CHAPTER 7
MEMBRANE TECHNIQUES FOR ANALYSIS, SAMPLING
AND SPECIATION IN ENVIRONMENTAL MEASUREMENTS
Jan Åke Jönsson1, Lennart Mathiasson1, Luke Chimuka2, Ewa Cukrowska3
1
Analytical Chemistry, Lund University, P.O.Box 124, 221 00 Lund, Sweden,
Department of Ecology and Resource Management, School of Environmental Science
and Engineering, University of Venda for Science and Technology, P/Bag X5050, 0950,
Thohoyandou, Limpopo Province, South Africa,
3
School of Chemistry, University of the Witswatersrand, Private Bag 3, Wits 2050,
Johannesburg, South Africa.
2
ABSTRACT
The techniques of membrane extraction permit the application of classical liquid-liquid
extraction (LLE) chemistry to instrumental and automated operation. Various
shortcomings of LLE are overcome by membrane extraction techniques as they use
none or very little organic solvents, high enrichment factors can be obtained and there
are no problems with emulsions. A three phase SLM system (aq/org/aq), where analytes
are extracted from the aqueous sample into an organic liquid, immobilized in a porous
hydrophobic membrane support, and further to a second aqueous phase, is suitable for
the extraction of polar compounds (acidic or basic, charged, metals, etc.) and it is
compatible with reversed phase HPLC. A two-phase system (aq/org) where analytes are
extracted into an organic solvent separated from the aqueous sample by a hydrophobic
porous membrane is suitable for more hydrophobic analytes and is compatible with gas
chromatography.
Chapter 7
1 INTRODUCTION
Many analytical techniques and methods have been developed for analysis of various
pollutants in environmental and biological samples. Despite these achievements in
analytical science, there are still challenges. The challenge lies in determining pollutants
in various complex matrices such as plant extracts, sediments and biological fluids.
Most developed analytical methods require several steps consuming time and solvents.
In ecological risk assessment for chemical pollutants, it is important to quantify the
concentrations of freely dissolved in aqueous samples for approximate characterisation
of the bioavailabe fraction. Determining of the concentration of pollutants of freely
dissolved in complex matrices as plant extracts and biological fluids is even more
challenging especially for metal ions. There are not many techniques available that can
be used for speciation studies of metal ions in biological fluids. The challenge of
chemical analysis especially speciation studies and determining the freely dissolved
pollutants in a complex sample is staggering. Moreover, components of interest exist at
trace levels. These challenges have made sample preparation become a key step in
modern chemical analysis. It is an essential part of any analytical procedure because of
the following reasons: sample preconcentration or enrichment and removal of
contaminants.
The most widely used sample preparation techniques are liquid-liquid extraction
(LLE) [1] and solid-phase extraction (SPE) [2]. LLE is the traditional technique for
extraction of organic analytes from aqueous solutions. The basis is the partitioning of
the dissolved analytes between the organic phase (extraction liquid) and the aqueous
solution (sample solution) according to their partition coefficients. Further shifting the
equilibrium towards the organic phase brings about increased enrichment in the organic
phase. This can be achieved by addition of a salt to the aqueous phase. Alternatively, or
additionally, the extraction is repeated several times. Ensuring that target analytes have
large partition coefficients compared to possible interferents controls the selectivity of
the extraction. Thus, selectivity can be fine-tuned by changing the polarity of the
organic solvent, using ion pairing or pH adjustments in the aqueous phase. The
technique is well known and still widely used though now is less attractive and is being
replaced by other techniques. This is because LLE (i) is tedious and time consuming
especially when extracting aqueous complex samples such as plant extracts and
sediments which demands many steps before a clean extract is obtained, (ii) is not easy
to automate, (iii) forms emulsions which at times makes it difficult to separate the two
phases and (iv) is environmentally unfriendly due to large volumes of organic solvents
used. It However, with LLE, large enrichment factors can be obtained despite the cited
drawbacks.
Solid Phase Extraction (SPE) techniques are perhaps the most popular in sample
preparation especially for organic analysis. However, most official methods still use
LLE like those published by the US Environmental Protection Agency (USEPA). The
principle of SPE is based on sorption of analytes on a sorbent. The aqueous sample
solution passes the SPE column and the analytes are first trapped on the sorbent and
then eluted with a suitable small volume of organic solvent. Extraction and enrichment
of the analytes is thereby simultaneously achieved. Most sorbents are now available as
disks, cartridges or precolumns [3, 4]. The use of SPE for environmental and biomedical
applications including details of its principles is well documented in many review
papers [2, 5-7] and books [1, 8]. Recent research has been directed towards developing
sorbents that are capable of trapping polar analytes (graphitised carbon blacks and
functionalised polymers) and selective sorbents (molecularly imprinted polymers and
immunosorbents) suitable for complex matrices such as wastewater, foodstuffs and
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Chapter 7
plant extracts [2]. This is because the common sorbents, especially n-alkyl bonded silica
sorbents are both nonselective and give low breakthrough volumes for polar analytes.
Another related technique to SPE which is now becoming wide-spread in many
laboratories is solid phase microextraction (SPME) which is easily connected to gas
chromatography in an automated way and uses little or no organic solvent. It has
recently been reviewed for environmental applications [9]. However, despite its
simplicity, it lacks selectivity when extraction analytes in complex matrices like plant
extracts, foodstuffs, and wastewater.
Membrane based extraction techniques in sample preparation are on the increase
as seen in the following review articles and book chapters [10-16]. Recently, a special
journal issue has been dedicated to membrane extraction in analytical chemistry [17].
An area enjoying much attention by various research groups is developing membrane
based extraction techniques that are simple, cheap and miniaturised [18-28].
2 MEMBRANE BASED SAMPLE PREPARATION
2.1 Introduction
The main membrane techniques (see Table 1) that have been used for analytical
applications can be classified based on whether the membrane is porous or nonporous
during the extraction of the sample solution [16]. A clear difference between these two
is that selectivity for porous membrane processes is mainly based on pore size and pore
size distribution. A nonporous membrane can be either a porous membrane impregnated
with a liquid or entirely a solid, like silicone rubber. In both of these cases, the
chemistry of the membrane material can influence the selectivity and the flux of the
process [18]. This review will focus on liquid membrane extraction techniques whereby
a porous membrane is impregnated with a liquid. These techniques are the SLM
extraction technique, which is a three-phase system, and which provides the most
versatile possibilities; and the MMLLE technique, which is a twp-phase system,
essentially with the same chemistry as classical liquid-liquid extraction, LLE. Before
going into the details of SLM and MMLLE, the other techniques which are mentioned
in table 1 will be briefly discussed.
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Chapter 7
TABLE 1
Different major membrane techniques used in analytical applications [10, 11].
Name
Abbreviation
Type
Dialysis
-
Electrodialysis
ED
Supported-liquid
membrane
extraction
Microporous
membrane liquidliquid extraction
Semi-permeable
membrane
devices
Polymeric
membrane
extraction
Membrane
extraction with
sorbent interface
SLM
Porous
membrane
Porous
membrane
Non-porous
membrane
Phase combinations used
Donor/membrane/
acceptor
Aq/membrane/Aq
Aq
Aq/org/aq
MMLLE
Non-porous
membrane
Aq/org/org
SPMDs
Non-porous
membrane
Aq/polymer/org
PME
Non-porous
membrane
MESI
Non-porous
membrane
Aq/polymer/aq,
Org/polymer/aq,
Aq/polymer/org,
Gas/polymer/gas,
Liquid/polymer/gas
2.2 Other membrane based extraction techniques
2.2.1 Dialysis and electrodialysis
In dialysis, solutes diffuse from the aqueous donor side of a porous membrane to the
aqueous receiving side as a result of a concentration gradient. Separation between the
solutes is obtained as a result of differences in diffusion rates arising from differences in
molecular size. Dialysis is therefore most effective in removing large molecules like
proteins from small ones [18, 29]. It is a technique that has few applications for
environmental analysis except in biological samples since in the former, both the
analytes and the sample matrix compounds are small molecules. The analytes are also
continually diluted in the acceptor unless a precolumn is incorporated [30]. The
electrodialysis technique tries to redress the drawbacks of dialysis in environmental
analysis, viz. its non-selectivity and the dilution of the analytes in the receiving phase. It
is a porous membrane technique whose separation power relies on differences in
molecular size of the compounds and also on their charges. The electrical potential
applied across the separation membrane makes sure that properly charged analytes in
the feed solution are drawn through the membrane to the receiving side [31]. With a
flowing feed solution and stagnant receiving phase, additional selectivity and
enrichment of charged analytes can be obtained. The basic principles of electrodialysis
for trace analysis have been reviewed by Debets et al. [19]. It has its own limitations
such as limited enrichment factors, pH change in the feed during extraction, thermal
degradation of the membrane at high potential [31]. The cited problems make
electrodialysis less attractive for analytical applications.
87
Chapter 7
2.2.2 Polymeric membrane extraction (PME)
In this case, instead of a porous membrane, an entirely solid membrane is used to
separate the donor and the acceptor solutions. Silicone rubber is mostly used because it
is hydrophobic and gives high permeability for small hydrophobic molecules [32]. The
difference in the solubility and diffusion of various analytes into the polymer is the basis
of selectivity. Changing conditions in the acceptor phase such as making sure that
analytes ionise can enhance the selectivity [20]. This condition is a similar requirement
as in supported-liquid membrane extraction technique as described below. Its major
advantage is that the solid nature of silicone rubber means that phase breakthrough is
minimised. The major disadvantage is that it does not allow for any room to incorporate
other functional groups (carriers) that can enhance both the mass transfer and the
selectivity of the compounds of interest. The solid nature of silicone rubber nonetheless
makes it a versatile technique as it allows aqueous [33], organic [20] and gaseous
samples to be processed (section 2.2.3). Especially it is an ideal method for extracting
analytes in complex samples with high amounts of organic materials such as lipids [34]
since the instability associated with liquid membranes does not exist. It also allows
various versions of phase combinations for extraction. In all the combinations, the
partitioning of the analytes into the polymer, diffusion through it and partitioning into
the receiving phase are important critical factors that influence the overall mass transfer
coefficient [32, 35].
2.2.3 Membrane extraction with a sorbent interface (MESI)
The technique is based on membrane extraction into a gas followed by trapping of the
analytes on a solid sorbent (cryofocusing) and subsequent thermal desorption into a gas
chromatographic system [36]. The technique is therefore suitable for volatile organic
compounds either in air or aqueous samples. The receiving phase is always a carrier gas
that continuously strips off and transports the analytes on the sorbent. The detailed
theory of the technique and the type of sorbents that are used to trap the analytes have
been described by Pawliszyn et al. [37] and Harper [38], respectively. The basis of
selectivity of the method is differences in solubility and diffusion of various analytes
into the nonporous polymer. The main drawback of the technique is that it has a narrow
application window for environmental analysis; only volatile organic compounds can be
extracted. As an example of an application of this technique, the research group of
Pawliszyn recently developed a method for analysis of volatile breath components using
a membrane extraction with a sorbent interface [39].
2.2.4 Semi-permeable membrane devices (SPMDs)
In SPMDs, hydrophobic organic analytes passively diffuse from aqueous donor phase
through a polymeric membrane such as polyethylene into the acceptor phase filled with
a thin film of a synthetic lipid such as triolein [40-43]. It is therefore used as a timeweighted passive field sampler. It is an important technique in exposure risk assessment
of pollutants since it provides truly dissolved and bioavailable time-weighted average
pollutant concentrations over longer periods. Generally, this technique is suitable for
extraction of hydrophobic nonpolar compounds such as polycyclic aromatic
hydrocarbons [44], chlorinated pesticides [40], and polychlorinated biphenyls [45] with
partition coefficients (logKow) in the range 3.0-6.0 [12]. SPMDs are not very selective
as they rely on the differences in solubility and diffusion of the various analytes into the
membrane and lipid. This necessitates additional clean-up of the extracts [46],
consuming time and a waste of organic solvents. For quantitation, very little data is
available for sampling rates of various pollutants into the SPMDs [44], making it
88
Chapter 7
difficult to estimate accurately the true concentrations of analytes in the original
environmental compartment. This setback is being investigated via the addition of what
are known as permeability reference compounds (PRC) to SPMDs lipid prior to
sampling. This provides an overall correction factor for variation in uptake sampling
rates
3 SUPPORTED-LIQUID MEMBRANE (SLM) EXTRACTION
3.1 Principles of SLM extraction
In an SLM extraction, an organic solvent is immobilised in the pores of an inert support
material separating the aqueous donor and the acceptor phases (Figure 1). The analytes
are partitioned from the aqueous sample stream into the organic membrane and are then
re-extracted into the aqueous acceptor phase. The driving force is the difference of the
analyte concentration between the donor and acceptor phases. In order to maintain the
concentration gradient across the two phases, the solutes must be able to exist in two
forms: in a non-ionic form on the donor side to be extracted into the membrane and in a
ionic form on the acceptor in order to be irreversibly trapped. This is most simply
achieved by pH adjustments in the two aqueous phases and the method is therefore
particularly well suited for ionisable compounds such as medium to weak acids and
bases. SLM extraction can provide very selective enrichment. Selectivity can be finetuned by proper choice of the conditions in the three phases as seen in Figure 1. This
creates a selectivity window such that by the time the analytes are enriched in the
acceptor phase, an indirect structural recognition is achieved and only analytes
belonging to the same family are generally trapped at a time. Macromolecules are
discriminated on the basis of their size while charged compounds are too polar to
dissolve into the organic liquid. Neutral molecules merely distribute between the three
phases without any enrichment.
Donor (aq)
Membrane (org)
RCOO -
Acceptor (aq)
RCOOH
RCOO -
Acid
Base
Neutral
RNH 3+
Neutral
RNH 2
RNH 3+
Base
Acid
∆C
Condition 1:
pH = pK a - 2
or
pH = pK a + 2
Condition 2:
~ 2 ≤ logKow ≤ ~ 4
Condition 3:
pH = pK a + 3.3
or
pH = pK a - 3.3
Figure 1. Principles of SLM extraction of ionisable organic compounds where the
transport mechanism is simple permeation.
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Chapter 7
Often, selective transport based on relative differences in solubility in the
membrane and trapping in the acceptor phase may be difficult to achieve. In another
case, the solubility of the analyte may be too low to give efficient extraction even when
the trapping in the acceptor can easily be realised. A good approach in such a case is to
incorporate a mobile carrier into the membrane that selectively binds the analytes. The
idea of incorporating a carrier also allows SLM extraction to be applicable to a variety
of compounds such as permanently charged chemical species like metal ions (See
below). It also gives different versions of carrier mediated transport mechanisms such as
simple carrier transport (with chemical reaction in the acceptor), coupled co-transport
[47, 48], and coupled counter transport [22, 48-52]. In simple carrier transport, the
carrier in the membrane forms a complex with the analyte in the donor that diffuses to
the acceptor, where the analyte is converted to a non-extractable form. This type of
transport was used in the extraction of short chain aliphatic carboxylic acids from acidic
donor solution to an alkaline acceptor solution with liquid membrane containing tri-noctylphosphine oxide (TOPO) as a neutral carrier [53]. Charged carriers can be used
such as the anionic di-(2-ethylhexyl) phosphoric acid (DEHPA) [22, 50]. In such a case,
dissolution of the analyte into the membrane occurs through ionic interactions with the
charged carrier. Once the analyte reaches the acceptor phase, it is exchanged for a
proton and converted to a non-extractable form. The proton gradient across the
membrane in this case is the driving force [50]. This is an example of a coupled counter
transport mechanism.
The various factors that influence the extraction process have been covered by
Jönsson et al. [14, 54]. These include the trapping in the acceptor phase, solvation
power of the membrane and donor flow rate besides other factors such as membrane
thickness, geometry of the donor and acceptor channels of the SLM module. The
trapping in the acceptor phase is seen as critical and it is desirable that analytes are
virtually completely ionised, otherwise extraction efficiency is not constant and
decreases with time [55]. The solvation power of the membrane should give large
analyte partition coefficients (KD) but studies has shown that too high KD values result
in slow release into the acceptor phase [56]. At lower donor flow rates, the contact time
of the analytes with the membrane is longer so highest extraction efficiencies are
obtained. The extraction efficiency, E, is defined as the fraction of analyte extracted
from the donor phase to the acceptor phase. It is a measure of the rate of mass transfer
through the membrane and is constant at specified extraction conditions. At high donor
flow rates, the extraction efficiency decreases as the contact time of the analytes with
the membrane reduces. However, for moderately polar analytes (with logKow > 2), high
donor flow rates results in an increase in the enrichment factors since the amount
extracted per unit time increases [57]. This reduces the extraction time needed to
achieve a certain enrichment.
Liquid membrane instability often cited as a major limitation of SLM extraction
technique is caused by decline of analyte flux or even leakage of one aqueous phase into
the other due to solvent or carrier loss during extraction. Factors such as differences in
osmotic pressure between the phases and emulsion formation have been identified as
causes of instability [58-61]. Gelation, is when a thin liquid film with properties of a
highly swollen cross-linked polymer rather than a liquid, has been applied on the
surface of the membranes to improve stability, but such an approach results in lower
diffusion coefficient [47, 62]. Another approach is forming a semi-permeable skin layer
on the surface of the supported-liquid membrane through either interfacial [63-65] or
plasma polymerisation process [62]. For analytical purposes, especially with di-nhexylether or n-undecane as membrane liquids, SLMs are stable from a week [66] to a
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Chapter 7
month [67, 68]. Convenient regeneration of the membrane using the same hydrophobic
porous support has also been demonstrated either in situ by Thordarson et al. [69] or by
Dzygiel et al. [51] after demounting the support.
3.2 Membrane holders (contactors)
Three different physical realisations of SLM modules have generally been reported [54,
70] i.e., the flat, spiral and tubular modules. One important property among these
modules is the ratio between membrane surface area and its volume [70]. This ratio
should be high to get large enrichments factors especially for analytical purposes. It is
highest for the tubular module (1000-10,000), followed by the spiral module (100-1000)
and lowest for the flat module (10-100) [70]. Most research groups have used either flat
module and/or hollow fibre modules with channel volumes in the range 10-1000 µL
[14] with exception of hollow fibre design where channel volumes of less than 1.3 µL
have been realised [69]. However, the area of modules and versions of SLM extraction
is currently enjoying much attention as seen from publications [22, 24-28]. Some
examples of such modules and their respective channel volumes are shown in Figure 2.
They are made of inert materials such as polytetrafluoroethylene (PTFE),
polyvinylidene fluoride (PVDF) or titanium. For flat modules, a groove is machined in
each block and liquid connections are provided at both ends. An impregnated membrane
is clamped between the blocks forming a channel at both sides of the membrane. The
choice of the membrane material has been discussed in previous works [68, 71, 72]. An
ideal membrane support is the one that gives high solute permeation and stable
membranes. Generally, thin membranes such as FGLP ( Millipore) or TE 35 (Schleicher
and Schuell) with small pores (~ 0.2 µm), and polyethylene backing have been used.
These have been found to facilitate mass transfer and at the same time able to retain the
impregnated liquid firm enough to avoid deformation due to small pressure changes in
the system.
M e m b ra n e
A cc e p to r c h a nn e l
Figure 2. (A) Membrane unit with 1 mL channel volume (A = blocks of inert material,
B = membrane). (B). Membrane unit with 10 µl channel volume. (C). Hollow
fiber membrane unit with 1.3 µl acceptor channel (lumen) volume (1 = Orings, 2 = polypropylene hollow fiber, 3 = fused silica capillaries, 4 = male
nuts).
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Chapter 7
3.3 Theory of SLM extraction
A thorough treatment of the theory and principles of the SLM process has been
presented earlier [54] and the following items are emphasized here:
Extraction is usually evaluated as the extraction efficiency, E. This is the
fraction of analyte amount input to the system that is collected in the acceptor i.e.,
E = nA / nI
(1)
where
nI and nA are the number of moles input during the extraction time and those collected
in the acceptor, respectively.
The extraction efficiency (fraction of analyte molecules that are transferred to
the acceptor) is a function of many parameters. These include the magnitude of the
partition coefficient of the uncharged analyte species between the aqueous phase and the
organic (membrane) phase, the trapping conditions in the acceptor, flow rate of the
donor, as well as the characteristics and dimensions of the membrane. In practice, its
experimental value will also be influenced by the efficiency of transferring the acceptor
phase to the analysing equipment.
The influence of the partition coefficient, K, is somewhat complex. At low values of K,
E is small, as the analyte is insufficiently extracted into the organic membrane and the
mass transfer is limited by the diffusion transfer through the membrane (membranecontrolled extraction conditions). At intermediate values, the mass transfer is limited by
the transport properties in the flowing donor phase (donor-controlled conditions), and in
this region, the most efficient extraction is obtained. At very high values of K, i.e., for
very hydrophobic species, the stripping of analyte into the acceptor phase becomes the
limiting factor, and the observed extraction efficiency will decrease, because significant
amounts of analyte will be left in the membrane. It was found [56] that the most
efficient extraction is obtained when the octanol-water partition coefficient (as a
measure of polarity) of the diffusing species is around 103.
The speed of extraction, i.e., the rate of mass transfer from donor to acceptor, is
proportional to the concentration difference, ∆C, over the membrane. With some
simplifications (especially related to activity effects at different ionic strengths) it
follows that [54, 55]:
∆C = αD CD - αA CA
(2)
where
CD and CA are the concentrations in the donor (sample) and acceptor phase,
respectively, and αD and αA are the fractions of the analytes that are in extractable
(uncharged) form in the actual phase. Typically, extraction conditions are set up such
that αD is close to 1 and αA is a very small value. CA is zero at the beginning of the
extraction and increases during the operation, usually to values well over CD.
As long as αA is sufficiently small, the second term in Equation (2) is negligible
and E will be constant during the course of the extraction, so the extracted amount will
be directly proportional to the volume that is extracted and also to the concentration of
analyte in the sample. This is the usually preferred situation and it is referred to as
complete trapping.
92
Chapter 7
If the trapping is not complete, the extraction efficiency will decrease with time,
leading to less precise quantitation. This was detailed in a recent paper [55]. However,
incomplete trapping conditions can, as will be described below, provide a means of
measuring free (non-bound) concentrations, as opposed to total concentrations.
As could be expected, extraction efficiency is highest for very low donor flow
rates, and decreases as the flow rate increases (see Figure 3). Therefore, the most
efficient extractions are obtained at low donor flow rates, as a low flow rate increases
the residence time of an analyte molecule in the donor channel.
1
12 0
10 0
0.8
80
E
60
0.4
Ee
0.6
40
0.2
20
0
0
0
0.05
φ
0.1 0
0.15
Figure 3. Extraction efficiency, E and enrichment factor Ee (arbitrary units) as functions
of a reduced donor flow parameter φ, (see text)
In practical work, time is an important issue, and it is therefore often more
relevant to maximise the concentration enrichment factor rather than to maximise the
extraction efficiency. Increasing the donor flow rate not only decrease E, but also
increases the amount of analyte that is introduced into the extraction system and the net
result often is an increase in the amount of accumulated analyte in the acceptor during a
given time.
This will lead to larger instrumental signals (e.g., peak areas) in the analysis of
the extract and thus to a more time-efficient analysis. The concentration enrichment
factor, Ee, is related to the extraction efficiency as:
Ee = CA/CS = E · VS /VA
(3)
CS and CA are the analyte concentrations in the extracted sample and the
acceptor channel, respectively, and VS and VA are the corresponding volumes. When the
donor flow rate is increased, E decreases, as shown in Figure 3, but this is compensated
for by an increasing amount of analyte being input to the system. The result is that,
under donor-controlled conditions, the enrichment factor typically increases with donor
flow rate for a given time, as seen in Figure 3. However, a high flow rate will obviously
consume a large volume of sample, which is usually no problem for extraction of
environmental samples (e.g., river water or similar samples). If the available sample
93
Chapter 7
volume is limited (e.g., biological samples), extraction could be better performed at low
flow rate to maximise the extraction efficiency.
In general, it is not necessary to strive for the maximum value of E, and this
parameter should not be confused with recovery. For good quantitative performance, the
important issue is to find conditions that lead to reproducible values of E and the value
of this parameter will be included in the calibration.
3.4 Equilibrium extraction in SLM
If the extraction is conducted for an increased time, the concentration in the acceptor CA
increases and eventually the second term in Equation (2) will become significant and
∆C decreases. This leads to a lower rate of mass transfer and a decrease in E. When ∆C
has reached zero, all three phases are in equilibrium and the maximum concentration
enrichment factor possible is reached. This is given by:
Ee(max) = (CA / CD)max = αD/αA
(4)
With αD ≈ 1 and αA ≤ 0.0005, signifying complete trapping, Ee(max) ≥ 2000, so it
is possible to enrich the analyte at least several hundred times until the E is appreciably
influenced. The maximum value is not attained in practice.
However, if the trapping conditions are set e.g. so αA = 0.1, the maximum
enrichment factor is only 10, which is easily reached, and the system reaches
equilibrium. The concentration of the acceptor is then 10 times higher than that in the
donor and will not increase if more of the sample is passed through the donor channel.
Equation 4 was experimentally verified [55] for the extraction of organic bases and also
for organic acids and for copper extraction using DEHPA (unpublished).
These acceptor-controlled conditions can be used to measure equilibrium
fractions of the analytes taking part in secondary equilibria. One example is the binding
of different environmental pollutants to humic compounds, particles, etc., in natural
waters. By setting up acceptor-controlled extraction conditions and carrying out the
extraction until equilibrium is attained, the analysis of the acceptor directly gives a
known factor times the free equilibrium concentration in the sample. As described
above, the factor is αD /αA, which can be set to a suitable value by selecting appropriate
conditions in the donor and acceptor phases.
In an unpublished work, 2,4-dichlorophenol was enriched to equilibrium from
pure water and from solutions with different concentrations of humic acid. As is seen in
Figure 4, the maximum enrichment factor in pure water solution agrees nicely with
theory (Equation 4) with αD = 1 and αA = 0.11. In solutions with humic acid, lower
responses are obtained, which can be interpreted as due to binding of the chlorophenol
with humic acid to various extent and that the membrane extraction in equilibrium mode
senses the free equilibrium concentration.
94
Chapter 7
1000
cacceptor (ppb)
cacceptor (ppb)
1000
750
a
500
250
750
b
500
250
Water, Enrichment: 9.1
10 ppm humic acid: 83 % of water
0
0
0
50
100
150
0
200
volume (ml)
50
100
150
200
250
volume (ml)
cacceptor (ppb)
750
500
c
250
20 ppm humic acid: 65% of water
0
0
50
100
150
200
volume (ml)
Figure 4. Equilibrium membrane extraction of 2,4-dichlorophenol (dcp) with pHA =
pKa + 1.0, leading to αA = 1/9. a: 100 ppb dcp in water (Ee = 9.1), b: 100
ppb dcp in 10 ppm humic acid (83% of a); c: 100 ppb dcp in 20 ppm humic
acid (65% of a) (unpublished student work; Christian Magnusson)
3.5 Chemical principles for metal extraction by SLM
Addition of ion-pairing reagents or chelating reagents to the donor phase permits the
SLM extraction systems to be used for various metal ions. Various carrier molecules or
ions can be incorporated in the membrane phase to enhance selectivity and mass
transfer, as well as trapping reagents in the acceptor phase preventing analytes to be
extracted back into the membrane. This was reviewed by Sastre et al [74] and by Buffle
et al [75].
As an analytical example of an addition of a reagent to the donor phase, we can
mention extraction of metals from solutions containing a complex former as 8Hydroxyquinoline. This forms extractable complexes with many metals [76]. See Figure
5a. The complex is transported through the membrane and the extracted analyte is
trapped in the acceptor by another ligand, in the cited work DTPA
(diethylenetriaminepentaacetic acid), which forms a stronger and charged complex.
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Chapter 7
Donor phase
2H2O
Membrane
2OH
2 Q-
Me 2+
Me(Q)2
4 SCN
Acceptor phase
-
2 HQ
Me(DTPA) 3-
Me(Q)2
H2DTPA 3-
-
4 SCN
2-
Me(SCN)4
2 R4NCl
-
Me(DTPA)3-
2+
Me
2 Cl
-
Me(SCN)4 (R 4N)2
DTPA
-
2+
2+
(HD) 2
Me
+
MeD 2
2H
2H
b
5-
2 Cl
Me
a
+
c
Figure 5. Membrane extraction schemes for of metal ions (Me). HQ = 8Hydroxyquinoline , DTPA5- = diethylenetriaminepentaacetic acid anion,
R4N+ = methyltrioctylammonium cation, HD = diethylhexyl phosphoric
acid. For a,b,c, see the text
The addition of a carrier to the membrane phase can be made in several ways. A
common carrier that has been used both for extraction of metals and for organic acids is
Aliquat-336 (methyltrioctylammonium chloride). It is a tertiary ammonium ion, i.e.
permanently positively charged in ion pair with chloride and it can be added to a
suitable membrane solvent. It has been used for extraction of Cu, Cd, Co, Zn [76] and
also for chromate [77]. With an addition of thiocyanate ions to the donor a negatively
charged metal-thiocyanate complex is formed in the donor and is extracted as ion pair
with the Aliquat-336 cation. The metal ion is eventually trapped in the acceptor using
DTPA as described above and presented in Figure 5b.
A commonly used extractant for metal ions is diethylhexyl phosphoric acid
(DEHPA) [49, 78]. See Figure 5c. In this case a pH gradient must exist over the
membrane, so the acceptor is kept more acidic than the donor (typically pH ≈ 1 and pH
≈ 3, respectively). Speciation of different chromium species (chromate and chromium
ion) has been performed by the combination of two membrane extraction systems, one
working with DEHPA for extraction of Cr3+ and the other with Aliquat-336 for
extraction of the chromate anions [77].
96
Chapter 7
4 THEORY AND PRINCIPLES OF MICROPOROUS MEMBRANE LIQUIDLIQUID EXTRACTION (MMLLE)
4.1 General
The MMLLE technique is a complement to the SLM extraction, permitting membranebased extraction to be extended to further classes of compounds and it can be performed
in similar membrane units as used for SLM. Also here, hydrophobic membranes are
used, typically PTFE or polypropene membranes. The acceptor phase is an organic
solvent, also filling the pores of the hydrophobic membrane. This forms an aqueousorganic two-phase system, with the organic phase partly in the membrane pores and
partly in the acceptor channel. MMLLE extends the concept of membrane extraction, as
it is best applicable to hydrophobic, preferably uncharged compounds i.e. those that
cannot be extracted with SLM. The extract is typically organic, not aqueous as with
SLM. Thus MMLLE is easier interfaced to gas chromatography and normal-phase
HPLC than SLM, which is best compatible with reversed-phase HPLC.
4.2 Differences from SLM
With SLM, the concept of trapping in a stagnant acceptor is central, but with MMLLE
and an organic acceptor, no trapping reactions are used. The maximum extraction
efficiency is directly given by the partition coefficient as in LLE, the only driving force
for the mass transfer being the attainment of distribution equilibrium between the
aqueous and organic phases. If the partition coefficient is very high, it is possible to
work with a stagnant acceptor and still get a considerable enrichment into a very small
extract volume. The extraction efficiency will be higher if the hydrofobicity of the
analyte is large; i.e. if the partition coefficient is large. With smaller partition
coefficients, the mass transfer can be improved if the acceptor phase is continuously or
intermittently flowing, removing the extracted molecules successively from the acceptor
and maintaining the diffusion through the membrane.
There is a recent example of MMLLE with the phases reversed, i.e. extraction of
phenolic compounds from an organic phase into an aqueous phase [79]. In this case the
phenols were trapped in a highly basic aqueous acceptor, which was kept stagnant
during the extraction.
4.3 Acceptor flow rate influence
To describe the influence of different experimental parameters, especially the donor and
acceptor flow rates (FD and FA, respectively) on the extraction efficiency E, a simplified
theory related to the SLM equations above has been derived [80] Assuming that the
partition is in equilibrium and that the flows in the channels are parallel, the following
equation for the extraction efficiency results:
E = 1 - FD / (FD + FA K)
(5)
As seen from this equation, compounds with large distribution coefficients K are
more efficiently extracted and the extraction efficiencies increase with acceptor flow
rate. A high acceptor flow rate maintains low concentrations of the analytes in the
acceptor phase and maximizes the concentration gradient leading to high mass transfer
rates.
97
Chapter 7
However, as the acceptor is flowing, the analyte is diluted, which leads to a
smaller concentration enrichment factor, Ee:
Ee = 1 / (1/K + FA/FD)
(6)
With a stagnant acceptor, the maximum value of the enrichment factors is:
Ee (max) = K
(7)
This equation for MMLLE should be compared to the corresponding equation
for SLM, Equation (4), which does not contain the value of the partition efficiency and
the maximum value of the enrichment factor is determined by the trapping conditions.
MMLLE is conceptually similar to continuous dialysis. Assuming equilibrium
between the phases and with the value of K equal to unity, Equations (5 - 7) are also
valid for that technique. To simplify the description it is assumed that the acceptor flow
is parallel to the donor flow. By reversing the acceptor flow (counter-current flow) the
concentration gradient between the two phases will be larger leading to more efficient
mass transfer. This situation is mathematically far more complex than when parallel
flow is assumed. For the corresponding problem in dialysis, numerical solutions to the
appropriate partial differential equation system have been derived [81].
In a recent paper [82], the MMLLE technique has been discussed in detail,
together with investigations of experimental conditions.
5 APPARATUS FOR LIQUID MEMBRANE EXTRACTION
5.1 Flow systems for membrane extraction
Simple and cheap membrane extraction flow systems for reasonably large sample
volumes (100 mL and up) can be built up around a peristaltic pump and membrane units
with channel volumes around 1 mL. An example of such a system is seen in Figure 6.
With this set-up, the acceptor phase is manually removed by the use of a syringe after
each extraction. Such systems have been used both for laboratory work [83,84] and for
sampling in natural waters [85] and in greenhouses [86].
Direct transfer of the acceptor phase from these extraction systems to an HPLC
can be arranged by means of a precolumn in order to inject as much as possible of the
extracted analyte, which will be contained in a volume of about 1-2 mL. See Figure 7.
Such systems can be completely or partially automated with pneumatically or
electrically actuated valves controlled by timers or by computer systems [67, 87].
It is also possible to use a heart-cut technique so a major part of the extract can
be accommodated in the injection loop for direct injection into the HPLC column
without a precolumn [53, 88]. This demands a more precise timing and pumping.
98
Chapter 7
Acid
Peristaltic
pump
Mixing
coil
Waste
Membrane
unit
Sample
Figure 6. Manual off-line instrument for liquid membrane extraction based on a
peristaltic pump After Knutsson et al [85]
Peristaltic
pumps
Membrane
unit
Water
Waste
Sample
Donor acid
pH 13 buffer
Mixing acid
Washing acid
Analytical
column
LC-pump
Precolumn
Figure 7. Apparatus for liquid membrane extraction with on-line connection to HPLC
for environmental studies After Nilvé et al [67].
99
Chapter 7
For smaller samples (<1 ml) the liquid delivery precision of peristaltic pumps is not
adequate and membrane extraction equipment based on syringe pumps connected to
what are called robotic liquid handlers can be applied [89,90]. A typical example is
shown in Figure 8. Here, a robotic needle connected to a syringe pump submits reagent
to adjust the pH of samples in vials, picks up an aliquot and passes it through the donor
channel of a small membrane unit (channel volume around 10 µl). The entire extract
collected in the acceptor channel is then transferred to an injection loop injector
connected to the HPLC system in the usual manner. Thus, the entire extract from, say, 1
mL sample ends up in one chromatographic injection. Typically, while one sample is
chromatographed, the next sample is extracted, so the cycle time of the system is
determined by the chromatogram time.
Membrane
unit
Donor buffer
Samples
LC-column
Acceptor buffer
LC-pump
Figure 8. Apparatus for SLM-HPLC determination of biomolecules in blood plasma or
urine. After Lindegård et al [90].
5.2 MMLLE-GC connection
For gas chromatography, the most suitable membrane extraction technique is MMLLE.
The organic acceptor is better compatible with GC than with LC, as are the analytes that
are best extracted in such a system, i.e. relatively hydrophobic compounds. One
approach is that the organic acceptor can be injected directly into a GC by means of a
large-volume injector [80]. A new development is the ESy instrument (Personal
Chemistry, Uppsala, Sweden) [23] where a MMLLE extraction in microscale (extract
volume ca 1.6 µl) is automatically performed and the organic extract is directly injected
into the GC by means of an injection needle, directly connected to the extraction cell.
This technology is currently under commercial development. See Figure 9.
100
Chapter 7
5
A
4
7
1
3
C
B
6
2
Gas Chromatograph
Figure 9. Schematic picture of the Extraction Syringe. For symbols, see the text. After
Norberg and Thordarson [23].
5.3 Off-line equipment
In contrast to the flow-system or robotic membrane extraction set-ups described above,
there are some examples of off-line devices utilising membrane extraction technology.
In this way a number of individual extraction units can be handled either manually or
with autosamplers in a parallel, off-line way. Most of these approaches are two-phase
systems.
Three-phase liquid membrane extraction in a porous hollow fibre was presented
by Rasmussen and Pedersen-Bjergaard [28, 91-93] under the name “Liquid Phase
Microextraction”. The fibre is impregnated with an organic phase to create both SLM
and MMLLE systems, depending on the nature of the acceptor phase, inside the fibres.
Physically, the fibre is arranged under the cover of an autosampler vial using two
hypodermic needles, thus creating a disposable extraction unit. See Figure 10. It has
been successfully applied to various drugs in biological samples. Similar devices have
been described by Lee and co-workers [94-96].
101
Chapter 7
Needle for injection
of acceptor solution
Screw top with
silicone septum
Needle for collection
of acceptor solution
Sample solution
(donor solution)
Porous hollow fibre
(impregnated with
organic solvent)
Vial
Acceptor solution
Figure 10. Off-line membrane extraction devices according to Grønhaug et al. [92].
6 APPLICATIONS
Membrane extraction has been applied in a number of analytes and matrices. For
bioanalysis, environmental, food, industrial analysis, a number of applications have
been published. In the review papers already mentioned above, for example [10, 16]
such applications are listed in detail and Table 2 gives some examples.
In bioanalysis, membrane techniques have been applied to the determination of
drugs but also to other compounds in biological fluids (blood plasma and urine). For
these applications selectivity is crucial as well as the possibility of automation. An
example is illustrated in Figure 11. Also for other applications in flow systems, similar
high degrees of selectivity have been demonstrated together with enrichment factors of
30-70 times. Off-line approaches have been applied to the extraction of sulfonylurea
and several other drugs in blood plasma, and other biological matrices.
102
Chapter 7
Figure 11. (a) Chromatograms of Amperozide (I), its metabolite (II) and homologue
(III) with the subsequent blank after enrichment from blood plasma. (b)
Corresponding chromatograms after enrichment from an aqueous buffer
solution. Concentrations 4µg/mL of I and II, 8 µg/mL of III. Reprinted from
Lindegård et al (90).
Pollutants and natural compounds have been determined in natural waters and
other environmental matrices. In these applications, various types of membrane
extraction provide high enrichment factors for determination of compounds in low
concentrations. Also the selectivity to discriminate against other naturally occurring
interferences, especially humic compounds, is important (see Figure 12). Compounds
that have successfully been extracted from natural waters with SLM include phenoxy
acids, sulfonylurea herbicides, phenolics, triazine herbicides, aniline derivatives, metal
ions and surfactants. MMLLE permits nonionizable compounds like toluene,
chlorobenzenes and naphthalene to be extracted.
103
Chapter 7
200
180
450
160
400
140
Ssignal (arb. units)
350
250
b
120
a
300
100
80
200
60
150
100
40
50
20
0
0
0
2
4
6
8
10
0
2
4
6
8
10
Tim e (m in)
Figure 12. Chromatograms (LC - UV) of methoxy-s-triazine herbicides. (a) SPEextraction of spiked river water (1.0 µg/l of each analyte); (b) SLMextraction of spiked river water (0.5 µg/l of each analyte). Peak designation:
1, Simetone; 2, Atratone; 3, Secbumetone; 4, Terbumetone. Reprinted from
Megersa et al. [103]
In food analysis, vitamin E was determined in butter and various pesticides in
eggs. Triazine herbicides were extracted from cooking oils by an MMLLE approach. In
an extension of the SLM extraction principle to solid or semi-solid samples it was
possible to successfully extract and quantify nicotine in snuff, vanillin in food samples
(e.g. chocolate) and caffeine in coffee and tea.
Some industrial applications membrane extraction, usually in the PMA or
MMLLE versions have been presented, mainly related to determination of phenols in
fuels, oils, gasoline, etc.
104
Chapter 7
TABLE 2
Selected applications of membrane extraction. Abbreviations are found in the text or are
generally used.
Analytes
Basic drugs
Organophosphate
esters
Lead
Phenoxy acids
Sulfonylurea
herbicides
Phenolics
Carboxylic
acids
Triazine
herbicides
Anilines
Metals
Anionic
surfactants
Cationic
surfactants
Fungicides
Semi-volatile
organics
Pesticides
Biogenic
amines
Vitamin E
Triazine
herbicides
Phenols
Matrices
Membrane
technique
Blood plasma SLM
Blood plasma, LPME
urine
Urine
SLM
Blood plasma MMLLE
Blood plasma MMLLE
Analytical
technique
HPLC
HPLC, GC,
CE
HPLC
GC
GC-MS
89, 98, 99
80
100
Urine
SLM
AAS
78
Natural water
Natural water
SLM
SLM
HPLC
HPLC
85, 101
67
Natural water
Nutrient
solutions
Soil liquid
SLM
SLM
HPLC
HPLC
87
86
SLM
IC
102
Natural water
SLM
HPLC
103-105
Natural water
Natural water
Natural water
Natural water
MMLLE
SLM
SLM
SLM
FIA
HPLC
AAS
HPLC
106
107
49, 76, 76
108
Natural water
MMLLE
HPLC
109
Natural water
Water
SLM, MMLLE
PME
HPLC
HPLC
110, 111
112, 113
Eggs
Wine
Wine
PME
MMLLE
SLM
HPLC
GC
HPLC
114
115
116
Butter
Cooking oil
PME
MMLLE
HPLC
FIA, HPLC
88
117
Mineral oil
Pyrolysis oil
PME
MMLLE
HPLC
LC-LC
118, 119
79
´
105
Ref
90
28, 94-96
Chapter 7
ACKNOWLEDGEMENTS
The membrane extraction work of the Lund group was supported by grants from
the Swedish Natural Science Research Council (NFR), the Swedish Environmental
Protection Agency (SNV), the Swedish Council for Forestry and Agricultural Research
(SJFR), the Swedish Institute, the Swedish International Development Co-operation
Agency (SIDA), the Crafoord Foundation and the European Community (DG XII). The
companies Pharmacia AB, Astra Draco AB and Astra Pain Control AB have contributed
with funds and interesting applications. The work would not have been possible without
a number of colleagues, graduate and undergraduate students as well as guest
researchers who have made important contributions
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