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February 1, 2010
Electromembrane Extraction: The Use of Electrical

Potential for Isolation of Charged Substances from
Biological Matrices
By Astrid Gjelstad,Ronald E. Majors
Recently, efforts have been devoted to the miniaturization of existing liquidliquid

extraction methods to make them more versatile and powerful. Reducing the use of
hazardous organic solvents due to environmental and cost concerns, time reduction,
ease of automation, possible online coupling, high-throughput capability, and the small
amounts of matrix available are major incentives that have motivated scientists working
towards miniaturization. In bioanalysis, which in this context is defined as the analysis of
small drug molecules and their metabolites in biological samples, the complexity of the
matrices requires selective and specific sample preparation methods to isolate the
analytes of interest. Macromolecules, salts, cellular material, fat, or lipids in biological
matrices such as urine, plasma, whole blood, and breast milk can disturb the separation
Ronald E. Majors
and data analysis steps. In addition, the analytes of interest can often exist in low
concentrations (pg/mLg/mL). Therefore, a sample preparation method with high degree of selectivity and
enrichment is crucial for a successful analysis.
With these considerations kept in mind, a totally new approach to sample preparation was proposed in 2006
(1). The innovative concept, called electromembrane extraction (EME), combined the technical setup for
hollow-fiber liquid-phase microextraction (HF-LPME) (2,3) with known principles for electroextraction (410).
This combination offers a highly selective sample preparation method using simple equipment and gains a
high degree of enrichment within a short period of time.
The EME method extracts charged substances from a small sample volume through a thin membrane of
organic solvent immobilized in the wall of a hollow fiber and into a receiver solution inside the lumen of the
hollow fiber. This extraction process is forced by an applied potential difference across the membrane, and
this combination of well-known liquidliquid extraction processes with electrokinetic migration yields a rapid
and selective sample preparation method for ionic substances. EME has shown to be compatible with a
wide range of biological matrices for example, plasma, whole blood, urine, and breast milk preparing
clean extracts in a short period of time with simple and inexpensive equipment.
This installment of "Sample Prep Perspectives" is a minireview of the EME technique and shows the
investigation of several parameters that affect recovery of charged analytes. In addition, a number of
application examples will illustrate its potential for the successful extraction of drugs from complex matrices.
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Experimental
The technical setup for the equipment used in EME is based upon earlier
experience with HF-LPME and is shown in Figure 1a. The hollow fiber used
is made of porous polypropylene, which is compatible with a broad range of
organic solvents. The thickness of the wall of the hollow fiber is 200 m,
with a pore size of 0.2 m and an internal diameter of 1.2 mm. A piece of
the hollow fiber is cut to a length of 25 mm and mechanically closed at the
lower end with a pair of pincers. The upper end is sealed to the end of a 22mm pipette tip by heating. To make the supported liquid membrane (SLM),
the fiber is dipped in an organic solvent for 5 s to fill the pores in the walls,
and the excess of organic solvent gently removed with a medical wipe.
Figure 1
The fiber connected to the pipette tip is guided through a punched hole in the sample compartment cap as
illustrated in Figure 1a. The pipette tip works as a mechanical support for a 0.5-mm-thick platinum wire
placed inside the lumen of the hollow fiber. Another platinum wire is introduced directly into the donor phase
through the sample compartment cap. When coupled to a power supply, these inert wires act as electrodes,
thus creating an electrical field across the SLM. In this way, the equipment makes a closed electrical circuit,
where the SLM functions as a resistor.
The volume of the sample varies between 150 L and 500 L, depending upon the sample compartment
size. However, the compartment is never filled more than half full because of the need for convection space.
The sample is shaken on a platform shaker during the extraction to increase the physical movement of the
analytes in the bulk donor phase and to reduce the thickness of the stagnant layer at the interface between
the donor phase and the SLM. The acceptor phase volume is set to 25 L and is introduced into the lumen
of the hollow fiber by a microsyringe. When the predetermined extraction period is finished, 20 L of the
acceptor phase is collected by the microsyringe and transferred to a vial for analysis in a capillary
electrophoresis (CE) instrument (1115) or by high performance liquid chromatography (HPLC) (16).
Results and Discussion
Extraction chemistry: The major driving force in EME is the potential difference across the SLM, in which
charged analytes are drawn from the donor phase, across the interface between the donor phase and the
organic membrane, through the organic membrane, across the interface between the organic membrane
and acceptor phase, and toward the electrode of opposite charge in the acceptor phase. The principle of the
extraction is shown in Figure 1b. To ensure full ionization of the model analytes, the pH in both the donor
phase and the acceptor phase should be well controlled. In the extraction of basic analytes from water, the
pH in the aqueous donor phase was adjusted to 2.0 with 10 mM hydrochloric acid (1116). The acceptor
phase also consisted of 10 mM hydrochloric acid, which increased the solubility of the analytes in the
aqueous solution compared to the SLM. In the case of acid extractions, the analytes were dissolved in 10
mM sodium hydroxide to a final pH of 12.0, which also was the pH in the acceptor phase (17).
Choice of organic solvent: The properties of the organic solvent
impregnated in the SLM are important for a functional EME system. Based
upon earlier experiences with HF-LPME, the nitro compound 2-nitrophenyl
octyl ether (NPOE) was tested as the organic solvent for extraction of basic
drugs with different polarities. An electropherogram representing the extract
after EME of five hydrophobic basic drugs with NPOE as the SLM is shown
in Figure 2. Hydrophilic model analytes (log P < 1.0) were unable to
penetrate the interface between the donor phase and the SLM with pure
Figure 2
NPOE. The high polarity of these analytes with low partition into the SLM
seemed to counteract the influence of the electrical field.
To increase the solubility of the analytes in the organic solvent, di-(2-ethyl-hexyl) phosphate (DEHP) was
added to the SLM as an ion-pair reagent (12). The results in Table I illustrate the importance of the nature of
the organic solvent. The addition of 10% DEHP to the membrane increased the recoveries of the hydrophilic
drugs tested from zero up to 2254%, while the recoveries of the more hydrophobic analytes tested were
reduced dramatically with DEHP presented in the SLM. Also, the hydrophobic drugs ion-paired with DEHP,
and were efficiently transported into the organic membrane. However, these ion pairs were highly
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hydrophobic, and the strong interaction partly prevented them from

liberating to the acceptor phase. The addition of 10% tris-(2-ethylhexyl)
phosphate (TEHP) to NPOE altered the situation, as shown in Table I.
Despite a similar structure to DEHP, TEHP did not function as an ion-pair
reagent, as it does not contain any ion-pairing seats. The hydrophilic drugs
were not extracted by the addition of TEHP to the SLM. However, the
Table I: The influence of organic solvent on
recoveries of the hydrophobic drugs increased with TEHP in the SLM,
recovery
probably caused by alteration of the polarity in the SLM. A combination
SLM consisting of 10% DEHP and 10% TEHP in NPOE led to extraction of almost all the basic drugs tested,
showing that development of a membrane that permitted electrokinetic transport of drug substances in a
very large logP window was possible.
Table II: List of

substances and
matrices tested in EME
Proper organic solvents for extraction of acids have been examined (17). The aromatic
nitro compounds were found to be inefficient for the acidic model analytes, and aliphatic
alcohols with different chain length were tested based upon experiences with HF-LPME
(18). The alcohols 1-octanol and 1-heptanol were found to be suitable solvents because of
their insolubility in water and easily impregnation in the fiber. In addition, the alcohols
showed an appropriate electrical resistance to the applied voltage that is, the current
was low enough to avoid bubble formation and excessive electrolysis but still sufficiently
high for the system to maintain the properties of a closed circuit. Recoveries between 8%
(hexobarbital) and 100% (fenoprofen) were obtained with 1-heptanol as the SLM (Table
II).
The effect of different organic solvents was strongly dependent upon the
potential difference applied across the SLM. The optimal potential
difference with NPOE as the SLM was 300 V, which showed recoveries between 16%
(pethidine) and 78% (methadone) for the nonpolar model analytes (12). To reduce the
applied potential difference, other organic solvents were tested (14). Different SLMs were
made with different nitro compounds including ethyl nitrobenzene (ENB) and 1-isopropyl-4nitrobenzene (IPNB). It was shown that 10 V was the potential difference that showed the
highest recoveries, which were in the ranges of 5480% (ENB) and 5993% (IPNB). The
results show that the chemical nature of the organic solvent in combination with a proper
potential difference can be used to design very selective extractions of different analyte
Table II: continued
classes.
Other parameters that were found to influence the recoveries in EME
included the convection speed, the ion balance between the donor and
acceptor phase and the temperature (16).
Table II: continued
Extraction by batteries: Because of the successful extractions with only

10 V as the driving power, a simple 9-V battery was tested as the only
power supply (14). This replacement of the power supply did not influence
the recoveries of the drugs tested, making the idea of a portable sample
preparation device possible.
Comparison with HF-LPME: The original idea for EME was to employ the
same setup that was used for HF-LPME. The only technical difference
between these two systems was the electrical circuit introduced in the EME
setup. The platinum electrodes coupled to a power supply created a
potential difference across the SLM, hence, changing the extraction
mechanism from passive diffusion to electrokinetic migration. A systematic
study of the differences between the two methods was performed regarding
to the extraction mechanisms, extraction times, recoveries, and selectivity
(11). Overall, the introduction of another transport force into the EME
system made a significant change in the extraction time compared to the Figure 3
existing HF-LPME model. Generally, the most time-consuming step in
bioanalysis is the sample preparation step. Therefore, the extraction speed is of great importance in
development of a new extraction method. The time to reach a steady-state level is determined by different
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parameters in the driving force of the extraction. The only differences between the two systems were the pH
in the donor phase, which was 2.0 in the EME system and 12.0 in the HF-LPME system, and the voltage
applied across the SLM in the EME system. The recoveries as a function of the extraction times were
examined, and the extractions were completed when a steady state level was reached. The graphs in Figure
3 compare the results from an EME extraction with an HF-LPME extraction of the same compound,
haloperidol. The time-saving effect obtained from the voltage application in EME was significant, with a
reduction in the equilibrium time from about 60 min with passive diffusion as the driving force to 510 min
with an electrical field as the major driving force. The speed advantage EME offered compared to HF-LPME
was most remarkable using the smallest sample compartment tested (300 L, 31 mm 4 mm i.d.). In the
small compartment, the short electrode distance created a relatively high electrical field strength, resulting in
a stronger driving force and hence increased extraction speed.
EME from biological matrices: A number of biological matrices have been tested in combination with
EME. The matrices covered a wide range of complexity from urine, to diluted and untreated plasma, to
whole blood, to breast milk. We will now look at some of these examples.
Human plasma: Extractions of basic drugs from both diluted plasma (14) and undiluted plasma (13) has
been tested. These experiments showed that the extraction recoveries were almost independent of the
dilution and the pH in the plasma. In other words, extraction from plasma samples without any pretreatment
was possible.
Figure 4
The time to reach a steady-state level from untreated plasma was

somewhat extended compared to extraction from phosphate buffer (pH 7.4),
as exemplified with nortriptyline in Figure 4. The slow extraction kinetics in
plasma was probably caused by two factors: the increased viscosity of
plasma samples, and drug interactions with plasma proteins. To check
whether the viscosity was a deciding factor or not, the plasma sample was
diluted with pure water in the ratio 1:1 in a subsequent experiment. The
results are shown in Figure 4. It can be seen that lowering the viscosity of
the plasma samples did not increase the extraction speed significantly. The
steady-state recoveries were comparable in phosphate buffer as well as
untreated and diluted plasma.
Whole blood: Experiments with EME from whole blood samples have been performed (13). The recoveries
obtained from extraction from untreated whole blood spiked with some basic drugs were in the range 19
51%. They were comparable to EME from untreated plasma, even though whole blood is a more viscous
and complex matrix than plasma. In some subsequent experiments, whole blood samples were diluted with
pure water in the ratio 1:1 or 1:3 to test if a less viscous matrix would increase the recoveries. For the
majority of the model analytes tested, the dilution step led to improved recoveries.
Urine: EME of diluted urine samples has been performed (14). The recoveries obtained after 5 min EME
from diluted urine were in the range of 3750%, which were significantly lower than EME from pure water
samples under otherwise identical conditions. To test the effect of variety in the water and salt content, urine
samples from different persons and collected at different times were spiked with the basic model analytes
and subjected to EME. The results showed no significant difference between the recoveries.
Breast milk: EME has been performed on breast milk (14). The recoveries of the drugs tested were in the
range 3955% from breast milk, and the resulting electropherograms showed very few interferences.
Extractions from samples collected from two different women and from the start and end of lactation showed
no significant differences in the recoveries.
Other applications: The repeatability for the extraction from human plasma was tested at two different
concentration levels. The repeatability of the extractions was 517% RSD (13). Other EME applications
have been performed simultaneously to the works briefly described in this article. EME of peptides (19,20),
lead ions (21), chlorophenols (22), nerve agent degradation products (23), and phenols (24) have shown the
potential of the technique. Table II gives a comprehensive overview of the substances and matrices that are
exposed to EME. The usage of electrical potential difference in sample preparation is also described by
Arrigan and his group (47). Their research focuses on the electrochemical properties of the interface
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between two immiscible electrolyte solutions (ITIES), which can be used in separation and detection of ionic
molecules from biological matrices.
Conclusion
A new idea about an electrical field as the extraction driving force combined with existing hollow-fiber
extraction technology is described briefly. It is shown that the technique offers rapid and selective isolation
and enrichment of ionic substances. The extraction recoveries are strongly dependent upon the nature of
the organic solvent. This dependence makes EME a highly selective extraction method that uses the nature
of the organic solvent and the applied potential to control the extractions.
The time-saving effect by using an electrical field as the driving force is demonstrated during a comparison
between EME and liquid-phase microextraction. The potential of EME is shown during extraction from
different biological matrices. Extractions from diluted urine, diluted and untreated plasma, diluted and
untreated whole blood, and diluted breast milk are noticed, showing promising results. Normally, existing
liquidliquid extraction methods require pH adjustment of the matrix before extraction of ionic analytes. In
that way, EME offers possible advantages, where ionic substances can be extracted directly from untreated
biological samples without disturbing the chemistry in the sample.
The high extraction speed and the simple equipment needed makes EME a promising tool for field sampling,
either from blood samples at the bedside in a hospital or in environmental analysis. The EME experiments
performed with a common battery as the only power supply were promising in that respect.
Ronald E. Majors "Sample Prep Perspectives" Editor Ronald E. Majors is business development manager,
Consumables and Accessories Business Unit, Agilent Technologies, Wilmington, DE, and is a member of
LCGC's editorial advisory board. Direct correspondence about this column to "Sample Prep Perspectives,"
LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail
lcgcedit@lcgcmag.com [lcgcedit@lcgcmag.com]
.
References
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(7) A. Berduque, A. Sherburn, M. Ghita, R.A.W. Dryfe, and D.W.M. Arrigan, Anal. Chem. 77, 73107318
(2005).
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(1996).
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(1995).
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341 (1994).
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(2007).
(12) A. Gjelstad, K.E. Rasmussen, and S. Pedersen-Bjergaard, J. Chromatogr., A 1124, 2934 (2006).
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(13) A. Gjelstad, K.E. Rasmussen, and S. Pedersen-Bjergaard, Anal. Bioanal. Chem. 393, 921928 (2009).
(14) I.J.O. Kjelsen, A. Gjelstad, K.E. Rasmussen, and S. Pedersen-Bjergaard, J. Chromatogr., A 1180, 19
(2008).
(15) T.M. Middelthon-Bruer, A. Gjelstad, K.E. Rasmussen, S. Pedersen-Bjergaard, J. Sep. Sci. 31, 753759
(2008).
(16) A. Gjelstad, K.E. Rasmussen, and S. Pedersen-Bjergaard, J. Chromatogr., A 1174, 104111 (2007).
(17) M. Balchen, A. Gjelstad, K.E. Rasmussen, and S. Pedersen-Bjergaard, J. Chromatogr., A 1152, 220
225 (2007).
(18) T.S. Ho, S. Pedersen-Bjergaard, and K.E. Rasmussen, J. Chromatogr. Sci. 44, 308316 (2006).
(19) M. Balchen, L. Reubsaet, and S. Pedersen-Bjergaard, J. Chromatogr., A 1194, 143149 (2008).
(20) M. Balchen, T.G. Halvorsen, L. Reubsaet, and S. Pedersen-Bjergaard, J. Chromatogr., A 1213, 1417
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(21) C. Basheer, S.H. Tan, and H.K. Lee, J. Chromatogr., A 1213, 1418 (2008).
(22) J. Lee, F. Khalilian, H. Bagheri, and H.K. Lee, J. Chromatogr. A 1216, 76877693 (2009).
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Astrid Gjelstad is with the Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo,
Oslo, Norway. She defended her PhD thesis about Electromembrane Extraction October 2009, and is
continuing her research regarding microextraction and membrane technology in Oslo.
astrid.gjelstad@farmasi.uio.no [astrid.gjelstad@farmasi.uio.no]
Ronald E. Majors
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Figure 1
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Figure 2
Table I: The influence of organic solvent on recovery
Table II: List of substances and matrices tested in EME
Table II: continued
Table II: continued
Figure 3
Figure 4
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Chromatography Online: Electromembrane Extraction: The Use of Electrical Potential ...

Electromembrane Extraction: The Use of Electrical

Recently, efforts have been devoted to the miniaturization of existing liquidliquid

hydrophobic, and the strong interaction partly prevented them from

Table II: List of

Table II: continued

Extraction by batteries: Because of the successful extractions with only

The time to reach a steady-state level from untreated plasma was

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