CHAPTER 7 MEMBRANE TECHNIQUES FOR ANALYSIS, SAMPLING

AND SPECIATION IN ENVIRONMENTAL MEASUREMENTS Jan Åke Jönsson1, Lennart Mathiasson1, Luke Chimuka2, Ewa Cukrowska3

1Analytical Chemistry, Lund University, P.O.Box 124, 221 00 Lund, Sweden, 2Department 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.

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.

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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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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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TABLE 1 Different major membrane techniques used in analytical applications [10, 11].

Name Abbreviation Type Phase combinations used Donor/membrane/ acceptor

Dialysis

- Porous membrane

Aq/membrane/Aq

Electrodialysis

ED Porous membrane

Aq

Supported-liquid membrane extraction

SLM Non-porous membrane

Aq/org/aq

Microporous membrane liquid- liquid extraction

MMLLE Non-porous membrane

Aq/org/org

Semi-permeable membrane devices

SPMDs Non-porous membrane

Aq/polymer/org

Polymeric membrane extraction

PME Non-porous membrane

Aq/polymer/aq, Org/polymer/aq, Aq/polymer/org,

Membrane extraction with sorbent interface

MESI Non-porous membrane

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.

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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 time-weighted 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

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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 fine-tuned 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.

Membrane (org) Acceptor (aq)Donor (aq)

Acid Base RCOO-RCOOH

RCOO-

RNH3+

Base Acid

RNH3+RNH2

NeutralNeutral

Condition 1:pH = pKa - 2

orpH = pKa + 2

Condition 3:pH = pKa + 3.3

orpH = pKa - 3.3

Condition 2:~ 2 ≤ logKow ≤ ~ 4

∆C

Figure 1. Principles of SLM extraction of ionisable organic compounds where the transport mechanism is simple permeation.

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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-n-octylphosphine 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-n-hexylether or n-undecane as membrane liquids, SLMs are stable from a week [66] to a

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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.

Accepto r channe lM em brane

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 = O-rings, 2 = polypropylene hollow fiber, 3 = fused silica capillaries, 4 = male nuts).

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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 (membrane-controlled 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.

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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.

0

0.2

0.4

0.6

0.8

1

0 0.05 0.10 0.15

E

0

20

40

60

80

100

120

E e

φ 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

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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.

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0 50 100 150 2000

250

500

750

1000

volume (ml)

c acc

epto

r (pp

b)

Water, Enrichment: 9.1

a

0 50 100 150 200 2500

250

500

750

1000

volume (ml)

c acc

epto

r (pp

b)

10 ppm humic acid: 83 % of water

b

0 50 100 150 2000

250

500

750

20 ppm humic acid: 65% of water

volume (ml)

c acc

epto

r (pp

b)

c

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 8-Hydroxyquinoline. 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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MembraneDonor phase Acceptor phase

2 HQ

Me(Q)2

2 Q -

Me(Q)2

2H O 2OH2-

Me2+

H DTPA

Me(DTPA)

23-

3-

2 R NCl

Me(SCN) (R N)

42-

2 ClMe2+

DTPA

Me(DTPA)

5-

3-

4 4 2

2 Cl -

4 SCN-

Me(SCN)4-

4 SCN-

(HD)

MeD

22+

2 H 2+

Me 2+

2 H+

Me

a

c

b

Figure 5. Membrane extraction schemes for of metal ions (Me). HQ = 8-

Hydroxyquinoline , 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].

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4 THEORY AND PRINCIPLES OF MICROPOROUS MEMBRANE LIQUID- LIQUID EXTRACTION (MMLLE) 4.1 General The MMLLE technique is a complement to the SLM extraction, permitting membrane-based 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 aqueous-organic 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.

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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.

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Waste

Mixingcoil

Membraneunit

PeristalticpumpAcid

Sample

Waste

Mixingcoil

Membraneunit

PeristalticpumpAcid

Sample

Figure 6. Manual off-line instrument for liquid membrane extraction based on a

peristaltic pump After Knutsson et al [85]

WasteMembrane

unit

Peristalticpumps

Donor acid

Sample

Water

Washing acid

Mixing acid

pH 13 buffer

Precolumn

LC-pump Analyticalcolumn

WasteMembrane

unit

Peristalticpumps

Donor acid

Sample

Water

Washing acid

Mixing acid

pH 13 buffer

Precolumn

LC-pump Analyticalcolumn

Figure 7. Apparatus for liquid membrane extraction with on-line connection to HPLC for environmental studies After Nilvé et al [67].

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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.

LC-pumpLC-column

Donor buffer

Acceptor buffer

Membraneunit

Samples

LC-pumpLC-column

Donor buffer

Acceptor buffer

Membraneunit

Samples

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.

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Gas Chromatograph

A

C B

174

6 2

3

5

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].

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Needle for injectionof acceptor solution

Needle for collectionof acceptor solution

Porous hollow fibre(impregnated withorganic solvent)

Acceptor solution

Sample solution(donor solution)

Screw top withsilicone septum

Vial

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.

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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.

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Figure 12. Chromatograms (LC - UV) of methoxy-s-triazine herbicides. (a) SPE-

extraction of spiked river water (1.0 µg/l of each analyte); (b) SLM-extraction 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.

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TABLE 2 Selected applications of membrane extraction. Abbreviations are found in the text or are generally used. Analytes Matrices Membrane

technique Analytical technique

Ref

Basic drugs Blood plasma SLM HPLC 90 Blood plasma,

urine LPME HPLC, GC,

CE 28, 94-96

Urine SLM HPLC 89, 98, 99 Blood plasma MMLLE GC 80 Organo-phosphate esters

Blood plasma MMLLE GC-MS 100

Lead Urine SLM AAS 78 Phenoxy acids Natural water SLM HPLC 85, 101 Sulfonylurea herbicides

Natural water SLM HPLC 67

Phenolics Natural water SLM HPLC 87 Nutrient

solutions SLM HPLC 86

Carboxylic acids

Soil liquid SLM IC 102

Triazine herbicides

Natural water SLM HPLC 103-105

Natural water MMLLE FIA 106 Anilines Natural water SLM HPLC 107 Metals Natural water SLM AAS 49, 76, 76 Anionic surfactants

Natural water SLM HPLC 108

Cationic surfactants

Natural water MMLLE HPLC 109

Fungicides Natural water SLM, MMLLE HPLC 110, 111 Semi-volatile organics

Water PME HPLC 112, 113

Pesticides Eggs PME HPLC 114 Wine MMLLE GC 115 Biogenic amines

Wine SLM HPLC 116

Vitamin E Butter PME HPLC 88 Triazine herbicides

Cooking oil MMLLE FIA, HPLC 117

Phenols Mineral oil PME HPLC 118, 119 Pyrolysis oil MMLLE LC-LC 79

´

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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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