analytes a) Direct immersion (DI)-SDME
from various samples rather than hundreds of In DI-SDME, a drop of a water-immiscible
ml needed intraditional LLE. It is compatible solvent is suspended directly from the tip of a
with capillary gas chromatography (GC), micro syringe needle immersed in the aqueous
capillary electrophoresis (CE) and HPLC. In sample.
LPME, extraction normally takes place into a
small amount of a water-immiscible solvent b) Headspace(HD)-SDME
(acceptor phase) from an aqueous sample In HS-SDME, a micro drop of appropriate
containing analytes (donor phase)48. solvent is placed in the headspace of the
sample solution or in aflowing air sample
Advantages stream to extract volatile analytes.Headspace
 It is a rapid, simple, solvent free and (HD) – SDME is shown in figure no. 3.
sensitive method for the extraction of
analytes; Advantage
 It is a simple, effective A wider variety of solvents to choose from.
adsorption/desorption technique;
 It is compatible with analyte Disadvantage
separation and detection by high- Extraction and injection have to be performed
50,51
performance liquid separately, using different apparatus .
chromatography withultraviolet 2. Dispersive liquid-liquid microextraction
detection (HPLC - UV); (DLLME)
 It provides linear results for a wide This technique that uses1Lit volumes of
range of concentrations of analytes; extraction solvent along with a few ml of
 It has a small size, which is dispersive solvents. Inthis method, a cloudy
convenient for designing portable solution is get formed when anappropriate
devices for field sampling; and, mixture of extraction and dispersive solventsis
 It gives highly consistent, quantifiable injected into an aqueous sample containing
results from very low concentrations of the analytesof interest. Hydrophobic solutes
analytes. are rich in the extraction solvent, which is

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dispersed into the bulk aqueous solution. After

centrifugation, analytes in the settled phase Applications
can be determined by using conventional All LPME techniques can be utilized effectively
analytical techniques. for extraction of target analytes from various
In DLLME, the dispersive solvent plays a key sample solutions54.
role that helps extraction solvent form fine
droplets in aqueous samples, representing
about 97–99% of the total volume of the 4. MOLECULARLY IMPRINTED
extraction mixture. Compared to other POLYMERS (MIPS)
methods, abundant surface contact between Molecularly imprinted polymers (MIPs) are
fine droplets and the analyte in DLLME speeds polymers prepared in presence of a template
55,56
up the mass-transfer processes of analytes that serves as a mould for the formation of
from aquatic phase to organic phase, which template complementary binding sites. Thus,
not only greatly enhances extraction efficiency MIPs can beprogrammed to recognize a large
but also overcomes the problem of the time variety of target structures with antibody-like
57
taken. affinities and selectivities .MIP is based on
the formation of a complex between an analyte
Advantages (template) and a functional monomer. In the
1. Simplicity of operation presence of a large excess of a cross-linking
2. Rapidity agent, a three-dimensional polymer network58
3. Low cost is formed. After polymerization process, the
4. High recovery template is removed from the polymer leaving
5. High enrichment factor specific recognition sites complementary in
6. Very short extraction time (a few shape, size and chemical functionality to the
seconds)52. template molecule. Usually, intermolecular
interactions like hydrogen bonds, dipole–
3. Hollow-fiber microextraction (HF-LPME) dipole and ionic interactions between the
They used the basic principle of supported template molecule and functional groups
liquid membrane (SLM), for the first time, in present in the polymer matrix drive the
simple, inexpensive, disposable extraction molecular recognition phenomena. Thus, the
units for the liquid-liquid-liquid microextraction resultant polymer recognizes and binds
(LLLME) utilizing polypropylene HFs as the selectively only the template molecules59.
membrane.
The sample vial is filled with the aqueous Molecular imprinting process
sample. A short piece of a porous HF may be Template design and monomer selection are
either a rod with a closed bottom or a u-shape two of the mostcritical features of the
where both ends are connected to guiding molecular imprinting process. For analytical
tubes. Prior to extraction, the HF is first dipped applications, the “MIP Rule of 6” should be
in the organic solvent for a few times to followedwhen possible:
immobilize solvent in the pores, and excess  Never use the analyte as a template
solvent is removed. The solvent is immiscible unless there is absolutely no
with water to ensure that it remains within the alternative
pores during the extraction with no leakage to  Make rational choices about which
the aqueous sample. The organic solvent regions of an analyte are likely to
forms a thin layer within the wall of the HF. command the best types of interaction
The extraction solvent must be compatible in a low dielectric medium (organic
with the HF so that the pores in the wall of the solvent) and then incorporate these
HF can be filled completely. elements in an analog of the analyte
The acceptor solution then fills the lumen of molecule
the HF. This acceptor solution can be an  Select monomers that are likely to
organic solvent (the same as that used for the form strong interactions in the chosen
organic solvent in HF pores) resulting in a two- solvent (e.g., Bronsted acids or
phase extraction system, or the acceptor bases/H-donors or acceptors/nonpolar
solution may be an acidic or alkaline aqueous groups, etc.)—this will increase
solution, resulting in a three-phase extraction capacity and influence homogeneity of
system. In the two-phase LPME system, the the binding cavities.
target analytes are extracted from the aqueous  Choose templates and monomers that
sample and into the organic solvent (acceptor will be soluble in the porogenic solvent
solution) present both in the porous wall and to be used in the polymerization-this
53
inside the lumen of the HF .

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may seem obvious but it sometimes

requires carryingout solubility tests c) Semi-covalent imprinting
 Ensure as far as possible that the This approach includes all the procedures in
template–monomer mixture is stable which the template is covalently bound to a
and does not undergo side reactions polymerizable group for polymer synthesis but
under the polymerization conditions. template rebinding takes place by noncovalent
 Consider the nature of the matrix from interactions (Fig. 10)71-73.
which the analyte will eventually be
extracted when selecting the cross- d) Metal-mediated interactions
linking monomer - a range of di- or tri- Metal ions can play different roles in
unsaturated cross linking monomers imprinting; they can be used as templates or
with varying chemistries are available as components of the template
to create the porous organic network functionalmonomer interaction. Metal ion
material60-64. Process of preparation of imprinting can be achieved by cross-linking
MIPs is shown in figure no. 4. preformed polymers bearing complexing
ligands, or polymerizing specific metal
Methodologies applied for the preparation complexes with polymerizable ligands74,75.
of MIPs
The main methodologies applied for the  Applications of MIPS
preparation of MIPs are based on covalent, 1. Their use as tailor-made separation
non-covalent,semi-covalent, and metal- materials,
mediated interactions66. 2. Their use in organic synthesis and
enzyme technology as catalytically
a) Covalent imprinting active polymers or enzyme mimics.
Covalent imprinting, or the pre-organized 3. As sensors in biosensor-like
approach67, involves the formation of configurations76.
reversible and easily cleavable covalent bonds 4. Scheme outlining the main
between the template molecule and one, or applications envisaged for MIPs are
more, polymerizable monomers prior to the shown in figure no.577.
polymer synthesis68.
5.Turbulent flow chromatography (TFC)
Advantages Turbulent Flow Chromatography (TFC)is a
1. The monomer/template complexes are technique that combines high-throughput and
stable and stoichiometric. high reproducibility by means of separating
2. wide variety of polymerization conditions analytes from various matrices with reduced
can be used. sample handling. The sample can be injected
Disadvantages directly onto a narrow diameter column (0.5 or
1. slow release and binding of 1.0 mm) packed with large particles (30–60 m)
69
templates . at a high flow rate (higher than 1 ml min−1)
helping creating a very high linear velocity
b) Non-covalent imprinting inside the turbulent flow column. Under
In non-covalent imprinting the prearrangement turbulent flow conditions the improved mass
between the template and the transfer across the bulk mobile phase allows
functionalmonomer(s) occurs by non-covalent for all molecules to improve their radial
interactions such as hydrogen bonding, ionic distribution, however, under these conditions a
interactions, π – π interactions, hydrophobic laminar zone around the stationary phase
70
interactions or Van der Waals forces . particles still exists, where diffusional forces
Advantages still dominate the mass transfer process78.
 Easy preparation of the Molecules with low molecular weight diffuse
template/monomer complex. faster than molecules with a high molecular
 Easy removal of the templates from weight, forcing large molecules to quickly flow
the polymers. to waste while retaining the small analytes.
 Fast binding of templates to MIPs. The retained compounds are then back-
flushed and focused on theanalytical column
Disadvantages for chromatographic separation. It is extremely
 The template/monomer complex is not important to effectively avoid interferences
stable and in order to minimize the from the matrix on the analysis of a
non-specific binding sites the contaminant. The optimization of the different
conditions ofpolymerization must be on-line extraction steps is crucial, as
carefully chosen69. parameters like mobile phase composition,

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flow rates and extraction time windows will binary pump, an on-line degasser, an auto-
affect recovery and extraction efficiency in plate-sampler, and a thermostatically
general79. controlled column compartment) was used to
deliver a gradient flow to elute the analytes
Principle of TFC from the extraction column and to perform the
Turbo Flow methods are based on the direct separation on a fast HPLC column. Two
injection of biological samples without previous Rheodyne six-port switching valves were used
extraction or treatment a column packed with for the column-switching purposes. The L and
large particles. These large particles have an E in the center of the each six-port valve
additional level of selectivity via the stationary designate “load” or “elute” positions for the
81
phase chemistry added to them. After the flow path .
sample is injected onto a TurboFlow column
the high flow rate (cf. 1.5 – 5.0ml/min) Applications of TFC
generates turbulent flow conditions inside the  In food and environmental analysis.
column. Since 100% aqueous mobile buffers  In the handling of biological samples
are used, the small analyte molecules are containing a large amount of proteins,
retained via diffusion into the particle pores, such as blood plasma82,83–88.
while the proteinaceous material is washed to  Isolation of veterinary drugs and
waste. Once the compounds of interest are growth promoters from food[89].
extracted from the biological matrix, they are  A solution that has gained wide use,
eluted from the Turbo Flow column onto the particularly in the clinical field, to
analytical column with a volume of solvent, increase throughput on such
which has been stored in a holding loop. The systems90.
holding loop should have a volume at least ten
times that of the Turbo Flow column and is CONCLUSION
typically filled with organic mobile phase (for Solid phase extraction is a widely used
reversed stationary phase) or pH buffered sample-preparation technique for isolation,
solutions (for ion exchangephases). As the concentration, clean-up and medium
analytes are released from the Turbo Flow exchange. SPME is a technique for extraction
column they are transferred with the pumping of organic compounds from gaseous,
solvent (at a considerably lower flow rate than aqueous, and solid matrices. All LPME
that used during loading) through the tee rotor- techniques can be utilized effectively for
seal in the second valve and mixed with the extraction of target analytes from various
pumping solvent from the analytical system80. sample solutions. Imprinted polymers are now
Schematic representation of on-line turbulent- well established as materials for molecular
flow column-switching is shown in figure no. 6. recognition, chromatographic separation, and
analytical sample enrichment but their use as
Online turbulent-flow column-switching active biomedical devices is still in the early
A schematic diagram of the on-line TFC- stages of development. The on-line TFC-
LC/MS instrument set-up based on column- LC/MS method was suitable for TCMs
switching and fast HPLC is shown in Fig. (12). pharmacokinetic study at a low dose level.
A pump was used to deliver a high flow
through a hydrophilic–lipophilic balanced ACKNOWLEDGEMENT
(HLB) reversed-phase column to load and We extend our sincere thanks to principal,
wash the sample, and subsequently to flush I.B.S.S. College of pharmacy, Malkapur
and equilibrate the extraction column. Solvent (Maharashtra), India, for critical review of
A was used as the solvent for this pump. An manuscript.
Agilent 1100 HPLC system (equipped with a
T abl e 1: Selecting SPE tube size
If Your Sample is Use Tube Size
< 1mL 1ml
1mL to 250mL and
the extraction speed 3ml
is not critical
1mL to 250mL and a
fast extraction procedure 6ml
is required
10mL to 250mL and
higher sample capacity 12, 20, or 60ml
is needed
< 1 liter and extraction
12, 20, or 60ml
speed is not critical

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T abl e 2: Selecting SPE disk size

If Your Sample is Use Disk Size
100mL to 1 liter 47mm
>1 liter and higher
sample capacity 90mm
is needed

Fig. 1: Steps of solid phase extraction process

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32
Fig. 2: SPME device

Fig. 3: Headspace single drop microextraction

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65
Fig. 4: Schematic representation of the molecular imprinting process

Fig. 5:Scheme outlining the main applications envisaged for MIPs77

Fig. 6: Schematic representation of on-line turbulent-flow column-switching

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