4

Microextraction Techniques as a Sample

Preparation Step for Metal Analysis
Pourya Biparva1 and Amir Abbas Matin2
1Department of Nanotechnology, Islamic Azad University, Langaroud Branch, Langaroud,
2Research Department of Analytical Chemistry, Iranian Academic Center for Education,
Culture & Research (ACECR), Urmia
Iran

1. Introduction
Analytical methods consist of several steps including; sampling, sample preparation,
analysis, calculations and statistical evaluation of the results. Each step has a direct impact
on accuracy, precision and sensitivity of the method. Among theses steps, sample
preparation is the most time consuming step. Result of studies showed that more than 60%
of analysis time is spent for sample preparation. Sample preparation follows two main aims;
sample clean-up and concentration. Sample Clean-up is carried out for isolating the target
analytes from matrix components which interfere on determination and concentration is
done for enrichment of the analytes in sample because despite advances in analytical
instrumentation, sensitivities are limited.
Characteristics of an ideal sample preparation technique are listed as below:
- Minimum loss of the sample and maximum recovery of the analyte
- Elimination of accompanying compounds with high yield
- Simple, fast and cheap method
- Capable with analytical instruments
- In agreement with green chemistry
In the case of atomic absorption spectrometry (AAS) which is the subject of this book, there
are two priciple systems which are familiar with readers of this book, flame and
electrothermal AAS. In continue of our discussions about microextraction techniques for
metal analysis by AAS, we will emphasis on reduced volumes of extracting phases in the
microlitre scale. It is clear that due to consumption of large volumes (in the mililitre scale) of
the sample in flame AAS, coupling of microextraction techniques with flame AAS is
difficult. But in the case of electrothermal AAS, this is so easy. Because volume of the
samples introduced to graphite furnaces are very low and in microlitre scale. So a review on
literature show that most of the microextraction methods are capable with electrothermal
AAS not with flame AAS.

1.1 Liquid-Liquid Extraction (LLE)

Liquid-Liquid Extraction (LLE) is a versatile classical sample preparation technique. LLE is
based on establishment of distribution equilibrium of the analytes between two immiscible

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62 Atomic Absorption Spectroscopy

phases, an aqueous and an organic phase. Apparatus for LLE is a separating funnel. If
distribution equilibrium constant is enough large, a quantitative extraction of the analytes
can be occurred in one step. But most of the LLEs are multistep.
LLE commonly is used for extraction of organic and inorganic compounds. In the case of
metal analysis, extraction of them as ammonium pyrrolidin dithiocarbamat (APDC)
complexes using methyl isobuthyl ketone (MIBK) as extraction solvent is known as
Standard method. Other examples for application of LLE in metal analysis are briefly
described here; Extraction of As (inorganic and organic) in urine and water samples as their
iodide salts extracted in chloroform and re-extracted in dilute dichromate solution for total
As determination by electrothermal atomic absorption spectrometry (ET-AAS) was
reported (Fitchett et al., 1975). Another method for extraction of As (inorganic and organic)
is based on extraction to toluene and back extraction with cobalt nitrate solution (Lauwerys
et al. 1979). A simple LLE method for extraction of methylmercury was introduced by using
toluene as extraction solvent before analysis by ET-AAS (Saber-Tehrani et al. 2007). Ease of
operation and simplicity of the method are advantages of LLE. But important disadvantages
such as consumption of large volumes of expensive and toxic solvents, emulsion formation
at the interface of the two phases and difficult phase separations and finally low
concentration factor lead the analytical chemists to introduce alternative methods for LLE by
decreasing volume of the extracting solvent at micro liter scale known as liquid phase
microextraction (LPME) which introduced in the late 1990s and early 2000s.

1.2 Solid Phase Extraction (SPE)

One of the alternative methods for LLE is solid phase extraction (SPE) which was
introduced in early 1970 and developed during 1980-90. SPE process is based on distribution
of analytes between solid sorbent packed in a cartridge and liquid sample which moves
through the solid phase. Solid phase usually consists of small porous particles of silica with
or without bonded organic phase, organic polymers and ion exchangers. Mechanisms of
extractions are based on adsorption, partitioning or ion exchange according to kind of solid
phase. SPE is used for extraction of both of the organic and inorganic compounds. A
wide group of chemicals which their SPE procedures were reported in the literature is metal
ions.
Main SPE methods for metal ions are summarized in Table 1 (Fritz, 1999).
Increasing development of SPE during 1990s continues until recent years by introducing
novel solid sorbents such as molecularly imprinted polymers (Lucci et al., 2010) and
nanostructured materials (Faraji et al., 2010). SPE has many attractive features in comparison
with classical solvent extraction methods. However, it has its limitations. Some of the main
limitations of SPE are listed below:
1. Clogging the pores of the solid phase by large biomolecules, oily materials and fine
solids in the sample.
2. Despite, decrease in solvent consumption in SPE in comparison with LLE, SPE needs at
least 100 L of the solvent.
3. It is time consuming method due to several steps of operation including; conditioning,
sample loading and elution
Despite popularity of the SPE, miniaturization of it caused to introduction of solid phase
microextraction (SPME) (Arthur et al. 1990).

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 63

Metal ion Extracted as Solid Phase

Mo(VI), W(VI), Ta(V), Hydrogen Peroxide Cation- exchanger
Nb(V), Ti(IV), V(IV), complexes
V(V)
+2, +3 and +4 cations Bromide complexes Strong-acid cation-exchanger
Al(III), Mo(VI), Nb(V), Fluoride complexes Cation- exchanger
Sn(IV), Ta(V), Ti(IV),
U(VI), W(VI), Zr(IV)
Fe(III) Chloride complexe Haloport-F
Mo(VI) Chloride complexe Granular polymer
impregnated with MIBK
Au(III) Chloride complexe Amberchrome resin
Cd(II), Co(II), Cu(II), pyrrolidin dithiocarbamat C18
Fe(II), Ni(II), Pb(II) complexes
Cu(II), Fe(III), U(VI), Chelate with solid phase Hydroxamic Acid resins
Al(III) (Chelating resin)
Ag(I), Au(III), Bi(III), Chelate with solid phase Thioglycolate resins
Cd(II), Cu(II), Fe(III), (Chelating resin)
Hg(II), Pb(II), Sb(III),
Sn(IV), U(VI), Zn(II)
U(VI) Chelate with solid phase Cellulose phosphate ion
exchanger
(Chelating resin)
Table 1. SPE techniques for metal ion analysis.

2. Microextraction techniques
In order to get rid of limitations of classical sample preparation methods and reach to an
ideal method, miniaturization of extraction techniques is necessary. The first step in
miniaturization is reducing volume of extraction solvent in LLE (Liquid Phase
Microextraction). Different ways of this miniaturization causes various modes of LPME like
single drop microextraction, dispersive liquid-liquid microextraction, hollow fiber based
supported liquid membrane microextraction and liquid phase microextraction based on
solidification of floating organic drop. Miniaturization was also applied to SPE. Product of
this process is known as solid phase microextraction. Review of scientific literature shows
that design; development and applications of microextraction techniques are growing
rapidly. Popularity and applicability of microextraction techniques requires discussing these
subjects in books.

2.1 Solid Phase Microextraction (SPME)

Solid phase microextraction as a solvent free alternative method for conventional sample
preparation methods was introduced (Arthur et. al., 1990). In SPME, a small volume of
extraction phase (usually less than 1 L) coated on fused silica support is mounted in a
modified Hamilton 7000 series syringe. Extraction phase could be a high molecular weight
polymeric liquid or a solid porous sorbent with high surface area. Fig. 1 illustrates the
structure of a SPME device manufactured by Supelco Company. A stainless steel tube was

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64 Atomic Absorption Spectroscopy

replaced with inner wire of syringe needle and fiber was installed on inner tube. With
pulling the syringe plunger in, the fiber is protected in the needle and with pulling out; the
fiber is exposed to the sample. SPME process is carried out in three modes. Headspace mode
which fiber is exposed into the headspace of the sample suitable for volatile analytes, direct
mode which fiber is immersed directly in the sample suitable for nonvolatile analytes and
direct mode with membrane protection suitable for biological or dirty samples.

Fig. 1. SPME device manufactured by Supelco Co. (Mester et. al., 2005)

2.1.1 Theoretical aspects of SPME

SPME is based on distribution of the analytes between the sample and the fiber. Based on
nature of fiber coating, mechanism of microextraction differs. Mechanism of microextraction
for polymeric liquid phases and solid sorbents are partitioning and adsorption, respectively.
Amount of extracted analytes depend on distribution constant (D) of them.

D  Kd
V2
(1)
V1

Fraction of extracted analyte from aqueous sample to the fiber is calculated from Eq. 2

Fex  
D K dV2
1  D K dV 2 V1
(2)

2.1.2 Factors aaffecting SPME

In order to reach equilibrium conditions in SPME, factors such as nature of fiber coating,
time and temperature of extraction, time and temperature of desorption, salt addition,

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 65

sample agitation and solution pH must be optimized. One of the most advantages of SPME
is ability for hyphenation with various analytical instruments. The most capable instrument
with SPME is gas chromatograph. But HPLC, CE and AAS had been coupled with SPME.
According to subject of this book, we will focus on coupling of SPME with AAS. Direct
coupling of SPME with AAS can be carried out using hydride generation apparatus,
including quartz tube equipped with electric heater as flow cell. So due to this system carrier
gas flow is another parameter which needs to investigate.
2.1.2.1 Fiber coating selection
The first step in SPME is selection of appropriate fiber. SPME fiber coatings with different
natures including polar, non-polar and semi polar are available. According to chemical
nature of the analytes, the best fiber must be selected. Polydimethylsiloxane (PDMS) is the
most useful coating for SPME fibers which is commercially available. Now a days, in
addition to commercial fibers, various coating compositions are made at laboratories.
Therefore analysts can select appropriate available fibers or design novel coatings suitable
for their aims.
2.1.2.2 Microextraction temperature
Temperature has a major effect on efficiency of SPME. Increasing the temperature causes an
increase in distribution coefficient of the analytes between the sample and the fiber. In the
case of headspace SPME, it causes increase in distribution coefficients between the sample
and the headspace and between headspace and the fiber. But temperature is a limiting
parameter, because increasing the temperature more than a certain value causes a significant
decrease in distribution coefficient of the analytes between the sample (direct SPME) or
headspace and the fiber and results decrease in amount of extracted analytes.
2.1.2.3 Microextraction time
Exposure time of the fiber in the headspace of samples is usually kept long enough to
achieve equilibrium between the headspace and the adsorbent in order to maximize the
extraction efficiency.
2.1.2.4 Desorption temperature and time
In order to transfer the analytes from the fiber to the analytical instruments like GC and
AAS, thermal desorption is the best method. So desorption temperature and time of fiber
duration in the desorption chamber (injection port and heated quartz tube for GC and AAs,
respectively) must be studied.
2.1.2.5 Sample aagitation
Various modes of agitation could be applied to the sample for faster achieving the
equilibrium. Magnetic stirring and sonication are usual methods for transporting the
analytes from bulk of the sample solution to the surface of the fiber. Power and time of
sonication or rate of magnetic stirrer must be adjusted at different levels for selection of
optimum condition.
2.1.2.6 Salting out effect
Addition of an inorganic salt has often been used in order to enhance the activity coefficients
of volatile components in aqueous solutions, increasing the concentration in the headspace

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66 Atomic Absorption Spectroscopy

vapor. The salts are also added to equalize the activity coefficients of analytes in different
matrices (Zuba et al., 2001). For this purpose, microextraction processes are carried out in
presence of various salt concentrations and also in salt less solution. Results demonstrates
role of salt addition in microextraction of target analytes.

2.1.3 Application of SPME for extraction of metallic analytes before atomic absorption
spectrometric determination
SPME can be coupled with AAS easily via heated quartz tube. Quartz tube flow cells
equipped with electric heater usually used for mercury determination and hydride
generation techniques. Quartz tube AAS (QT-AAS) not only is used for direct coupling of
SPME with AAS but also it can be used as gas chromatographic detector (SPME-GC-QT-
AAS). This technique is mostly reported for determination of organometallic compounds or
derivatized metals as organometallics. A method for determination of organomercury
species based on solid phase microextraction after hydride generation using KBH4 and
determination using GC-QT-AAS was reported. As mentioned above, in the case of mercury
hydrides commercial fibers are not suitable and researchers had to design a novel fiber
based on acid treated fused silica. Suitable capillary column for separating mercury
hydrides is CPL-SIL 5CB (10 m × 0.25 mm) under 40 ˚C isothermal condition (He, et al.,
1998). This method was applied for determination of methylmercury in biological samples
and sediments. Another similar method was reported for determination of methyl, ethyl
and phenyl mercury species in soil samples using above mentioned method (He, et al.,
1999). Recently successful efforts were done for direct coupling of SPME with QT-AAS
(Fragueiro et al., 2004). In this method direct coupling between headspace SPME and QT-
AAS was evaluated for speciation of methylmercury in seafood after volatilization of its
hydride or chloride derivates. The best limit of detection (LOD) for methyl mercury
obtained by using PDMS/DVB fiber coating as 0.06 ng mL-1.

2.2 Single Drop Microextraction (SDME)

The LPME is a miniaturized version of the LLE in which the extraction solvents, volume
reduced to about 1-10 L. Various techniques are known as LPME. Single drop
microextraction (SDME) is one of these methods in which extraction solvent is a single drop.
Jeannot and cantwell was reported the SDME for first time by suspending an 8 L organic
solvent drop at the end of Teflon rod immersed in the stirring sample. After extraction,
solvent drop was removed from the end of the Teflon rod using a micro syringe and injected
to analytical instrument (Jeannot and cantwell, 1996). Fig. 2 shows the schematic SDME
system.
Some modifications were made by He and Lee on primary reported method. In newer
version of SDME, teflon rod was replaced by a micro syringe (Fig. 3). A one L immiscible
extracting solvent drop is exposed into the sample (liquid or gaseous) from a micro syringe.
After establishment of distribution equilibrium the organic drop is retracted
back into the micro syringe and is injected to the analytical instrument for determination
of the analytes (He and Lee, 1997). SDME is suitable for coupling with various instrumental
methods such as gas and liquid chromatography (GC & HPLC), atomic
absorption spectrometry (AAS) and inductively coupled plasma atomic emission
spectrometry (ICP).

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 67

Fig. 2. Illustration of SDME method reported by Jeannot and cantwell (Jeannot et. al., 1996)

Fig. 3. Schematic diagram of SDME using micro syringe (He et. al., 1997)
Some of the advantages of the SDME are:
- SDME is a cheap technique
- SDME needs simple equipment
- Its operation is easy
- Use of minimum amounts of solvents which introduces the technique as a green
approach to sample preparation
- In situ derivatization of the analytes is possible
In addition to above mentioned advantages, instability of the drop, small surface of the drop
and slow kinetics of extraction are disadvantages of the method (Dadfarnia and Haji
Shabani, 2010). Like SPME, SDME can be operated in various modes known as direct
immersed SDME (DI-SDME), headspace SDME (HS-SDME) and three phases SDME.

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68 Atomic Absorption Spectroscopy

2.2.1 Theoretical aspects of SDME

Extraction of the analytes via SDME process is an equilibrium phenomenon. Microextraction
conditions must be optimized for establishment of thermodynamic equilibrium of analyte
partitioning between the sample and extracting phase. In order to better understanding of
microextraction process a model was introduced. This model is based on dynamic mass
balances for the analytes in each phase or both. Mass balance equation for SDME is
presented as equation (3).

C AaqV aq  C Ao V o  C Aaq, oV aq (3)

Where C Aaq and C Ao are the concentrations of the analyte in the sample and the microdrop
respectively; V aq is the sample volume and V o is the microdrop volume, and C Aaq, o is the
initial concentration of the analyte in the aqueous sample. The dynamic mass balance of the
analyte in the microdrop is given by following equation:

 ktot Ai [K AC Aaq  C Ao ]
d(C Ao V o ) o
(4)
dt
Where Ai is the interfacial area (the surface area of the microdrop). K A is the equilibrium
o
partition coefficient and ktot is the total mass transfer coefficient of the analyte with respect
o
to the organic phase. If the two-film theory is considered, ktot is given by:

 o  aqA
1 1 K
o
(5)
ktot k k

Where k o and k aq are the mass transfer coefficients for the analyte in the film of the organic
and the aqueous phases respectively. If we consider the volume of microdrop constant, then
Ai is also constant. So we can obtain Eq. 4 as result of combination of Eq. 1 and 2. Eq. 6
presents C Ao as a function of time.

C Ao (t )  C Ao , aq [1  e   t ] (6)

Where C Ao , aq is the analyte concentration in the microdrop at equilibrium and is the rate
constant.

2.2.2 Factors affecting SDME

In order to reach equilibrium status, all the factors affecting microextraction process must be
optimized. Kind and amount of organic solvent drop, extraction time and temperature,
salting out and agitation rate are important factors.
2.2.2.1 Kind and volume of extraction solvent
In SDME solvent selection is the most important parameter. Selectivity and extraction
efficiency directly depends on solvent’s nature. Solvents with different polarities must be
examined for extraction of studied analytes. Volume of the selected solvent is also
important. Solubility of the analytes and partitioning of them in extraction solvent depends
on volume of solvent microdrop. Increase in solvent volume, increases the amount of

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 69

extracted analytes but after reaching quantitative recoveries, increasing the solvent volume
causes a significant decrease in concentration factor due to dilution of the analytes. On the
other hand, hanging of large volumes of organic solvents made the microdrop unstable. So
in SDME solvent volume is a limited parameter.
2.2.2.2 Extraction time
Exposure time of the microdrop to the samples is an important parameter in achieving
distribution equilibrium of analytes between solvent drop and sample; it is a decisive factor
for improving the extraction efficiency. So it is necessary to be optimized. But according to
difficulty of microdrop expose to the sample in this technique, long extraction times are not
preferred.
2.2.2.3 Extraction temperature
As mentioned before, SDME process is a thermodynamic equilibrium, so effect of
temperature is not negligible. But extraction temperature in SDME, strongly limited by
solvent’s boiling point.
2.2.2.4 Salt addition
Addition of salt to the sample increases the ionic strength of the solution. Depending on the
solubility of the target analytes, extraction is usually enhanced with increased salt
concentration (salting out effect). But in the case of SDME, presence of salt changes the
physical properties of the extraction film and reduces the diffusion rates of the analytes into
the solvent drop. So, salt addition has a negative effect on method efficiency.
2.2.2.5 pH Adjustment
Effect of pH on extraction efficiency, depends on analyte nature. Extraction of analytes with
weak acidity and basicity is strongly pH sensitive. Solution pH easily adjusted by
appropriate buffers according to the pKa values of the analytes.
2.2.2.6 Sample agitation
Similar to the other extraction methods, sample agitation has a significant effect on
enhancement of extraction yield. Increase in agitation rate, decreases the extraction time due
to faster establishment of the distribution equilibrium. But despite positive effect of higher
agitation rates, it is so critical parameter in SDME because instability of microdrop on
syringe tip.

2.2.3 Application of the SDME for extraction of metal ions before atomic absorption
spectrometric determination
Due to low volumes of extraction solvents in SDME, electrothermal atomic absorption
spectrometry is suitable for determination of metals after extraction with SDME
technique. The first coupling of SDME with ET-AAS was reported for extraction of As in
aqeous samples. Total arsenic species were converted to As (III) using NaBH4 and
extracted by a 4 L organic drop consisting pyridine and benzyl alcohol containig silver
diethyldithiocarbamate (AgDDC) as complexing agent with arsine (Chamsaz et al., 2003).
Palladium is a good sorbent for arsenic and act as matrix modifier in ET-AAS. So a green
headspace SDME method for extraction of As (III) and total As was presentd using a 3 L
aqeous drop of Pd (30 mg L-1) as extraction solvent prior to determination by ET-AAS

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70 Atomic Absorption Spectroscopy

(Fragueiro et al, 2004). This method is faster than peviously reported method by Chamsaz
et al, but both of the concentration factor and detection limit of previouse method is
better.
Another green method using aqeous micro drop of Pd (II) and Pt (IV) was reported for
determination methylmercury in fish samples. Volatilization of methylmercury was
performed using hydride generation reaction and after extraction analysis was done by ET-
AAS (Gil et al., 2005). Microextraction of Se using SDME prior to ET-AAS analysis was
reported by photogeneration of volatile hydride and alkyl Selenium derinatives. Aqeous
microdrop containing Pd (II) was used as extracting phase (Figueroa et al., 2005). A series of
SDME methods were reported for lead determination in various samples by ET-AAS. A
microdrop of benzene containing 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) was
used for extraction of Pb (II) from biological samples and its determination by ET-AAS
(Liang et al., 2008). Dithizone is another extractant in SDME which was used for
microextraction of lead from water samples prior to assay by ET-AAS (Liu and Fan, 2007).
Application of ionic liquids as novel extractants in SDME was reported for determination of
manganese and lead. Complex of manganese with 1-(2-thiazolylazo)-2-naphtol (TAN) was
extracted into a microdrop of [C4MIM][PF6] (Manzoori et al., 2009). The same group
extracted lead ions after complexation with ammonium pyrrolidine
dithiocarbamate (APDC) into the same ionic liquid (Manzoori et al., 2009). Simulteneous
direct SDME of Cd and Pb after complexation with dithizone in aqeous samples into toluene
microdrop followed by determination with ET-AAS was reported (Jiang and Hu et al.,
2008). Ag, Tl, Cr and Sb are another metals which the SDME-ET-AAS method was reported
for them.

2.3 Dispersive liquid-liquid microextraction (DLLME)

Dispersive liquid-liquid microextraction (DLLME) is a newer mode of LPME (Rezaee, et al.,
2006). DLLME is a ternary solvent system consisting of aqeous sample solution, extraction
solvent and disperser solvent. Extraction solvent must be immisible with aqeous sample
solution and disperser solvent must solouble in both of the extraction solvent and aqeous
sample solution. In DLLME, 5-10 mL of sample solution is placed in a test tube with conical
bottom, then optimized volumes of extraction solvent ( L) and disperser solvent (mL) are
mixed and mixture of these solvents is injected rapidly in the sample solution. After
injection of solvents mixture a stable cloudy suspension consisting of fine droplets of
extraction solvent is appered which can be easily separated by centrifugation (Fig. 4).
Aqeous phase is discarded and sedimented organic phase is transfered to an analytical
instrument using a micro syringe. Early works on DLLME were based on denser solvents
than water (mostly chlorinated solvents) but later DLLME methods based on lighter
extraction solvents than water were reported (Farajzadeh, et al., 2009).
Some of the advantages of DLLME are listed below:
- Simple, fast and cheap
- High preconcentration factor
- High recovery of the analytes
- Small volumes of the sample are needed
- Minimum volumes of the organic solvents ( L) are used
- Easy coupling with most of the instrumental methods

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 71

Fig. 4. Shematic illustration of a DLLME process: a) prior to injection of extraction and

disperser solvents b) starting the injection c) end of injection, cloudy suspension was made
d) fine droplets of solvents in solution bulk e) after centrifugation f) conical bottom of the
test tube with sedimented phase (Rezaee et. al., 2006)

2.3.1 Theoritical aspects of DLLME

Inorder to design an accurate DLLME process, some terms must be identified:
Preconcentration or enrichment factor is the ratio of the analyte concentration in sedimented
phase (C sed ) to analyte concentration in the sample (C 0 )

EF 
C sed
(7)
C0

Extraction recovery of the analyte (%) is the percentage of extracted analyte in sedimented
phase, where n0 is the amount of analyte in the sample prior to extraction and nsed amount
of analyte in sedimented phase.

C  Vsed
ER   100  sed  100
nsed
C 0  V0
(8)
n0

ER  ( )EF  100
Vsed (9)
Vaq

2.3.2 Factors affecting DLLME

Like every other methods several experimental factors affect DLLME process. For
development of a sensitive and accurate DLLME method these parameters including Kind
and volume of extraction solvent, kind and volume of disperser solvent, extraction
temperature, solution pH and salting out must be optimized.

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72 Atomic Absorption Spectroscopy

2.3.2.1 Kind and volume of extraction solvent

Its clear that extraction solvent is the most important parameter in a LPME method. As we
disscussed above, extraction solvent must be immicible with water and must be a good
solvent for the analytes. Soloubility of the extraction solvent in water has an inverse relation
with stability of cloudy suspension. Volume of extraction solvent is important too, because it
is obvious that with increasing extraction solvent volume, amount of extracted analytes
increase. But when we reach to quantitative recoveries, inrease of extraction solvent volume
causes a significant decrease in an anlytical signal due to dillution and reducing enrichment
factor. Extraction solvent can be a denser or lighter solvent than water. Usuall denser
solvents are tetrachloroethylene, trichloroethylene, carbon tetrachloride and etc.
2.3.2.2 Kind and volume of disperser solvent
Disperser solvents usually are polar solvents such as aceton, acetonitril and methanol.
Disperser solvent acts as a bridge between two immicible phases. The role of an ideal
disperser solvent is making more fine droplets of extraction solvent and dispesion of
extraction phase in sample bulk. Amount of disperser solvent is also important. Increasing
in disperser solvent volume, increases the volume of sedimented phase and naturally lowers
the analytical signal due to dillution. So, optimization of disperser solvent volume is as
important as extraction solvent volume.
2.3.2.3 Extraction temperature and time
Temperature is an important parameter in all of the equilibrium systems. But in DLLME
temperature is a critical parameter. Because boiling point of the extraction and disperser
solvents are limited the process. Extraction time is contact time between the sample and
extraction solvent. It is clear that extraction time directy depends on extraction efficiency.
But in the case of DLLME, time is not so important parameter due to high surface contact
between the sample and fine droplets of extraction solvent. So in most of the reports, time
has no effect on extraction efficiency.
2.3.2.4 Salting out
Despite other extraction methods, in DLLME salting out has dual effect. Similar to other
extraction techniques addition of an inorganic salt, causes an increase in amount of
extracted analytes. But in DLLME salt addition causes a significant increase in volume of
sedimented phase. So effect of salt addition on extraction efficiency are controlled by
these two factors.
2.3.2.5 Complexation of metal ions prior to extraction
Similar to SDME, extraction of metal ions with organic solvent needs to convert them to
suitable form. Appropriate chealating agents must be selected and complexation
conditions such as solution pH, concentration of ligand and etc, must be adjusted. It is
clear that extraction of metal ions after conversion of them to organometallic compounds
is possible.

2.3.3 Application of DLLME for extraction of metal ions before determination by AAS
A simple and powerful microextraction technique was used for determination of selenium
in water samples using dispersive liquid–liquid microextraction (DLLME) followed by ET-
AAS (Bidari et al., 2007). In this study, complex of Se and APDC extracted by a mixture of

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 73

ethanol (disperser solvent) and carbon tetrachloride (extraction solvent) from water
samples. The concentration of enriched analyte in the sedimented phase was determined by
iridium-modified pyrolitic tube graphite furnace atomic absorption spectrometry.
Determination of trace levels of lead is possible with DLLME followed by ET-AAS (Liang
and sang, 2008). In the proposed approach, 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone
(PMBP) was used as a chelating agent, and carbon tetrachloride and ethanol were selected
as extraction and dispersive solvents. Another simple DLLME-Flame AAS method was
reported for determination Pd(II) as complex with thioridazine HCl (Ahmadzadeh Kokya
and Farhadi, 2009). Ethanol as disperser solvent and chloroform as extraction solvent were
used in this study.
Extraction of Co(II) as its complex with Br-TAO via a DLLME method was reported (Baliza
et al., 2009). The procedure is based on a ternary system of solvents, where appropriate
amounts of the extraction solvent, disperser solvent and the chelating agent Br-TAO are
directly injected into an aqueous solution containing Co(II). A cloudy mixture is formed and
the ions are extracted in the fine droplets of the extraction solvent. After extraction, the
phase separation is performed with a rapid centrifugation, and cobalt is determined in the
enriched phase by FAAS.
Use of ionic liquids is a novel development in DLLME. A new ionic liquid based DLLME
method was developed for preconcentration and determination of Pb (II) and Cd (II) in
aqueous samples containing very high salt concentrations (Yousefi and Shemirani, 2010).
This is believed to arise from dissolving of the ionic liquids in aqueous samples with high
salt content. In this method, the robustness of microextraction system against high salt
concentration (up to 40%, w/v) is increased by introducing a common ion of the ionic liquid
into the sample solution. The proposed method was applied satisfactorily to the
preconcentration of lead and cadmium in saline samples. After preconcentration, the settled
IL-phase was dissolved in 100 L ethanol and introduced to Flame-AAS.

2.4 Liquid phase microextraction based on solidification of floating organic drop

(LPME-SFO)
Liquid phase microextraction based on solidification of floating organic drop (LPME-SFO) is
one the newest versions of LPME (Khalili-Zanjani et al., 2007) in which the extraction
solvent with lower density than water, low toxicity and proper melting point near room
temperature (in the range of 10–30 ˚C) was used. In this method, small volume of an
extraction solvent is floated on the surface of aqueous solution. The aqueous sample
solution is agitated for an optimized time. After the extraction, tube containing the sample is
transferred in the ice bath and the floated extractant droplet solidified at low temperature.
The solidified organic solvent can be separated from the sample and melted quickly at
room temperature, which is then determined by appropriate analytical methods (Fig. 5).
In comparison with other methods, LPME-SFO has some advantages which is listed below:
- Simplicity of operation
- Small amount of low toxic solvent used
- Good repeatability
- Low cost
- High preconcentration factors
- More suitability for the analysis of complex matrix samples.

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74 Atomic Absorption Spectroscopy

LPME-SFO is only applicable for the analytes with high or moderate lipophilic property and
can not be used to those neutral analytes with high hydrophilic property and this is the
main disadvantages of the method.

Fig. 5.1 Floated organic solvent, 2) Wter bath, 3) Sample, 4) Stirring bar, 5) Heater-Stirrer, 6)
Conical vial, 7) Spatula (Ying-Ying et. al., 2010)

2.4.1 Thepritical aspects of LPME-SFO

LPME-SFO is based on the distribution of the analytes between floated extraction solvent
and the aqueous sample matrix. As mentioned previously, the organic extraction solvent of
LPME-SFO must have the melting point near room temperature (in the range of 10–30 ˚C)
and lower density than water. It should be readily solidified at low temperatures, and thus
its droplet can be collected easily. Maximum sensitivity and precision were obtained by
stirring the sample solution until the equilibrium was obtained.
Distribution coefficient (K) is defined as the ratio between the analyte concentration in
extraction solvent and sample solution. The enrichment factor (ER) and extraction recovery
(ER) are calculated as follows:

EF 
C o ,F
(10)
C aq

Where C o ,F and C aq are the analyte concentration in the organic solvent, and the initial
concentration of the analyte in the aqueous sample, respectively.

ER(%)  100 
C o ,FVo ,F
(11)
C aqVaq

Where Vo ,F and Vaq are the volume of the organic phase and the volume of the aqueous
sample, respectively.

2.4.2 Factors affecting LPME-SFO

The partitioning of the analytes between two phases (extraction solvent and the aqueous
solution) was affected by various parameters, such as the type and volume of the organic
solvent, aqueous sample volumes, the extraction time, the stirring rate, and salt addition. In
order to design an accurate method, every parameter must be optimized.

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 75

2.4.2.1 Selection of extraction solvent

The selection of an appropriate extraction solvent is of major importance for the
optimization of the LPME-SFO process. The selected extraction solvent must satisfy several
requirements. First, it should be immiscible with water, have low volatility, low density, low
melting point near room temperature and be able to extract the desired analytes. According
to these considerations, limited number of the organic extraction solvents commonly used in
LPME-SFO which are listed below. 1-Undecanol (13-15 ˚C), 1-Dodecanol (22-24 ˚C), 2-
Dodecanol (17-18 ˚C), 1-Bromo hexadecane (17-18 ˚C), n-Hexadecane (18 ˚C) and 1,10-
Dichlorodecane (14-16 ˚C).
2.4.2.2 Effect of extraction temperature
Generally, increasing sample solution temperature has a positive effect on extraction
efficiency. Based on the extraction kinetics, higher temperatures would facilitate the
diffusion and mass transfer of the analytes from sample solution to the organic solvent, and
the time required to reach the equilibrium would be decreased. However, at high
temperatures, the over-pressurization of the sample vial could make the extraction system
unstable. On the other hand in LPME boiling point of the solvents is a limiting factor.
2.4.2.3 Effect of organic solvent volume
Similar to other LPME methods, increasing organic solvent volume increases extraction
recovery. But after reaching quantitative recoveries, increasing solvent volume causes a
significant decrease in enrichment factor. Usually in LPME-SOF, 5–100 L of extraction
solvent is selected.
2.4.2.4 Effect of stirring rate
For the SFO-LLME, sample agitation is an important parameter that influences the
extraction efficiency. According to the film theory of convective-diffusive mass transfer for
LPME system, high stirring speed can decrease the thickness of the diffusion film in the
aqueous phase, so the aqueous phase mass-transfer coefficient will be increased with
increased stirring speed (rpm). But incresing stirring rate must be controlled, because it may
be cause to sputtering of the solvent drops and influence the extraction efficiency.
2.4.2.5 Effect of extraction time
LPME is not an exhaustive extraction, so extraction time has great effect on extraction
efficiency, precision, sensitivity, and the repeatability of the LPME-SFO. It is necessary to
choose an appropriate extraction time to guarantee the equilibrium between aqueous and
organic phases and the maximum extraction of analytes.
2.4.2.6 Salting out
As described before, salt addition can improve the extraction efficiency of the analytes due
to salting out effect. But higher salt concentration can be affect physical properties of the
extraction film. This causes reducing in the diffusion rates of the analytes into the organic
phase. Therefore, the amount of salt should be optimized in LPME-SFO.

2.4.3 Application of LPME-SFO for extraction of metal ions before determination by

AAS
LPME-SFO can be used for extraction of metal ions from aqeous solution in combination
with ET-AAS. A LPME-SFO technique was used for the extraction of lead in water samples
(Dadfarnia et al., 2008) 1-undecanol containing dithizone as the chelating agent was used as

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76 Atomic Absorption Spectroscopy

floated organic drop for extraction of lead ions. After stirring the sample for a certain time,
the sample vial was cooled in an ice bath for 5 min. The solidified extract was transferred
into a conical vial where it melted immediately, and then 10 L of it was analyzed by ET-
AAS. Another LPME-SFO method was designed for extraction of Co(II) and Ni (II) as
complexes with 1-(2-Pyridylazo)-2-naphthol (PAN) as chelating agent (Bidabadi et al., 2009).
A highly efficient LPME-SFO method for the determination of arsenic by electrothermal
atomic absorption spectrometry (ETAAS) was reported (Ghambarian et al., 2010). In this
method extraction of As(III) as its complex with APDC was carried out in ppb level.
Another literature reported the same method for the determination of trace lead and
cadmium in water samples (Rivas et al., 2010).
When LPME-SFO is coupled with flame atomic absorption spectrometry (FAAS), direct
injection analysis cannot be performed, since the volume of the extraction solvent is too little
for Flame-AAS. For this purpose a LPME-SFO method was proposed for extraction of
cadmium ions in different water samples for determination by Flame-AAS (Dadfarnia et al.,
2009). In the method, the extraction solution was first diluted with ethanol to 250 L and then
100 L of it was analyzed by flow injection flame atomic absorption spectrometry (FI-FAAS).

2.5 Hollow fibre based liquid phase microextraction

An alternative concept for LPME was developed using hollow fiber membranes (Pedersen-
Bjergaard, et al., 1999). This technique is based on the use of hollow fibers, typically made of
polypropylene. This form of LPME consists of a donor phase (the sample), an acceptor
phase (in the lumen of the hollow fiber) and the hollow fiber between them. Pores of the
hollow fiber membrane are impregnated with organic solvent and the system is called as
supporting liquid membrane. This configuration can be used in two modes; two phase and
three phase. In two phase system donor phase is aqueous; acceptor phase is organic with
oraganic solvent in hollow fiber pores. But in three phase system, donor phase and acceptor
phase are both aqueous with hollow fiber impregnated with organic solvent (Fig. 6)

Fig. 6. Illustration of Hollow fiber based LPME (i) two phase (ii) three phase system
(Psillakis et. al., 2003)

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 77

2.5.1 Theoretical aspects of hollow fiber based LPME

Two phase extraction mode: Extraction through hollow fiber membranes by passive
diffusion of analytes from donor phase (the sample) into acceptor phase (extractant) is based
on a partitioning equilibrium. Partition coefficient is presented by:

K a /d  C eq , a / C eq , d (12)

Where C eq , a and C eq , d are analyte concentration at equilibrium in acceptor and donor

phases, respectively. Extraction recovery and enrichment factor are given by:

R  (100K a /dVa ) /( K a /dVa  Vd ) (13)

E  (Vd R ) /(100Va ) (14)

Where Va and Vd are volume of acceptor and donor phase solutions.

Three phase extraction mode: Similar to two phase mode, this system is based on passive
diffusion of analytes through the membrane, too. But two partitioning equilibrium system
must be established. The first equilibrium between donor phase and organic phase
(impregnated in membrane pores) and second one between organic phase and donor phase.

K org /d  C eq , org / C eq , d (15)

K a /org  C eq , a / C eq , org (16)

K a /d  C eq , a / C eq , d  K org /d .K a /org (17)

Where C eq , org is the analyte concentration in organic phase at equilibrium also K a /d as

partition coefficient of the analytes between acceptor and donor phases is the main force for
performing the microextraction. Extraction recovery (R) in three phase system can be given as:

R  (100K a /d .Va )(K a /d .Va  K org /d .Vorg  Vd ) (18)

2.5.2 Factors affecting hollow fiber based LPME

Several parameters affects hollow fiber based LPME. Kind of hollow fiber, nature of organic
phase, volume of donor, acceptor and organic phases, pH of donor and acceptor phases, salt
addition and sample agitation must be optimized.
2.5.2.1 Kind of hollow fiber
The hollow fibers used in LPME should be compatible with the organic solvent.
Polypropylene hollow fibers are the most popular fibers for this purpose. Common
dimensions of employed hollow fibers are as listed below. Inner diameter 600 m, wall
thickness 200 m, nominal and maximum pore sizes are 0.2 and 0.64 m, respectively.
Length of the hollow fiber is based on optimum volume of acceptor phase according to
volume of fibers lumen.
2.5.2.2 Organic solvent
An important step in method development for different modes of hollow fiber based LPME
is organic solvent selection. Water solubility and polarity are the main characteristics of the

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78 Atomic Absorption Spectroscopy

organic solvent which must be considered. To prevent dissolution of it in aqueous phase, it

should have minimum solubility. Low volatility is also another important characteristic of
the selected organic solvent which will restrict solvent evaporation during extraction.
Solubility of the analytes in organic solvents and high partition coefficients of the analytes
between two phases are other principle considerations in solvent selection.
2.5.2.3 Agitation of the sample
In order to improve extraction kinetics, use of factors increasing convection of the analytes
from solution bulk into the extracting phase is usual. In this method sample and extracting
phase are not in direct contact with each other and sample agitation facilitates transfer of the
analytes through the supported liquid membrane. Stirring, vibration and sonication are
popular agitation techniques. Suitability of the selected agitation method must be
considered. Rate and power of agitation must be optimized to prevent air bubble formation
around the membrane or accelerate solvent evaporation which causes repeatability and
imprecision problems in analysis.
2.5.2.4 Salt addition
In both of the two phase and the three phase LPME methods, salting out effect must be
investigated by adding optimized amounts of selected salt to donor phase. Positive effect of
salt addition on extraction efficiency depends on nature of the analyte and ionic strength of
the sample solution.
2.5.2.5 Volumes of donor and acceptor solutions
It’s clear that, the main aim of all preconcentration techniques is reaching to higher
enrichment factors. This is available with increasing the ratio of donor phase volume to
acceptor phase volume. In this case kind of analytical instrument which LPME is coupled is
important. Because various volumes are required for analyte introduction. In the case of the
atomic absorption spectrometry (AAS) it depends on the mode of the instrument. For
Flame-AAS appropriate minimized volume of the analyte solution is about one mL which is
large enough in comparison to the volume of membrane lumen. But electrothermal-AAS is
more compatible with this technique due to lower volumes ( L scale) of the samples need to
be introduce to graphite furnace.
2.5.2.6 Adjustment of pH
pH of donor and acceptor phases must be adjusted according to the nature of the analytes.
In both of the two phase and three phase modes of this technique extraction efficiency has
great dependence to solution pH. If the analytes are dissociable in different pHs (weak acids
and bases) effect of pH on extraction efficiency must be investigated.
2.5.2.7 Extraction time
In all of the extraction methods, extraction time is an important parameter. Because sample
preparation is rate determining step of an analytical method. Mass-transfer of the analytes
between two phases is a time consuming process. As discussed before sample agitation is
one of the methods which is applied for faster extraction and lower extraction times.

2.5.3 Application of the hollow fiber based LPME for extraction of the metal ions
before atomic absorption spectrometric determination
One decade after introduction of hollow fiber based LPME for enrichment of the organic
compounds, the first application of this technique for extraction of inorganic compounds

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 79

was reported (Xia, et al., 2006). In this study extraction of Se (IV) and Se (VI) from water
samples before determination by ICP-MS was investigated. The first report on use of hollow
fiber based LPME before AAS is about determination of Cd (II) in sea water using
electrothermal AAS (Peng, et al., 2007). In this three phase extraction system dithizone/oleic
acid in 1-octanol – HNO3 was used as extraction phase 0.8 ng L-1 and 387 are LOD and
preconcentration factor of the method, respectively. Other two phase and three phase
methods for extraction of organomercury and As before electrothermal AAS were reported
and summarized in a review article (Dadfarnia, et al., 2010).

2.6 Cloud point extractin (CPE)

Cloud point extraction (CPE), known also as phase separation extraction and surfactant (or
micelle)-mediated phase separation is based on the phase behavior of non-ionic surfactants
in aqueous solutions, which exhibit phase separation after an increase in temperature or the
addition of a salting-out agent. Separation and preconcentration based on cloud point
extraction (CPE) are becoming an important and practical application of surfactants in
analytical chemistry. The technique is based on the property of most nonionic surfactants in
aqueous solutions to form micelles and to separate into a surfactant-rich phase of a small
volume and a diluted aqueous phase when heated to a temperature known as the cloud
point temperature.
The small volume of the surfactant-rich phase obtained with this methodology permits the
design of extraction schemes that are simple, cheap, highly efficient, fast and environmental
friendly in comparison with classical methods. CPE might be an interesting and efficient
alternative, once it eliminates or reduces consumption of organic solvents significantly.
Trace elements can be extracted to the surfactant-rich phase usually after formation of a
hydrophobic complex with an appropriate chelating agent (ghaedi et al., 2009).

2.6.1 Surfactants and micelles

Surfactants are compounds that lower the surface tension of a liquid, the interfacial tension
between two liquids or between a liquid and a solid. The term surfactant is a blend of
surface active agent (Rosen, 2010). Surfactants are amphiphilic molecules, one of whose
parts (the head) is polar or hydrophilic in nature and the other (the tail) hydrophobic (Fig.
A). This latter part is generally a hydrocarbon chain with different numbers of carbon atoms
and may be linear or branched. It may also contain aromatic rings. Therefore, a surfactant
molecule contains both a water insoluble (oil soluble component) and a water soluble
component. Surfactant molecules migrate to the water surface, where the insoluble
hydrophobic groups may extend out of the bulk water phase, either into the air (Fig. B) or if
water is mixed with an oil, into the oil phase, while the water soluble head group remains in
the water phase. Surfactants in dilute aqueous solutions arrange on the surface. With
increasing the concentration of the surfactant the solution surface becomes completely
loaded with surfactant and any further additions must arrange as micelles (Fig. C).
Therefore, when the surfactant concentration is increased above a certain threshold, called
the critical micellar concentration (CMC), the surfactant molecules become dynamically
associated to form molecular aggregates of colloidal size. These aggregates containing 60 to
100 monomers are at equilibrium with surfactant molecules in solution with concentration
near to CMC.

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80 Atomic Absorption Spectroscopy

Fig. 7. Schematic representation of the surfactant and formation of a micelle from its
monomers beyond its critical micellar concentration (CMC).

2.6.2 Phase separation in CPE

When a micellar solution of a non-ionic surfactant is heated, it becomes turbid over a
narrow temperature range, which is referred to as its cloud-point temperature (Hinze &
Pramauro, 1993). Above the cloud-point temperature, the system initially in an isotropic
phase is separated into two isotropic phases, one of them surfactant-rich phase which is
separated from the bulk aqueous solution; and the other aqueous phase, in which the
surfactant concentration will be approximately equal to the critical micelle concentration.
The phenomenon is reversible and upon cooling, a single phase is again obtained. The
mechanism by which separation occurs is poorly understood but some authors have
explained the cloud point phenomenon on the basis of the dehydration process that occurs
in the external layer of the micelles of non-ionic surfactants when temperature is increased
(Hinze, 1987).

2.6.3 Experimental procedure of CPE in metal analysis

The extraction process of CPE for metal ions is very simple and is shown in Fig. 7. First, a
few ml of the surfactant or a concentrated surfactant solution is added to some tens to
hundreds of millilitres of an aqueous sample containing the metal ions. The final
surfactant concentration must exceed its CMC in order to ensure formation of micelle
aggregates. The chelating agent is added, where necessary, along with the surfactant,
dissolved in an organic solvent or directly to the water, depending on its solubility. Next,
the solution is heated above the cloud point and separation of the phases usually takes
place after centrifugation. Any analyte solubilized in the hydrophobic core of the micelles,
will separate and become concentrated in the small volume of the surfactant-rich phase.
After cooling in an ice bath, the surfactant-rich phase became viscous and retain at the
bottom of the tube. The supernatant aqueous phases can readily be discarded by inverting
the tube.
Finally, a volume (microliter) of nitric acid in aqueous or organic solvent can be added to the
surfactant-rich phase to reduce its viscosity and to facilitate sample handling prior to AAS
assay.

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 81

Fig. 8. Experimental schemes for cloud point extraction of metal ions prior to atomic
absorption spectrometry.

2.6.4 Calculations
The CPE of metal ion from micellar solutions was evaluated in terms of extraction recovery
(ER), distribution coefficient (D), selectivity SX /A and the concentration factor CF that are
defined as follows:
The distribution coefficient is a parameter used to describe the degree of analyte partitioning
from the aqueous to the surfactant-rich phase, is given by

D
[ A]S
(19)
[ A]W

where [ A]S and [ A]W are the final analyte concentrations in the surfactant-rich phase and in
the aqueous phase, respectively. [ A]W can be calculated from the mass balance equation:

[ A]O VO  [ A]W VW  [ A]S VS (20)

where [ A]O refers to the analyte concentration in the original aqueous solution prior to the
extraction step, and VO is the volume of original aqueous solution. VW and VS are the
volumes of aqueous solution and surfactant rich phase obtained after the extraction step,
respectively.
The extraction recovery (percent of analyte extracted) can be expressed as:

[ A]O  [ A]W
ER   100 (21)
[ A]O

Selectivity of analyte in the CPE process is described as:

SX /A 
DX
(22)
DA

Where DX and DA are distribution coefficient of foreign ion and analyte, respectively.
Finally the concentration factor is given by:

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82 Atomic Absorption Spectroscopy

[ A]O VO  [ A]W VW
CF   
mS [ A]S VS
(23)
mW [ A]W VW [ A]W VW

Where mS and mW are total mass of analyte in the surfactant rich phase and aqeous phase,
respectively.

2.6.5 Factors affecting the CPE efficiency

In CPE, extraction needs to be carried out under optimal conditions in order to maximize
the preconcentration factor and extraction recovery. It is well known that the
extraction/preconcentration process can be altered by pH, complexing agent, the types and
concentration of surfactant, ionic strength (additive), equilibration temperature and
incubation time (Quina & Hinze, 1999).
2.6.5.1 Effect of solution pH
The separation of metal ions by CPE involves prior formation of a complex with sufficient
hydrophobicity to be extracted into the small volume of surfactant-rich phase. The
formation of metal-ligand comples and its chemical stability are two important factors for
CPE. The pH, which plays a unique role on formation of the complex and subsequent
extraction, is proved to be a main parameter for CPE. Extraction yield depends on the pH at
which complex formation is carried out (Biparva & Hadjmohammadi, 2007).
2.6.5.2 Effect of the complexing agent
Generally, CPE of a metal ion is taken place via a complex formation of the analyte with a
lipophilic ligand. These complexes interact with the micellar aggregate and can be extracted
from the aqueous solution into the surfactant-rich phase. The selectivity and efficiency of the
method depend directly on the hydrophobicity of the ligand and the complex formed, the
apparent equilibrium constants in the micellar medium, the kinetics of the complex
formation, and the transfere of the complex between the phases (Constantine, 2002;
Carabias-Martinez, et al., 2000).
2.6.5.3 Effect of surfactant type and concentration
Depending on the nature of the hydrophilic group, surfactants are classified as non-ionic,
zwitterionic, cationic, and anionic. Up to now, non-ionic, zwitterionic and anionic
surfactants are most widely used for CPE of metal ions. However, it is very important to
select an appropriate surfactant for a successful CPE analysis since it can directly affect the
extraction and preconcentration, and accuracy of the final analytical results. Reports showed
that Triton X-114 and PONPE-7.5 (cloud point temperature, near room temperature)
(Paleologos et al., 2000, 2001) are proper surfactants to perform CPE for trace elements
because of its commercial availability in a high purified homogeneous form, low toxicity
and cost. Also, low cloud point temperature (23–26 °C) and high density of the surfactant-
rich phase facilitates phase separation by centrifugation. A successful cloud point extraction
V
should maximize the extraction efficiency by minimizing the phase volume ratio ( S ) . This
VW
shows that the smaller surfactant concentration ptovides higher preconcentration factor; but
when the volume of surfactant-rich phase is small, the extraction process becomes more
difficult and the accuracy and reproducibility probably suffer (Eiguren Fernandez et al.,
1999; Moreno Cordero et al., 1993). However, since the volume of the surfactant- rich phase

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Microextraction Techniques as a Sample Preparation Step for Metal Analysis 83

must be manageable, a compromise must be reached so that the surfactant concentration

will allow a high phase ratio and a manageable surfactant-rich phase.
2.6.5.4 Effect of Ionic strength
The cloud point of micellar solutions can be controlled by addition of salts, alcohols, non-
ionic surfactants and some organic compounds (salting-out effects). To date, most of the
studies conducted have shown that ionic strength has no appreciable effect on the extraction
efficiency. An increase in the ionic strength in the CPE does not seriously alter the efficiency
of extraction of the chemical forms. Moreover, the addition of a salt can markedly facilitate
the phase separation process. As demonstrated with some non-ionic surfactant systems, it
alters the density of the bulk aqueous phase.
2.6.5.5 Effects of equilibration temperature and incubation time
Optimal incubation time and equilibration temperature are necessary to complete the
reaction, and to achieve easy phase separation and preconcentration as efficient as possible.
The greatest analyte preconcentration factors are thus expected under conditions where the
CPE is conducted using equilibration temperature that are well above the cloud point
temperature of the surfactant. It was desirable to employ the shortest equilibration time and
the lowest possible equilibration temperature, which compromise completion of the reaction
and efficient separation of phases.

2.6.6 Applications of CPE in atomic absorption spectrometry

The use of CPE coupled with atomic absorption spectrometry (AAS) offers a conventional
alternative to more traditional extraction systems and permits the design of extraction
schemes that are simple, cheap, of high efficiency, reducing in extraction time and
environmentally clean methodology due to low consumption of a solvent, apart from the
results that comparable to those obtained with other separation procedures. CPE technique
has been successfully employed for the preconcentration of micro amounts of several metals
in different matrices, as prior step before their determinations by Atomic Absorption
Spectrometry.
Watanabe et al. used this unconventional liquid–liquid separation to extract metal ions for
the first time (Watanabe & Tanaka, 1978). They extracted Ni using Triton X-100 but this
surfactant has a relatively high cloud point, around 70 ˚C. Later, to extract Zn, they used
PONPE 7.5, which requires a lower temperature (around 5 ˚C) for the phase separation. This
surfactant and Triton X-114 which has a very convenient cloud point were used in other
studies of metal ion extraction. Cu (Kulichenko et al., 2003), Mn ( Doroschuk et al., 2004), Co
(Nascentes & Arruda, 2003), Cd and Ni (Manzoori & Karim-Nezhad, 2004), Ag and Au
(Mesguita da Silva et al., 1998) were determined by FAAS. CPE was also applied for
extraction of Fe(III) (Ohashi et al., 2005) and As(III) and As(V) (Shemirani et al., 2005) before
determination by ET-AAS. Cd, Cu, Pb, and Zn (Chen & Teo, 2001) were simultaneous
extracted as complex with 1-(2-thiazolylazo)-2-naphthol (TAN) using TritonX-114 prior to
determination by FAAS. Several ligands such as 1-(2-pyridylaso)-2-naphthol (PAN), 2-(2-
thiazoylazo)-4-methylphenol (TAC), dialkyldithiophosphates (DDTP), 4-(2-pyridylazo)
resorcinol (PAR), 2-(5-bromo-2-pyridylazo)-5-diethilaminophenol (Br-PADAP) have been
used in CPE of metal ions.

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84 Atomic Absorption Spectroscopy

3. Conclusion
Analysis of metallic analytes is so important in environmental, biological and food samples.
Due to complex matrix of real samples and trace concentration of them use of sample
preparation methods is necessary. Development of efficient sample preparation methods in
agreement with green chemistry is one of the most exciting research fields for analytical
chemists. Growing number of scientific publishing in this area shows emerging needs for
newer methods with higher concentration factors, higher recoveries, more cheap and simple
and environmental friendly methods. All of the techniques discussed in this chapter have
some advantages and some limitations. An analyst must be able to choose the best method
according to his problem. Kind of analyte and sample, complexity of the sample, range of
concentration which analyte exist in the sample are some of the parameters which must be
considerd.

4. Acknowledgment
Financial supports of Iranian Academic Center for Education, Culture and Research
(ACECR) from our research projects gratefully acknowledged. Also the authors express
their deep sense of gratitude to INTECH publishing company for their efforts in order to
development of science and technology.

5. References
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Microextraction for the Selective Determination of Trace Amounts of Palladium by
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Arthur, C. L. & Pawliszyn, J. (1990). Solid Phase Microextraction with Thermal Desorption.
Analytical Chemistry, Vol 62, pp. 2145-2148.
Baliza, P. X., Teixeira, L. S. G. & Lemos, V. A. (2009). A Procedure for Determination of
Cobalt in Water Samples after Dispersive liquid–liquid Microextraction.
Microchemical Journal, Vol 93, pp. 220-224.
Bidabadi, M. S., Dadfarnia, S. & Shabani, A. M. H. (2009). Solidified Floating Organic Drop
Microextraction (SFODME) for Simultaneous Separation/Preconcentration and
Determination of Cobalt and Nickel by Graphite Furnace Atomic Absorption
Spectrometry (GFAAS). Journal of Hazardous Materials, Vol 166, pp. 291-296.
Bidari, A., Zeini Jahromi, E., Assadi, Y. & Milani Hosseini, M. R. (2007). Monitoring of
Selenium in Water Samples using Dispersive liquid–liquid Microextraction
Followed by Iridium-modified Tube Graphite Furnace Atomic Absorption
Spectrometry. Microchemical Journal, Vol 87, pp. 6-12.
Biparva, P. & Hadjmohammadi, M. R. (2007). Cloud Point Extraction Using NDTT Reagent
for Preconcentration and Determination of Copper in Some Environmental Water
Samples by Flame Atomic Absorption Spectroscopy. Acta chimica Slovenica, Vol 54,
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 Atomic Absorption Spectroscopy
Edited by Dr. Muhammad Akhyar Farrukh

ISBN 978-953-307-817-5
Hard cover, 258 pages
Publisher InTech
Published online 20, January, 2012
Published in print edition January, 2012

Atomic Absorption Spectroscopy is an analytical technique used for the qualitative and quantitative
determination of the elements present in different samples like food, nanomaterials, biomaterials, forensics,
and industrial wastes. The main aim of this book is to cover all major topics which are required to equip
scholars with the recent advancement in this field. The book is divided into 12 chapters with an emphasis on
specific topics. The first two chapters introduce the reader to the subject, it's history, basic principles,
instrumentation and sample preparation. Chapter 3 deals with the elemental profiling, functions, biochemistry
and potential toxicity of metals, along with comparative techniques. Chapter 4 discusses the importance of
sample preparation techniques with the focus on microextraction techniques. Keeping in view the importance
of nanomaterials and refractory materials, chapters 5 and 6 highlight the ways to characterize these materials
by using AAS. The interference effects between elements are explained in chapter 7. The characterizations of
metals in food and biological samples have been given in chapters 8-11. Chapter 12 examines carbon capture
and mineral storage with the analysis of metal contents.

How to reference
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Pourya Biparva and Amir Abbas Matin (2012). Microextraction Techniques as a Sample Preparation Step for
Metal Analysis, Atomic Absorption Spectroscopy, Dr. Muhammad Akhyar Farrukh (Ed.), ISBN: 978-953-307-
817-5, InTech, Available from: http://www.intechopen.com/books/atomic-absorption-
spectroscopy/microextraction-techniques-as-a-sample-preparation-step-for-metal-analysis

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