Phospholipids and liposomes in liquid chromatographic and capillary electromigration techniques

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Trends in Analytical Chemistry, Vol. 23, No. 8, 2004

Phospholipids and liposomes in liquid chromatographic and capillary electromigration techniques Susanne K. Wiedmer , Minttu S. Jussila, Marja-Liisa Riekkola The structural resemblance of liposomes to natural cell membranes has caused them to be employed in liquid chromatographic and capillary electromigration techniques to study the interactions between analytes and phospholipid membranes. Phospholipids and liposomes are immobilized in stationary phases for liquid chromatography by a variety of techniques, to which we give special attention. We present selected applications to the separation of analytes. Though still few, applications in capillary electromigration are particularly attractive because of the small amounts of liposomes and samples that are required. We demonstrate the promising use of liposomes as carriers and coating materials in capillary electrophoresis with reference to recent applications. We note future directions for the utilization of phospholipids and liposomes in liquid chromatographic and capillary electromigration techniques. ª 2004 Elsevier Ltd. All rights reserved.

perazin-1-ium iodide; MLV, multilamellar vesicle; MVV, multivesicular vesicle; NSAID, non-steroidal anti-inflammatory drug; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PED, N0 -(1,2-dimyristoyl-sn-glycero3-phosphoethyl)-N-[m-[3-(trifluoro-methyl)-diazirin3-yl] phenyl]-thiourea; PG, phosphatidylglycerol; PI, phosphatidylinositol; PLA2 , pancreatic phospholipase A2 ; POPC, 1-palmitoyl-2-oleyl-sn-glycero3-phosphocholine; PS, phosphatidylserine; SEC, size exclusion chromatography; SMUBS, standardized modified universal buffers; STA, stearylamine; SUV, small unilamellar vesicle; Tm , main phase-transition temperature; TNBS, 2,4,6-trinitrobenzene sulfonic acid

Keywords: Capillary electromigration techniques; Liposomes; Liquid chromatography; Phospholipids

1. Introduction

Susanne K. Wiedmer*, Marja-Liisa Riekkola* Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, 00014 University of Helsinki, Helsinki, Finland Minttu S. Jussila The Finnish National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland

*Corresponding authors. Tel.: +358-9-19150253; E-mail: susanne.wiedmer@ helsinki.fi; [email protected]

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Abbreviations: BGE, background electrolyte; BiotincPE, 1,2-dioleylphosphatidylethanolamine-N-(cap biotinyl); Calcein, 3,3-bis[N,N-di(carboxymethyl)aminomethyl]fluorescein; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG, 1,2dimyristoyl-sn-glycero-3-phosphatidyl glycerol; DPBS, Dulbecco’s phosphate-buffered saline solution; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; EPL, egg phospholipid; EYP, egg yolk phospholipid; Glut 1, glucose transporter; HEPES, N-(2-hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid); HPLC, high-performance liquid chromatography; IAM, immobilized artificial membrane; x IAM.PCy , PC derivatives immobilized on silica propylamine particles – x indicates the linkage between the acyl chains and the glycerol backbone of the lipid – y indicates endcapping of residual amino groups; ILC, immobilized liposome chromatography; LDL, low-density lipoprotein; LECC, liposome electrokinetic capillary chromatography; LSER, linear solvation energy relationship; LUV, large unilamellar vesicle; M1C4, 1-(4-iodobutyl)-1,4-dimethylpi-

Within the last 25 years, a promising new field of chromatography has opened up with the immobilization of phospholipids and liposomes in columns and their use as biomimetic membranes to study interactions between analytes and natural membranes. Phospholipids are the main components of natural membranes [1]. In eukaryotic cells, the most common phospholipids are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI). Most natural phospholipids are either zwitterionic or negatively charged. Liposomes are vesicles formed by aggregation of amphiphilic phospholipid molecules [1,2]. Structurally, the liposomes closely resemble natural cell membranes, and they are extensively used

0165-9936/$ - see front matter ª 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2004.03.001

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in medical and pharmaceutical research as models for mimicking the structure and the function of cell membranes. One important application is the determination of membrane distribution coefficients of drug molecules to assess the ability of drugs to penetrate the cell membrane and be absorbed in the body. Interactions between analytes and phospholipid membranes depend on the characteristics of both analytes and membrane. Temperature is an essential parameter to consider when dealing with biological membranes since, above the main phase-transition temperature (Tm ), membrane bilayers exist in a fluid state and, below Tm , in a wellordered gel state [2]. Under normal conditions (i.e., at body temperature), biological membranes will generally be in the fluid state. The length of the acyl chains, the degree of saturation, the structure of the polar head group, and the activity of water all influence the phasetransition temperature of the phospholipids. Liposomes are divided into groups depending on their size and the physical structure of the membrane. Small unilamellar vesicles (SUVs) are in the range 25–100 nm and large unilamellar vesicles (LUVs) in the range 100–1000 nm. Multilamellar vesicles (MLVs) are in the size range 100 nm–20 lm [3]. In addition, there are multivesicular vesicles (MVVs, 100 nm–20 lm), which are composed of several small vesicles. In this review, we describe the use of phospholipids and liposomes in liquid chromatography (LC) and capillary electromigration techniques. Phospholipid vesicles are denoted liposomes throughout the text. In both techniques, phospholipids are commonly used as SUVs and LUVs in the fluid state. MLVs have generally been avoided because of their heterogeneous properties. Most attention is paid to LC, where phospholipids and liposomes are most widely used. In immobilized artificial membrane (IAM) chromatography, single phospholipids are covalently bound to propylamino silica particles, whereas, in immobilized LC (ILC), the stationary phase is prepared by immobilizing preformed liposomes into hydrophilic gels. The major advantage of IAM over ILC is that the stationary phases are commercially available. At the moment, stationary phases containing immobilized liposomes must be prepared in the laboratory because their stability and the reproducibility of the preparation are so poor that commercialization is not yet profitable. IAM particles differ structurally from liposomes and their field of application is limited. Interactions of analytes with phospholipid membranes have been extensively studied. IAM chromatography and ILC have also been employed for purification and refolding of proteins and for investigating the interactions between analytes and proteoliposomes. The focus of this review is the immobilization of liposomes and the characterization of phospholipid- and liposome-based stationary phases in LC. In addition, we describe the promising new applications of liposomes in capillary

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electromigration techniques and note the most innovative applications.

2. Phospholipids and liposomes in stationary phases for LC The first LC phospholipid-containing stationary phases were introduced some 20 years ago. Typically, they were prepared by immobilizing phospholipid molecules onto various stationary materials. PC and sphingomyelin were immobilized on agarose gel [4], PS and cholesterol were immobilized on polyacrylamide [5], egg PC was coated onto polystyrene-divinylbenzene particles [6], and PC was immobilized on silica gel [7]. The 1990s saw the emergence of several new techniques for the preparation of liposome stationary phases, and various applications were demonstrated. Today, stationary phases containing phospholipids or liposomes are prepared in two ways essentially. IAMs are prepared by the covalent binding of single phospholipids or their analogues to propylamino molecules on the surface of silica particles [8]. The surface area of one propylamino molecule on a silica particle is approximately 100
A2 , but only 60% of the silicapropylamino groups are available for binding of phospholipids [9]. Commercial IAM phases typically contain silicapropylamino particles with diameters of 12, 7, or 5 lm and pore sizes of 300 or 100
A [10–12]. Immobilized liposomes, in turn, are prepared by immobilizing liposomes in hydrophilic gels, such as those commonly used in size exclusion chromatography (SEC). Most of the common SEC gels are agarose derivatives, but agarose–dextrane mixtures and methacrylate-based gels are also used [13]. The pore size of the gel depends on the gel and the gel composition, and there is wide variation in the pore size (i.e., 7–500 nm). The first liposome stationary phases were based on hydrophobic or steric interactions between liposomes and the gel. Later, other techniques were developed, such as immobilization through covalent interactions and the avidin–biotin method. Dynamic coating of columns with phospholipids has also been employed. 2.1. Immobilized artificial membranes Although the hydrophobic chains of immobilized phospholipids may differ considerably from those of natural membranes, usually the polar head groups are similar, allowing immobilized artificial membranes to mimic cell membranes. The most common phospholipid in natural membranes is PC, and most IAMs have been based on PC. However, because of the zwitterionic character of PC, better models for cell membranes are IAMs that are slightly negatively charged, such as those containing PS. The first IAM stationary phase in high-performance LC (HPLC) was introduced in 1989 [14]. PC was bound http://www.elsevier.com/locate/trac

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separation of a large number of acidic, basic, and neutral drugs, including b-blockers, steroids, and arylpropionic acids [15–18,53]. The highest stability has been obtained by endcapping amino groups first with decanoic (C10) anhydride and then with propionic (C3) anhydride. Unfortunately, the resulting ester IAM.PCC10=C3 phase is highly hydrophobic and does not mimic natural membranes very well [8]. Not only are the particles poorly soluble in water [9], but they are also highly unstable under acidic conditions [19]. In ether IAM.PCC10=C3 phases, there is only one fatty acid chain, which means that the particles are less hydrophobic and can be immobilized at higher surface density. Ether IAM.PC phases are very stable, but their preparation is time-consuming and laborious. It needs to be added that endcapping of IAM phases protects the stationary phase in two ways: not only is the chemical stability improved by decrease in the basicity of the surface, but also the dense packing of alkyl chains between the phospholipids and the endcapping ligands hinders the solvent from eroding the silica surface [11]. In general, IAMs are highly stable and they are commercially available. IAM phases have been shown to

to spherical silicapropylamino particles and the synthesized chromatographic support was used for the separation of hydrophilic cysteine-containing peptides and three acidic proteins (trypsin inhibitor, albumin, and ovalbumin). Later, various PC derivatives were immobilized on silicapropylamino particles. In the beginning, these phases were denoted IAM.PC, but later the abbreviation x IAM.PCy was introduced (Fig. 1), where the left superscript (x) describes the linkage between acyl chains and the glycerol backbone of the phospholipids and the right superscript (y) the endcapping of residual amino groups. Unreacted primary amino groups on IAM particles make the surface basic, causing acidic compounds to be strongly retained on the surface and decreasing the chemical stability of the phase [11]. Various endcapping reagents (silylating reagents, acetyl analogues, glycidol, methylglycolate, and anhydrides) have been used to eliminate the effect of amino groups, but all induce loss of phospholipids from the silica surface. Endcapping with methylglycolate (MG) generates hydroxyl groups on the silica surface, making the particles less like natural biological membranes. Despite this, IAM.PC.MG-phases have been used for the

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Figure 1. Structures of phosphatidylcholine immobilized artificial membrane phases: (a) (c) dG IAM.PCC10=C3 .

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last for hundreds of runs. Variations in their stability are because of the structure of the phospholipid ligand and the different endcapping groups linked to the material. The lowest stability has been observed for nonendcapped columns. However, the values reported for column stabilities vary widely [16,17,53]. The performance of IAM phases has been studied by measuring the loss of phospholipids during chromatographic runs, or during storage. Also, changes in retention times or retention factors can be used to measure the stability of the columns. The silica propylamino particles used as IAM support are structurally stable and rigid [11], and chemically stable in solutions with pH values lower than 8. Theoretically, IAM stationary phases could be stable over the pH range 2–8, because PC liposomes are stable in the pH range 2–11 [20]. Nevertheless, the stability of phases containing different ligands varies widely. As noted above, the PC stationary phase ester IAM.PCC10=C3 cannot withstand acidic solutions [19], whereas ether IAM.PCC10=C3 is highly stable under acidic conditions. Unfortunately, synthesis of the latter is too complex to allow commercial preparation. More commonly used is dG IAM.PCC10=C3 , which is much easier to synthesize. The superscript dG denotes the deletion

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of the glycerol backbone from PC (Fig. 1). The ether IAM.PCC13COOH=C3 phase containing carboxylic acid end groups has been shown to be stable at pH 8.5 [21]. Endcapped IAM phases are stable in aqueous buffer solutions, but can also withstand several organic solvents (e.g., methanol, chloroform, acetonitrile, and acetone [25]) and surfactants [22]. On the other hand, an IAM.PC phase that is not endcapped is stable in acetone and acetonitrile. Chloroform may cause great loss of phospholipids from the phase [10]. Most loss of phospholipids from IAM phases is explained by breakage of the ester bond, and not by hydrolysis of silica or the amide bond. IAM phases have also been prepared with anionic PG, PS, phosphatidic acid (PA), or zwitterionic PE [8] (Fig. 2). In the preparation of mixed phospholipid IAM phases, PC is first bound to the silica particles and the second phospholipid is then immobilized. Usually, the surface density of the second phospholipid is about 6–10%, corresponding well to the density of natural phospholipids [23]. Elimination of possible interactions between the polar head groups of the phospholipids and the silicapropylamino particles, requires protection of the phospholipid functional groups during immobilization

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[9,19,24]. Since the later deprotection step is carried out under acidic conditions, the immobilized phospholipids must contain an ether linkage between the glycerol backbone and the acyl chain to prevent them from breaking down during synthesis or the deprotection step. In the case of IAM.PC, the protection step is not needed. The structure and characteristics of IAM phases have been studied by several non-chromatographic methods: elemental analysis [9,11]; surface area measurements; Fourier transform infrared spectroscopy [10]; and, nuclear magnetic resonance spectroscopy [9,11,14,25]. An important topic has been determination of the density of the phospholipids immobilized on the silica particles [9]. The surface density of phospholipids in natural membranes is approximately 67–77
A2 /molecule [14], whereas the densities in immobilized artificial membranes have been about 66–105
A2 /molecule [9,12]. Hence, most prepared IAM phases have phospholipid densities close to the density of natural membranes. The density of the phospholipids on IAM particles is mostly determined by the characteristics of the phospholipids (i.e., the amount of phospholipid chains and the structure and size of the polar head group of the immobilized phospholipid) and by the properties of the silicapropyl particles. The density can be increased by repeating the immobilizing process several times. 2.2. Liposomes immobilized into gel matrix Liposome-containing stationary phases can also be prepared by incorporating phospholipid vesicles, (i.e., liposomes) directly into the gel of LC stationary phases. We describe the several different ways of preparing these phases and their characteristics in the following sub-sections. 2.2.1. Immobilization by hydrophobic interactions The first liposome stationary phases were prepared by hydrophobic interactions [26] where hydrophobic ligands able to interact with the hydrophobic parts of the phospholipid membrane were added to a gel matrix. In 1987, Sandberg et al. [27] described a technique based on the immobilization of liposomes on hydrophobic alkyl derivatives of agarose gel beads. The liposomes, which were small or large unilamellar vesicles, consisted of mixtures of PC and PE (80/20 mol%), while the ligands were mainly butyl-, octyl-, or dodecyl-sulfides. The density of immobilized liposome phases is typically expressed as the amount of phospholipids in the gel (i.e., as lmol phospholipid/ml gel). The amount of phospholipids in the stationary phases is usually determined by the method introduced by Bartlett in 1959 [28], where total phosphorus is calculated from the amount of reduced phosphomolybdate in eluates from chromatographic columns. The reduction involves the formation of a blue color, the intensity of which depends on concentration. 566

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The internal volume of immobilized liposomes can be determined through incorporating an easily detected compound, such as calcein (3,3-bis[N,N-di(carboxymethyl)aminomethyl] fluorescein) in the aqueous part of the immobilized vesicles. Calcein is a self-quenching fluorescing compound, the yellow-greenish fluorescence of which is observed under acidic conditions. A known concentration of calcein is added to the phospholipid suspension [26] and, after vesicle formation, excess calcein is removed by flushing, and the calcein-containing liposomes are immobilized in the gel. For determination of the liposome internal volume, the membrane structure of the immobilized liposome is broken up by rinsing with a surfactant solution (including cholate or octyl glucoside). Calcein is released and detected by fluorometry. The liposome internal volume (reported as ll/ml gel) is calculated from the amount of released calcein, the original concentration of calcein, and the amount of immobilized phospholipids [46]. In work by Sandberg et al. [27], the density of alkylsulfide ligands in the gel was 8–10 lmol/ml. The immobilization process took 1–20 h, depending on the gel and the size of the liposomes. The density of the immobilized liposomes was only 1–3 lmol of phospholipids/ml gel with LUVs, and 20–100 lmol of phospholipid/ml gel with SUVs. The highest phospholipid densities were obtained using 4% agarose gel with octylsulfide ligands. The amount of immobilized phospholipid was shown to depend on not only the size of the liposome but also the concentration of the ligand [29]; the highest phospholipid densities were obtained with small-sized liposomes and gels with high alkylsulfide ligand concentrations. However, in studies on membrane penetration using calcein as a marker, the largest calcein leakage occurred with small liposomes and high alkylsulfide ligand density in the gel. Accordingly, in immobilization by hydrophobic interactions, the best alternative for obtaining stable membrane structures would appear to be relatively low ligand densities and mid-sized liposomes. Immobilization of small PC liposomes on a polymer gel support by hydrophobic anchors containing a disulfide linkage was studied by Khaleque et al. [30]. The liposomes could be released from the gel matrix for further study, through reductive cleavage of the disulfide linkage. Up to 58% of the immobilized phospholipid was released in this way. The immobilization of egg PC liposomes in gels with covalently linked octyl or propyl ligands was investigated by Zhang et al. [31]. Fusedsilica capillaries (ID, 320 lm) were polymerized with piperazine diacrylamide/methyl acrylamide and the gel was derivatized with alkyl chains by adding epoxy alkanes and boron trifluoride ethyletherate. Liposomes were then immobilized by hydrophobic interactions. Recently, LUVs of DMPC were adsorbed onto C-18-RP and C-30-RP packed columns and utilized for the

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Figure 3. Separation of anions using 10.0 mM NaCl (a) and 5.0 mM CaCl2 (b) as eluent. C18 /DMPC stationary phase, C18 -packed column (250
4.6-mm i.d.) coated with DMPC; flow-rate: 1.0 ml/min. Sample: 0.1 mM each of NaIO3 , NaI, NaBrO3 , NaNO2 , NaNO3 , NaSCN, and Na2 S2 O3 ; injection volume: 20 l. Detection: UV absorbance at 210 nm. Peaks: 1, S2 O32 ; 2, IO3 ; 3, BrO3 ; 4, NO2 ; 5, NO3 ; 6, I ; and 7, SCN (reprinted from [32], with permission).

determination of iodide and thiocyanate in highly saline water samples (Fig. 3) [32]. The separation depended on both the type of RP material and the eluent employed. Liposome bilayer permeability has usually been studied with calcein. The permeability is studied during the immobilization process, during use of the immobilized phase or during storage. In some immobilization techniques, the liposomes may undergo strong mechanical stress. For example, when liposomes are immobilized by hydrophobic interactions the liposome structure may change because of alkyl ligands (in the gel) penetrating the membrane. However, after immobilization, the phospholipid bilayer structure usually recovers and calcein leakage ceases [26]. 2.2.2. Immobilization by steric interactions In immobilization of liposomes by steric interactions, the liposomes are entrapped in the gel-bead pores by dialysis or by freeze-thawing [26]. In the first case, liposome formation takes place in the gel beads during dialysis and the liposomes that are formed are immobilized in the gel [33]. In the second case, small liposomes are introduced into the gel, are allowed to fuse and to grow, and thereafter are immobilized in the gel [34]. 2.2.2.1. Dialysis. The first steric immobilization of liposomes by dialysis was demonstrated in 1989 [33]. When

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a mixture of surfactants, phospholipids, and gel particles was dialyzed, liposomes were formed both inside and outside the gel. The success of the technique is based on the steric entrapment of large liposomes in the gel [26]. Liposomes have been immobilized by dialysis in several different gels, using many different phospholipid mixtures and even liposomes containing membrane proteins [26,33,35,36]. Before immobilization, phospholipids are solubilized in a surfactant (e.g., cholate or octyl glucoside) solution where the concentration of surfactant exceeds the critical micelle concentration [26,33]. Typical gels used for the immobilization are agarose and copolymers of allyldextrane and N,N0 methylenebisacrylamide. The phospholipid solution is pumped through a gel-filled column, after which the gels are transferred to a dialysis vessel [33]. Mixing phospholipids and gel can also be done by vortexing [37]. At first, both free surfactant monomers and phospholipid–surfactant micelles are entrapped in the gel, but, during the dialysis, the surfactant monomers are removed from the vessel. As the phospholipid–micelle molar ratio changes, large phospholipid aggregates begin to form. A part of the liposomes form in the pores of the gel and the largest will be entrapped inside and be immobilized. As a final step, non-immobilized liposomes are removed by washing and centrifugation, and the immobilized liposome gel is packed into a column. The whole process is carried out at temperatures above the main phase-transition temperature of the phospholipids (i.e., in the fluid state of the phospholipid [33]). The immobilization capacity can be increased through the addition of calcium, which increases the fusion of vesicles [36]. The dialysis process is relatively slow and may take several days. The amount of immobilized liposomes depends on phospholipid concentration, liposome diameter, and the pore size of the gel. 2.2.2.2. Freeze-thawing. In 1994, Yang and Lundahl [34] investigated steric immobilization of liposomes by three different methods: freeze-thawing; freeze-drying and rehydration; and, reverse-phase evaporation. All techniques are common in the preparation of liposomes, but this was the first time they were applied for the immobilization of liposomes into gels. Only freezethawing has been applied in later immobilization studies. In the freeze-thawing technique, small phospholipid vesicles of desired phospholipid composition are prepared, after which the liposome suspension is concentrated up to 20-fold. The concentrated liposome suspension is mixed with the dry gel and left to swell for 0.5–3 h, depending on the dryness of the gel. As the gel swells, small liposomes are entrapped in the gel pores. The mixture is then frozen at )70 to )75
C for 10–15 min, thawed, and kept at room temperature for an hour. The process may be repeated several times. During the freeze-thawing process, the liposomes will grow in size and cannot escape the gel. Non-immobilized liposomes are removed by washing and centrifugation. Several liposome compositions have been used and the amount of immobilized liposomes depends greatly on http://www.elsevier.com/locate/trac

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the phospholipid concentration, the composition of the liposomes, the gel, and the method by which the gel is dried. The amount of immobilized liposomes is increased at higher phospholipid concentration and with use of freeze-dried gel instead of moist gel. In an investigation of different lipid compositions, Yang and Lundahl [34] observed that addition of cholesterol to the phospholipid mixture (70% PC, 21% PE, 9% other phospholipids and lysophospholipids) increased the amount of immobilized liposomes, whereas charged phospholipids, such as PS and stearylamine, had the reverse effect. Brekkan and Lundahl [38] found that a part of the liposomes are immobilized in the gel even without the freeze-thawing cycle (about 20 lmol phospholipid/ml of gel, independent of the concentration of the phospholipid suspension). The freeze-thaw process increased the amount of immobilized phospholipids only if the concentration of the phospholipid suspension exceeded 45 mM. With the freeze-thaw technique, the internal volume of the liposomes depends greatly on the phospholipid concentration and on the characteristics of the gel [38]. When a PC/PE phospholipid mixture (70% PC, 21% PE, 9% other phospholipids and lysophospholipids) was immobilized in an allyldextrane-N,N0 -methylenebisacrylamide crosslinked copolymer gel, both the internal volumes and the specific internal volumes depended on the phospholipid concentration and the dryness of the gel [34]. (The specific internal volume is the liposome internal volume relative to the amount of immobilized phospholipids.) When a moist gel was used, the liposome internal volume increased rapidly with phospholipid concentration, but, at the same time, the specific internal volume of the liposome decreased. Even though larger amounts of liposomes are immobilized with freeze-dried gels than with moist gels, the liposome internal volumes are clearly smaller with freeze-dried gels. Usually large liposome internal volumes are preferred and especially large specific internal volumes, because, if the specific internal volume is very small, there may be undesirable multilamellar liposomes in the solution. When liposomes are immobilized into gels it needs to be kept in mind that, even though the immobilized amount of liposomes usually increases with the phospholipid concentration, the size and the lamellarity of the liposomes increase as well. Determination of the external surface area of immobilized liposomes is particularly important when immobilization is done by steric interactions, because in that case liposomes are fully formed or partially increase in size in the gel during immobilization. With other techniques, the external surface area can be determined before immobilization. One technique for studying the external liposome surface area, applicable to liposomes containing primary amino groups (e.g., PE), involves the use of 2,4,6-trinitrobenzene sulfonic acid (TNBS) [38]. The reaction between the liposome and TNBS is carried out without and with detergent. In the absence of 568

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detergent, TNBS will react with phospholipids only on the surface of the membranes, whereas, in the presence of detergent, it will react with phospholipids also on the inner side of the membrane. It is assumed that TNBS is evenly distributed between the external and the internal surfaces of the membrane. The ratio between the liposome external surface and the total liposome surface can then be determined photometrically. Part of the TNBS may penetrate the phospholipid membrane, leading to results that are slightly too large, but, with different incubation times, the external surface areas plotted as a function of time can be extrapolated to time zero, and a more correct value for the external liposome surface is then obtained. The stability of columns containing liposomes immobilized by dialysis is usually rather poor [35,36]. The loss may be over 20% after a few chromatographic runs. In practice, the same column can be used for 2 or 3 days only. Stationary phases containing liposomes immobilized by freeze-thawing are considerably more stable. When PC-containing liposome phases prepared by freeze-thawing were used for interaction studies between drugs (including b-blockers and steroids) and membranes, the stationary phases could be used for 2–3 weeks for over 100 chromatographic runs, and the € phospholipid loss was less than 5% [38,59]. Osterberg et al. [60] used immobilized liposome phases prepared by freeze-thawing in drug analyses for close to a year without significant change in the retention of compounds. Various drugs, including b-blockers, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, benzodiazepines, local anesthetics, and peptides, were investigated. The amount of phospholipids retained in the gel beads after 9–12 months use was 55–65% of the phospholipids present before packing; however, half or more of the losses occurred during packing of the columns. Liposomes composed of a photoreactive negatively charged phospholipid (N0 -(1,2-dimyristoyl-sn-glycero3-phosphoethyl)-N-[m-[3-(trifluoromethyl)diazirin-3-yl] phenyl]-thiourea (PED)) and PC (2:1) were immobilized by freeze-thawing in copolymer (crosslinked allyldextrane and N,N0 -methylenebisacrylamide) gel beads and the phase was then irradiated in the hope of improving the stability through the formation of covalent bonds between the PED phospholipids and the gel matrix [34]. The stability of this phase was indeed improved, but the amount of immobilized liposomes was less than with a corresponding non-irradiated stationary phase. In addition, the polar head groups of PED seemed to couple to neighboring phospholipids upon photoactivation, leading to some disappearance of the negative charge of PED. 2.2.3. Immobilization by covalent interactions In the presence of appropriate ligands in the gel, liposomes can also be covalently bound to gel matrices [39].

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Knowledge of the covalent binding of compounds containing primary amino groups (proteins, peptides) to gels activated with certain chemicals suggested that lipsomes containing primary amino groups (e.g., PE) could be mmobilized in gels by the same technique [29,40]. The immobilization process starts with activation of the gel with 4-nitrophenylchloroformate, cyanobromide or N-hydroxysuccinimide. Mostly, gels of methacrylate or agarose have been used, with 4-nitrophenylchloroformate as the activator [41–44]. The covalently bound immobilized liposome columns have been prepared by mixing liposomes containing a primary amino group with the activated gel, or by flushing the liposome suspension through the activated gel column. After covalent binding, excess activated groups were protected by treating the gel with 0.5 M ethanolamine. If 4-nitrophenylchloroformate was used in the activation of the gel, the excess groups could be removed by hydrolysis with base. The amount and the yield of liposomes immobilized in activated gels depend on the liposomes (size and composition) and on the characteristics of the gel and activator [30]. Temperature is also important; the immobilization efficiency was better when immobilization was carried out at lower temperatures (4–10
C). When liposomes of PC (no primary amino group) were used, the best results were obtained with gels activated with 4-nitrophenylchloroformate [39]. When the phospholipid concentration was increased, the amount of immobilized liposomes increased. In general, at the same phospholipid concentration, SUVs were immobilized with higher efficiency than LUVs. The presence of primary amino groups was found to be unnecessary with a chloroformate-activated gel, and the addition of 1,2-dioleylphosphatidyl-ethanolamine-Ncaproylamine (am-ino groups) to the phospholipid mixture had no influence on the amount of liposomes immobilized by covalent linkage. Egg PC liposomes have been immobilized on Sephacryl (crosslinked copolymer of allyldextrane and N,N0 methylenebisacrylamide) gel particles through the in situ formation of disulfide linkages [45]. Both the gel particles and the liposomes contained thiol groups, and the liposomes could be efficiently adsorbed and released. The amount of immobilized phospholipids varied between 61% and 93%, depending on the density of the mercapto moieties on the liposome surface and on the ratio of the liposome thiol to the gel particle pyridinethiol groups. Within 6 h, 80–85% of the immobilized PS was released, and, in double the time, the release was almost complete. The stability of covalently immobilized liposomes is good. Egg PC liposomes immobilized in methacrylatebased gels activated with 4-nitrochloroformate were stable during storage for a month [39]. However, there was a 4% decrease in the retention volumes of 15 studied drugs after 30 chromatographic runs. More-

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over, the structure of the liposome membrane changed during the coupling and washing steps, as evidenced by a strong leakage (over 67%) of calcein (used as a marker) from the liposomes. But, covalently immobilized liposomes are very stable at high osmotic pressures, and elution with denaturing guanidine chloride released only 30% of liposomes immobilized in Superdex 200 (dextrane covalently bound to highly cross-linked porous agarose beads) over a period of 200 runs, and no phospholipid leakage was observed after 50 runs on a liposome-immobilized methacrylate-based column [42]. The amount of phospholipids immobilized in ILC phases by covalent binding depends on the phospholipid concentration and composition, the size of the liposome, the gel matrix, the density and the chemical characteristics of the active groups in the gel, the gel pore size, and the immobilization time. 2.2.4. Immobilization by avidin–biotin technique In 1998, Yang and co-workers [46] presented a technique by which unilamellar vesicles can effectively be immobilized in gel matrixes by avidin–biotin binding. The technique yielded stable liposome-immobilized gels, partly because of the narrow size distribution of liposomes that was obtained, and the resultant homogeneous stationary phase. The liposomes contained phospholipids with attached biotin molecules, and these biotin-modified liposomes were immobilized in avidin- or streptavidin-derivatized gels. The avidin–biotin technique relies on the very strong interaction between biotin (a B-vitamin that acts as a coenzyme in several carboxylation reactions) and avidin (a glycoprotein in raw egg white with a molar mass of 66 kDa). The gel that was used (usually agarose, a methacrylate derivative, or a crosslinked copolymer of allyldextrane and N,N0 -bisacrylamide [46–48]) was first activated, typically with 4-nitro-phenylchloroformate. The ligand density varies with the gel and values between 5 and 60 lmol choloroformate/g gel have been reported. Avidin was covalently bound to the activated gel with densities of approximately 3 mg of avidin/ml of gel. Unreacted chloroformate could be protected by rinsing the gel with 0.5–1 M ethanolamine [46,47,49]. Liposome immobilization was achieved by mixing the avidin-activated gel with biotin–liposomes. The immobilization process is simple and reproducible, and the chromatographic phase obtained is stable and homogeneous [46]. The phospholipid densities have typically been about 35–47 lmol/ml using SUVs, and about 24–43 lmol/ml using LUVs. With gel of very small pore size, the amount of LUVs immobilized remained below 5 lmol/ml. Also, liposomes other than PCs have been investigated, and slightly smaller amounts of immobilized phospholipids have been obtained using charged liposomes. The biotin moiety http://www.elsevier.com/locate/trac

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needs to be linked to PC by a short hydrocarbon chain, because, in the absence of this chain, the amount of immobilized liposomes in gel was found to decrease by 50% [46]. Of all liposome immobilization techniques developed so far, the most stable phases have been prepared by the avidin–biotin technique. Immobilized liposome stationary phases prepared from egg yolk phospholipids (EYPs) (70% PC, 21% PE, and other phospholipid components) could be stored for a year without leakage of phospholipids [13]. In use, fewer than 20 chromatographic runs had no practical effect on the phospholipid concentration of the phase [47,48,50]. Phases prepared by the avidin–biotin technique are also relatively stable at different temperatures [46,50]. Still more important is the stability of the structure of the phospholipid membrane in ILC. The membranes proved stable during both immobilization and runs. Less than 1% of calcein was released from liposomes after storage for a week [47,49]. 2.3. Dynamic coating with phospholipids and liposomes While closely related to the immobilization techniques, dynamic coating differs in the looser, less permanent binding of phospholipids and liposomes. Dynamic coating of columns with phospholipids was described in 1999 by Krause et al. [51], who named the technique non-covalent immobilized artificial membrane chromatography. The phospholipid column was prepared by pumping a phospholipid suspension through a reversedphase (RP-18) column, where the hydrophobic parts of the phospholipids were adsorbed to the hydrocarbon chains of the reversed-phase material. In another study [52], SUVs were pumped through a reversed-phase RP-8 column, leading to breakdown of the vesicles and to the adsorption of phospholipids onto octyl chains of the column. Different phospholipids (including PC, PE, PS, and sphingomyelin) have been applied for dynamic coating of columns, but the most common one has been PC, typically with use of C-8 or C-18 columns [32,51,53]. The preparation of dynamically coated columns with phospholipids is simple and repeatable, and the major differences between studies have been the saturation time for the column and the amount of phospholipids adsorbed. Stationary phases coated dynamically with phospholipids are usually fairly stable. Dynamically prepared phases have been shown to withstand up to 60% of acetonitrile in the mobile phase without changes in the retention or the peak symmetry of the analytes [53]. Retention properties remained constant during storage of columns for 12 months. An attractive feature of dynamically coated stationary phases is that simple flushing with the phospholipid solution will restore the original retention properties of the column. 570

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2.4. Immobilization of proteoliposomes and cell membranes Stationary phases containing integral membrane proteins can be prepared by several immobilizing techniques. The most common technique has been freeze-thawing [34,54–57], but also dialysis [33] and immobilization by hydrophobic interactions [29] have been applied. Membrane proteins can be immobilized in artificial membranes by non-covalent interactions, as proposed by Pidgeon et al. in 1989 [14]. All published studies on immobilized proteoliposomes have involved ILC techniques. Small proteoliposomes are usually used when liposomes containing integral membrane proteins are immobilized by freeze-thawing. Immobilization occurs during the freeze-thawing cycles [54–57]. With the freeze-thawing technique, proteins can also be included after the liposomes have been immobilized [34] by adding integral membrane proteins dissolved in an appropriate surfactant solution (cholate) to a moist gel containing immobilized liposomes. The surfactant disorders the double layer of the liposome membrane, allowing the proteins to be incorporated into the phospholipid bilayer. The use of surfactant for the incorporation of proteins leads at the same time to a loss of liposomes from the gel; on average over 20% of the immobilized liposomes will be released by the surfactant treatment. However, rather large amounts of proteins can be bound to the liposomes – 0.12–0.42 mg/ml of gel depending on the concentration of the protein and the surfactant. Besides proteoliposomes, stationary phase materials in ILC have been prepared by freeze-thawing from human red cell membranes (containing phospholipids and transmembrane proteins, spectrin, and other cytoskeleton proteins) and cytoskeleton-depleted human red cell membrane vesicles (containing mainly human red cell membrane phospholipids, the anion transport protein, glycophorins, and the glucose transport protein Glut 1). With the dialysis technique [33], the immobilized liposomes were formed during the dialysis process, and subsequent addition of integral proteins to the phospholipid suspension resulted in immobilization of proteoliposomes. About 10% of the added proteins were incorporated into the liposomes. When proteoliposomes were immobilized by hydrophobic interactions, the reaction mechanism was similar to that for liposomes. The proteins most commonly used have been integral membrane proteins from human red cells [58]. The internal volume of immobilized proteoliposomes can be determined according to the selective penetration of the proteoliposomes by certain compounds [26]. For example, the internal volume of membrane liposomes from human red cells containing the glucose transporter (Glut 1, a transmembrane protein) can be

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determined from the difference in the retention volumes of D - and L -glucose. Glut 1-proteins transport D -glucose stereospecifically through human red cell membranes. The reaction is fully selective in that D -glucose interacts by hydrogen bonding with Glut 1, leading to its penetration of the cell membrane; L -glucose does not react with Glut 1 and does not penetrate the cell membrane. With proteoliposome-immobilized phases, the important thing is the amount of integral membrane proteins incorporated or the activity of these. In the very first columns containing proteoliposomes immobilized with hydrophobic ligands, the activity of Glut 1 persisted for only 2 or 3 days; 4 days after the immobilization the difference in the retention volumes of D - and L -glucose had already disappeared [29]. The stability was better for proteoliposome-immobilized phases prepared by freezethawing; the elution profiles of D - and L -glucose were then repeatable for up to 10 days [22]. Yang and Lundahl [54] have studied the stability of immobilized proteoliposome stationary phases containing Glut 1 and EYPs. The amount of phospholipids was decreased by just 2% after 50 chromatographic runs carried out over a period of 3 weeks. However, the loss of proteins was much higher (around 16%), and the retention volume of the transport inhibitor cytochalasin B, which interacted with Glut 1, decreased by 30%. With bovine PS used in the proteoliposomes, the corresponding loss of phospholipids was 20% and that of proteins about 25%. However, the volume of cytochalasin B that was eluted was decreased by only 4%. The highest loss of proteins (about 75% in 10 days) was observed with egg PC. Human red cell membrane vesicles (stripped of peripheral proteins) and proteoliposomes with reconstituted Glut 1 were sterically immobilized in gel beads by freeze-thawing, and the stability of immobilized Glut 1 was investigated by determination of the amount of cytochalasin B binding sites [55,56]. In the presence of dithioerythritol, over 80% of original cytochalasin B binding capacity of Glut1 was preserved even after 23–40 chromatographic runs. In the absence of dithioerythritol, the binding capacity depended on the amount of Glut 1 in the

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vesicles. With purified Glut 1 in proteoliposomes, the cytochalasin B binding capacity decreased by 50% in a week; however, with non-purified Glut1 in proteoliposomes or Glut 1 in immobilized membrane vesicles (egg phospholipids), the binding capacity remained constant, reflecting the high density of Glut 1 in the human red cell membrane vesicles. 2.5. Evaluation and comparison of techniques As described above, there are several different techniques by which liposomes can be immobilized in chromatographic stationary phases. Table 1 shows typical immobilized liposomes, phospholipid densities in the gel, and recoveries obtained with the different immobilization techniques. The choice of immobilization technique will depend on the phospholipid concentration and composition, liposome size, and lamellarity. Other important criteria are the stability of the gel and the application. In practice, the cost, speed, and ease of the technique will also influence the choice of method. As seen in Table 1, with steric immobilization techniques (i.e., dialysis and freeze-thawing), the immobilized liposomes are always multilamellar. With other techniques, the immobilized liposomes are formed beforehand, and their size and structure are more easily adjusted. SUVs are used with most techniques. The density of the immobilized liposomes much depends on the preparation technique, and amounts and the recoveries of immobilized phospholipids are lowest with dialysis. The highest phospholipid densities have been achieved by freeze-thawing and hydrophobic interactions, but the best recoveries with the avidin–biotin technique. Despite the high phospholipid densities obtained, some problems arise in immobilization by hydrophobic interactions. First, when the hydrophobic ligands of the gel used in the immobilization penetrate the liposome membrane, a part of the bilayer is damaged. Secondly, free (unbound) ligands may interact by hydrophobic interactions with the analytes. Although problems can be diminished by decreasing the ligand density in the gel, this will also decrease the efficiency of the liposome immobilization.

Table 1. Typical immobilized liposomes, phospholipid densities in the gel, and recoveries obtained by different immobilization techniques Immobilization technique

Immobilized liposomesa

Density of phospholipids in the stationary phase (lmol phospholipid/ml of gel)

Recovery (%)

Hydrophobic interactions Dialysis (steric) Freeze-thawing (steric) Covalent bonding Avidin–biotin technique

SUV, LUV MLV MLV SUV, LUV SUV, LUV

20–120 3–10 50–100 5–40 35–50

– 12–20 – 10–70 40–70

a

SUV, LUV, and MLV stand for small unilamellar, large unilamellar, and multilamellar vesicles, respectively.

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Normally, phases with immobilized liposomes of large internal volume are preferred in ILC. However, liposomes of very large diameter are difficult to immobilize in pores of the gel. In addition, the larger the liposome size, the less the amount of phospholipids immobilized. Achievement of equilibrium between the phospholipid membranes and the analytes of interest during the chromatographic run usually favors the use of unilamellar liposomes. The degree of lamellarity can be estimated from the internal volume and the diameter of the liposome [46]. The specific internal volume (reported as ll/lmol) of immobilized liposomes can be calculated from the ratio of the internal volume of the liposome to the amount of immobilized phospholipids in the gel [38]. The smaller the liposome specific internal volume, the larger is the degree of multilamellarity. The surface accessibility of immobilized liposomes has been studied by electrostatic binding of proteins onto the surface [34,35]. In the work of Yang and Lundahl [34], liposomes with various surface charges were allowed to interact with ferritin [35], lysozyme, and cytochrome C. The immobilized liposome column was overloaded with proteins and the surface accessibility was determined from the surface areas of the bound protein and the immobilized liposomes. Large variations in the amount

of bound proteins were observed; large ferritin molecules (440 kDa) were able to cover only 30% of the immobilized liposomes [35], whereas smaller lysozyme (10 kDa) covered 90% of the liposome surface [34]. As presented in Table 2 for dialysis, freeze-thawing, avidin–biotin, and covalent interaction techniques, the stability of liposome immobilized phases varies with the composition of the liposomes, the gel used for immobilization, the eluent and the run conditions (pH, ionic strength, temperature), and the analyzed compounds. Even though the most stable immobilized liposome stationary phases can withstand chromatographic runs for several months, even under harsh conditions, immobilized phospholipids are released from the column over the long run [37,38,59]. Comparison of the stabilities of liposome suspensions and immobilized liposomes has shown that often the stability of immobilized liposomes is very much higher, mainly because of the hindrance of aggregation of liposomes in the gel matrix [47]. As noted above, even the analytes may affect the stability of immobilized liposome columns. Hydrophobic compounds tend to induce greater losses of liposomes from the phase than do hydrophilic compounds, especially where liposomes have been immobilized by steric

Table 2. Stabilities of liposome-immobilized stationary phases prepared by different techniques

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interactions [37,38,59]. Hydrophobic compounds can penetrate the hydrophobic phospholipid bilayer, leading to leakage of phospholipids from the membrane [37]. Proteins, by changing the structure of the phospholipid bilayer, are another group of compounds that may induce strong leakage of phospholipids from the phase [36]. Where elution of proteins from the columns is accomplished by increasing the ionic strength of the mobile phase, phospholipid leakage may also occur during the elution step [35,36]. The advantage of immobilization by covalent interaction or the avidin–biotin technique is that the immobilized liposomes can be unilamellar, and so correspond better to the structure of real cell membranes. Relatively high phospholipid densities and recoveries are obtained by these methods. The major advantages of the avidin–biotin technique are the high stability of the immobilized phase and the high recovery. The use of eluents of strong ionic strength will usually be the main cause when liposomes are leached from immobilized liposome columns [26,35]. The strong osmotic pressure leads to the shrinkage of liposomes and their release from pores of the gel. The largest loss of phospholipids from immobilized liposome columns occurs during the first chromatographic runs; after that, the column is stabilized and the leakage diminishes [34–37]. If leakage of liposomes is strong during the first two or three runs, a few pre-chromatographic runs are recommended before injection of the analytes [36]. It has been observed that, when the columns are filled with gels containing immobilized liposomes prepared by freeze-thawing, the leakage of liposomes is already marked during the column-packing process [38,58,60]. In addition, phospholipid leakage is stronger from liposome columns containing PS than from those containing PC or EYPs [34,36,58,60,61]. Steric immobilization by freeze-thawing leads to high phospholipid concentrations and the immobilized stationary phases are relatively stable and easy to prepare. Proteoliposome-containing stationary phases are usually prepared by the freeze-thawing technique. An advantage of all immobilized liposome phases prepared by steric interactions is that there are no ligands present in the gel to interfere with the interaction between analytes and the stationary phase. Dynamic coating of stationary phases with liposomes is simple, and stationary phases can easily be recoated with liposomes. However, even though simple flushing with the phospholipid solution will restore the original properties of the column, the major disadvantage of dynamic coating is the large amount of phospholipids needed. 2.6. Application of stationary phases In the application of the stationary phases described above, consideration will have to be paid to the com-

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position and the flow rate of the mobile phase and to the detection technique. Both the properties of analytes and the stability of phospholipid- and liposome-immobilized phases are relevant to the choice of mobile phase. In order to keep the system as natural as possible, the use of organic modifiers has usually been avoided in drug and protein liposome membrane interaction studies. Stationary phases also tend to last longer in aqueous solutions. The pH of the mobile phase has typically been close to physiological pH 7.4. In ILC, it is recommended that the buffer used as mobile phase be similar to that used in the preparation of the liposomes. In the case of liposomes immobilized by steric interactions, the stability of the phase has sometimes been improved with use of a mobile phase of slightly lower ionic strength than that used in the original immobilization of the liposomes [26,37]. However, if the ionic strength of the mobile phase is much lower than that of the liposome buffer, the liposomes may swell and the bilayer membrane will be destroyed [42]. If, in turn, the ionic strength of the mobile phase is much higher than that used for the liposome preparation, the immobilized liposomes may shrink because of osmotic pressure, leading to leakage of liposomes from the gel beads. By far the most frequently employed detector in both IAM chromatography and ILC has been the UV detector. Even though small amounts of immobilized liposomes may leak out of the column, this has seldom had a significant effect on detection of compounds. Immobilized calcein-entrapped liposomes have been used in the study of weak solute-membrane interactions [47,49]. The release of the fluorescent calcein was monitored by fluorescence detection, where the fluorescence intensity was directly proportional to the amounts of released calcein. Hence, with fluorescence analysis, the interaction of solutes with membranes can be determined from both the retention times of the solutes and the leakage of liposomes caused by solute binding and/or penetration on or into membranes. On-line coupling of IAM chromatography to mass spectrometry using atmospheric pressure chemical ionization and electrospray ionization has been applied successfully in the analysis of neuropeptide FF antagonists [62,63], b-blockers and imidazoline derivatives (Fig. 4) [64], and low-molar-mass drugs [65]. IAM chromatography and ILC have mainly been applied to the determination of distribution coefficients between analytes and membranes and much less so to the separation of analytes. Some separations by IAM chromatography are noted in Table 3, and separations of proteins and peptides by ILC are listed in Table 4. In IAM chromatography, proteins can be purified through the affinity mechanism. IAM phases are highly selective and typically 50–80% of non-target proteins in the sample will be eluted without any interaction with the phase. The adsorbed protein is eluted by change of salt, surfactant, or organic modifier content in the eluent. http://www.elsevier.com/locate/trac

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Figure 4. Overlaid selected ion chromatograms of seven b-blockers and five imidazoline derivatives obtained by IAM.PC–LC/ESI–MS. The inset in the upper right corner is an expansion of the ion chromatograms of early-eluted peaks. The IAM.PC column was 1.0 cm long, 3.0 mm i.d., and had a void volume of 30–40 ll. The flowrate was 0.2 ml/min and the mobile phase comprised 5.0 mM PBS buffer (pH 7.4) (reprinted from [64] with permission).

Table 3. Selected separations by immobilized artificial membrane chromatography

IAM phase

Analytes

IAM.PC.DD a) 12-OH-silica b) 12-MO-silica ester C10/C3 IAM.PC , ether C10/C3 IAM.PC , G C10/C3 IAM.PC ether

IAM.PC

C10/C3

IAM.PC.MG

-Blockers, imidazoline and imidazolidine derivatives -Blockers, imidazoline- and imidazolidine derivatives, phenol derivatives, phenethylamine derivatives -Blockers, steroids, anti-inflammatory drugs Nonsteroidal anti-inflammatory drugs (NSAIDs)

Column dimensions 0.46 x 3.0 cm (5 m) 0.46 x 3.0 cm (12 m) 0.46 x 3.0 cm (12 m) 0.46 x 15.0 cm 0.46 x 3.0 cm 0.46 x 15.0 cm 0.46 x 3.0 cm 0.46 x 15.0 cm

IAM.PC.MG

Arylpropionic NSAIDs

0.46 x 15.0 cm

IAM.PC.MG IAM.PC.DD IAM.PC.DD

Local anesthetics and -blockers -Blockers -Blockers Warfarin, salicylic acid, lidocaine, propranolol and diazepam (p-Methylbenzyl)alkylamines, -blockers, other acidic and basic drugs

0.46 x 15.0 cm 0.46 x 10.0 cm (5 m) 0.46 x 10.0 cm (5 m)

Synthetic hydrophilic peptides

0.4 x 10.0 cm

Synthetic oligopeptides Phenols and anilines

10 cm 0.46 x 3.0 cm (5 m)

IAM.PC.DD IAM.PC.DD2

c)

Nucleosil-lecithin (IAM PC) IAM.PC.DD IAM.PC.DD a)

0.46 x 10.0 cm (5 m) 0.46 x 10.0 cm (12 m)

Eluent

Ref.

d)

0.01 M PBS , pH 5.4 or 7.4

0.01 M PBS, pH 7.4

[67,68]

0.01 M PBS, pH 7.4

[69]

0.10 M PBS / ACN at different ratios, pH 7.0 or 5.5 0.016 M phosphate, pH 7.4 / ACN at different ratios 0.1 M PBS / ACN at different ratios, pH 7.0 0.01 M PBS, pH 3.5–7.4 e) 0.017 M DPBS pH 7.0 f) 0.23 M SMUBS + MeOH / EtOH / ACN, pH 2.5–8.0 0.02 M KH2PO4 / 0,15 M KCl, + MeOH at different ratios, pH 7.0 0.1% TFA, PBS, H2O / ACN at different ratios 0.01 M PBS pH 7.4 0.010 M phosphate + KCl pH 2.5–7.5

C12-fatty acid with hydroxy groups immobilized onto the surface of the silicapropylamino particles. The phase is endcapped with C-3. As in a) but with methoxy groups on the C-12 chains. ester C10/C3 Commercial name for IAM.PC . d) PBS = phosphate-buffered saline. e) DPBS = Dulbecco´s phosphate-buffered saline solution. f) SMUBS = standardized modified universal buffers. b) c)

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[66]

[70] [71] [15] [16] [83] [72] [73] [14] [74] [75]

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Table 4. Separation of proteins and peptides by immobilized liposome chromatography ILC-phase preparation method, phospholipids and gels Dialysis, a EYP + PS/or stearylamine, Sepharose 6B Dialysis, PC, PC+PS, Superose 6 Freeze-thawing, PC, PC/PE (4:1) EPL, Sepharose CL-4B, Superdex 200 Freeze-thawing EYP, Superdex 200 Covalent bonds, POPC + 1 mol% egg yolk phosphatidylethanolamine, TSK G6000PW Avidin–biotin, c) EYP + biotin-cPE, Sephacryl S-1000, TSK G6000PW, Superdex 200, Sepharose 4B Avidin–biotin, EYP + biotin-cPE, Sephacryl S-1000, TSK G6000PW, Superdex 200, Sepharose 4B Dynamic coating, DMPC on RPLC C18-column a) b) c)

Column dimensions

Analytes studied

Eluent

Ref.

Ferritin, citraconylated myoglobin, human plasma proteins (MW 16–300 kDa)

2.8 x 1 cm

Ribonuclease A, lysozyme, cytochrome C

4 or 5 × 0.5 cm

Tr is-HCl or citric acid + Na2EDTA, mercaptoethanol and D-glucose, constant NaCl concentration or gradient 5 mM Tr is-HCl (pH 7.0) 0.1 mM Na2EDTA, NaCl-gradient

Amino acids and synthetic hydrophilic peptides

5, 10, 10.5, 11 or 20 × 0.5 cm

10 mM sodium phosphate 150 mM NaCl, 1 mM Na2EDTA, pH 4.5, 6.0, or 7.4

[76]

Synthetic oligopeptides

4 ml column (HR 5/20)

PBS, pH 7.4

[74]

-Galactosidase, α-glucosidase, carbonic anhydrase from bovine

5-5.5 × 0.5 cm

50 mM Tr is-HCl, pH 4 or 7.5

[44]

Synthetic amphiphilic peptides, polylysines, bovine carbonic anhydrase

5 x 0.5 cm

10 mM HEPES , 150 mM NaCl, pH 7.5 + 0–2.5 M guanidium HCl

Bee venom phospholipase A2

0.5-cm i.d. column 10 mM HEPES, 150 mM NaCl, with 0.2–1 ml gel pH 7.5 + 0–10 mM calcium

[49]

Helical antibacterial magainin-2-amide peptides

15 × 0.46 cm

[51]

[35] [36]

b)

Water/2-propanol, 85:15 (v/v)

[47]

Cationic vesicles were obtained by the addition of STA to the phospholipid solution. HEPES = N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid). biotin-cPE = 1,2-dioleylphosphatidylethanolamine-N-(cap biotinyl).

Examples of protein purification by IAM chromatography are listed in Table 5. Protein refolding has been carried out by ILC [42,43]. This type of ILC has been called refolding chromatography and it allows simultaneous refolding and purification of target proteins from insoluble inactive protein aggregates (inclusion bodies) [43]. Fig. 5 illustrates protein refolding by ILC [42]. As

can be seen in Fig. 5, the molten globule state has a similar secondary structure to the native state but its tertiary structure is not as closely packed, and hydrophobicity is relatively high compared to the native state. Proteins that have been refolded by ILC include bovine carbonic anhydrase, lysozyme, ribonuclease A, and a-lactalbumin [41,43].

Table 5. Purification of proteins by immobilized artificial membrane chromatography

IAM phase

IAM • PC ether ether

C10/C3

IAM.PE

Purified proteins Cytochrome P450 isozymes, NADH oxidase, Ferricyanide oxidoreductase N-acylphosphatidylethanolamine synthase

Amount of proteins injected into column

3, 25, 50, or 100 mg

a)

b)

About 5 mg

Bovine pancreatic c) phospholipase A2 (PLA2) from 0.5 mg PLA2 of total protein protein mixtures 0.8 mg protein/g IAM

C10/C3

IAM.PC C10/C3 δG IAM.PC

a) b) c)

Ref.

60–70% for cytochrome P450

[77]

close to 100%

[24]

70–100%

[23]

C10/C3

IAM.PC C10/C3 ether IAM.PE C10/C3 ether IAM.PG C10/C3 ether IAM.PS ether

Protein recovery

~70% [78]

PLA2 3.21 mg protein/g IAM

48%

Column dimensions 15 cm × 0.46 cm i.d. or 10 cm x 2.11 cm i.d. Column dimensions 6.5 cm × 1.0 cm i.d. Column dimensions 10 cm × 0.46 cm i.d.

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Figure 5. Schematic presentation of protein refolding by ILC. U, MoGl, N, and Agg stand for unfolded state, molten-globule state, native state, and aggregates, respectively (reprinted from [42], with permission).

3. Liposomes in capillary electromigration techniques Interest in the use of liposomes in capillary electromigration techniques is increasing rapidly. An essential advantage of capillary electromigration over LC techniques is the smaller amounts of liposomes and samples required. Though the same kinds of applications as LC can be envisaged, separation and purification processes promise to be particularly important. 3.1. Liposomes as carriers With liposomes used as carriers, or a pseudostationary phase, in electrokinetic capillary chromatography, the separation is based on the interaction of analytes with the liposomes and on the electrophoretic mobility of the analytes. The technique is called liposome electrokinetic capillary chromatography (LECC) and usually negatively charged liposomes are used. The separation mechanism is the same as in micellar electrokinetic capillary chromatography (MECC) and will not be discussed here. The first report on the use of liposomes as carriers in capillary electromigration techniques was pusblished in 1996 by Zhang et al. [84]. The technique was described as liposome capillary electrophoresis, but later on, in view of the electrokinetic separation mechanism, LECC (or LEKC) has been the preferred name. Polyacrylamidecoated capillaries and liposomes composed of PC phospholipids at very high concentrations were used in this first work. 576

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The possibility of applying liposomes as pseudostationary phase in ECC was noted by Roberts et al. [82] (the retardation of riboflavin in the presence of liposomes was shown), and soon thereafter the separation of nitrobenzene derivatives with DMPC/DMPG liposomes was demonstrated by Nakamura et al. [79]. Liposomes adsorb onto uncoated fused silica capillaries [80], and especially strongly at neutral pH [81]. Liposome-analyte interactions in LECC have been studied by saturating the capillary with liposomes before runs [82] or with use of coated (e.g., polyacrylamide-coated) capillaries [81,84]. Table 6 summarizes some of the work done on liposomes as carriers in LECC. In one application, lysophospholipids with only one fatty acid chain were used as micelle-forming reagent, and the technique was called lysophospholipid micellar electrokinetic chromatography [83]. Our group has shown the influence of phospholipid composition, liposome concentration, and buffer on the separation of analytes [80,81,86]. The liposomes that we used comprised POPC or DPPC with smaller amounts of anionic phospholipids. The separation was improved by increasing the amount of negative charge on the liposomes, as well as by increasing the total phospholipid concentration. Significant differences in selectivity were achieved through the addition of cholesterol to the system [81]. In general, unilamellar vesicles have been used as carriers in LECC; multilamellar vesicles, with their broad size distribution, are unsuitable because of noisy background and low sensitivity [80]. Most of the analytes studied by LECC have been drugs, steroids, or various substituted aromatic model compounds. UV detection and injection by both pressure and siphoning have been applied. Uncoated fused silica capillaries (length 50 cm and with 50-lm ID) and positive run voltages are typical [79,87,88], but coated capillaries have also been applied [81,89]. The temperature selected is important and will depend on the characteristics of the phospholipid; fluid–gel transition occurs when the temperature increases above the main phase-transition temperature of the phospholipid. In most studies, the temperature has been about 25
C, and the phospholipids have been in their fluid state; in our studies on DPPC (main phasetransition temperature 41
C), we showed that the separation of analytes is improved when phospholipids are in their fluid state [80]. Vesicle affinity capillary electrophoresis was recently applied to study the interactions between apolipoproteins (apoCIII) and unilamellar vesicles (DMPC) used as a model system for lipoproteins [90]. The studies were carried out under physiological conditions (temperature 37
C, BGE; 50 mM phosphate, 150 mM NaCl, pH 7.4). The binding constants of whole apoCIII protein (1–79) and three peptide fragments (1–19, 1–40, and 41–79) to

Type of liposome

Phospholipid compositon

Liposome concentration

Electrolyte solution

Capillary dimensionsa

Capillary coating

Run voltage

Temperature

UV-detection wavelength

Analytes separated

References

Not mentioned

Egg L -a-PC

25–30 mM

25/35 cm

PAA-coated

10 kV

22
C

[84]

DMPC/DMPG (80:20 mol%)

3.75 mM

50/70 cm

Uncoated

25 kV

RT

225, 280 nm 100 lm i.d.214, 280 nm 215 nm

Drugs Peptides

SUV

POPC/DPPC + PG/ PA/PS/CL (100:0 –70:30 mol%)

0.5–4.5 mM

50 lm i.d. 50/58.5 cm

Uncoated

15 kV 20 kV

30
C 25 or 43
C

210 nm 245 nm

Napthalene, biphenyl Nitrobenzenes Corticosteroids

[79]

LUV and MLV

25 mM phosphate + 125 mM cholate, pH 7.4 10 mM Tris–HCl + 50 mM NaCl, pH 7.0 50 mM AMPSO, borate, CHES, glycine and tricine, pH 9

LUV

POPC/PS (80:20 mol%)

3 mM

50 mM AMPSO, pH 8.3 or 50 mM CHES, pH 9

50 lm i.d. 60/68.5 cm

Uncoated

20, 30 kV

25
C

210, 215, 245 nm

Benzene [86] derivatives, phenols, steroids

LUV

POPC/PS-cholesterol (80:20:0, 60:20:20 and 40:20:40 mol%)

3 mM

20 mM HEPES, pH 7.4

Uncoated, PAA- or AMPS-coated

20, )30 kV

25
C

200, 245 nm

Steroids

SUV

DPPG/DPPC-cholesterol (24:46:30 mol%) DPPG/DPPC-cholesterol (24:46:30 mol%) and DPPG/ DPPC (30:70 mol%) POPC

Not mentioned

25 mM HEPES, pH 7.5

Uncoated

25 kV

36
C

Not mentioned

Not mentioned

25 mM HEPES, pH 7.5

Uncoated

25 kV

36
C

Not mentioned

Aromatic [87] compounds, b-blockers, drugs Various organic [88] compounds

3.25 mM

50 mM phosphate, pH 7.5 or 9.2 20 mM phosphate, pH 7.0

19.5/30 cm, 50 lm i.d.

Polyimidecoated

)10 kV

Not mentioned

200 nm

Drugs

[89]

50/58 cm 75 lm i.d.

Uncoated

15 kV

25
C

200 nm

b-Blockers

[83]b

25 mM TES, pH 7.4

32/40 cm, 25 lm i.d.

Uncoated

16 kV

20
C

225/210 nm

b-Blockers/ myoglobin + two peptides

[85]

SUV

SUV http://www.elsevier.com/locate/trac

Micelle

Bicelle

a b

1-Lauroyl-2-hydroxy-sn-glycero-3phosphocholine DMPC/DHPC (1:1, 2:1 mol%)

10 mM

90 mM

50 lm i.d. 40/48.5 cm, 50/58.5 cm

50 lm i.d. Length not mentioned, 50 lm i.d. Length not mentioned, 50 lm i.d.

[80]

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Table 6. Separations in liposome electrokinetic capillary chromatography

[81]

Capillary length to detector/total length of capillary, and inner diameter. Lysophospholipid micellar ECC – phospholipids with only one fatty acid as micelle-forming reagent.

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3.2. Liposomes as coating materials Liposomes can also be used in electromigration techniques as coating material. In principle, the technique resembles affinity electrochromatography. Only a few reports have been published on the use of liposomecoated capillaries for the separation of analytes under the influence of an electric field. Biotinylated liposomes (small and large unilamellar vesicles), composed of egg PC and biotinylated phosphatidylethanolamine, self-assemble and can be immobilized in the presence of avidin in fused-silica capillaries. Yang et al. [93] modified fused-silica capillaries with 3-aminopropyl triethoxysilane and activated the resulting aminopropyl silica capillary with glutaraldehyde. Avidin was coupled to the hydrophilic coating at room temperature, and non-reacted aldehyde residual groups were removed by washing with phosphate pH 7 buffer. Biotinylated liposomes without avidin were coupled to the avidin-capillary by flushing with the liposome solution. Next, a suspension of the liposomes in the presence of avidin was sucked into the capillary, allowed to react and washed with avidin solution. This procedure was repeated two to three times, after which the capillary was washed with the buffer without avidin to remove non-immobilized liposomes. Several layers of biotinylated liposomes were assembled in the capillary. The number of layers was estimated from the mean size of the liposomes and the unilamellarity determined from the size and the internal volumes of the liposomes. In the case of LUVs, the estimated number of liposome layers was 15 whereas, with SUVs, it was 4 or 5. The number of liposome layers was shown to depend on both the type of liposomes and the inner diameter of the capillary. Acebuterol was injected into the liposome-coated capillary and its migration time was increased from that in an uncoated capillary. € Ornskov et al. [94] immobilized liposomes in capillaries for electrophoresis through electrostatic interaction with derivatized agarose. The negatively charged 578

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liposomes comprised POPC/PS (80/20 mol%) liposomes in phosphate buffer at pH 7.4. The immobilization comprised two steps: coating of the fused silica capillary with positively charged agarose; and, liposome immobilization by electrostatic interaction. In recent work by Deyl’s group [95], a fused silica capillary was flushed with a POPC liposome suspension, the capillary was air-dried, and excess liposomes were removed by rinsing with alkali hydroxide. The liposomecoated capillary was used for the separation of charged drugs. In other work [96], cationic liposomes (Lipofectamine and Escort) were used for quantitative determination of liposome-oligonucleotide interactions by affinity capillary electrophoresis (through the determination of binding constants). The cationic liposomes in the buffer solution coated the fused silica capillary, resulting in a reversal of the electroosmotic flow (EOF). We recently demonstrated the coating of fused silica capillaries with anionic liposomes comprising POPC and PS and/or cholesterol (all phospholipids in fluid state) [97]. The buffer played an important role and, of several buffers studied (HEPES, phosphate, Tris, and Tricine), HEPES clearly improved the coating efficiency and stability. The liposome coating procedure was simple and fast; after preconditioning, the capillary was rinsed with the liposome solution for 10 min (at 930 mbar pressure) and left to stand for 15 min. Unbound liposomes were removed by rinsing with the background electrolyte (BGE) solution. Between runs, the liposome-coated capillary was rinsed with BGE and, under optimal conditions (coating with POPC/PC/cholesterol 80/20/0 or

-2.5 phenol

-3.0 p-cresol

-3.5 mAU

DMPC vesicles increased with the hydrophobicity and charge of the peptides. The results were comparable to those obtained by other techniques, demonstrating the utility of vesicle affinity capillary electrophoresis for the study of apolipoprotein–lipoprotein interactions. In another recent study, anionic and zwitterionic liposomes were used as models in an investigation by high-performance frontal analysis/capillary electrophoresis of the binding between basic drugs (S-verapamil and S-propranolol) and low-density lipoprotein (LDL) [91]. The binding affinity was dominated by electrostatic interactions between the model liposomes and the basic compounds, confirming earlier findings [92] that the drug-binding affinity of LDL is enhanced by LDL oxidation.

2,6-dimethylphenol

-4.0 eugenol

-4.5 -5.0 -5.5 4

5

6

7

8

9

Time (min)

Figure 6. Separation of phenols (50 lg/ml of phenol, p- cresol, 2,6-dimethylphenol, and eugenol in 20/80% v/v MeOH/water) in liposome-coated capillary. 3 mM 80/20 mol% of POPC/PS LUVs in HEPES at pH 7.4 (ionic strength of 20 mM) + 10 mM CaCl2 solution was used for coating. The BGE solution contained HEPES at pH 7.4 (ionic strength of 20 mM). Running conditions: fused silica capillary with total length of 60 cm (51.5 cm to detector) and i.d. of 30 lm (o.d. of 375 lm), temperature of capillary 25
C, injection 5 s at 50 mbar, running voltage 20 kV, detection at 214 nm [97].

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80/20/20 mol%), the RSD of the electrosmotic flow was less than 3% after 65 injections. The coated capillaries were used for the separation of neutral steroids. Use of calcium as a fusogenic agent has a strong influence on phospholipid coatings in capillary electrophoresis, as recently demonstrated [98,99]. Cunliffe et al. [98] used calcium in the coating of fused silica capillaries with zwitterionic phospholipids, and they found that, without calcium, the coating time was prolonged almost 20-fold. Efficient separations of proteins were achieved with the coated columns. Our studies on anionic phospholipids similarly demonstrated the importance of calcium; with calcium, the anionic coating performed well and independently of the buffer [99]. The anionic coating was applied to the separation of low-molar-mass uncharged compounds (Fig. 6). The retention of neutral analytes in capillary electromigration techniques with liposomes as carriers or with liposome-coated capillaries is directly proportional to the liposome–water partition coefficient. Burns and

Trends

coworkers [87,88] characterized the solvation properties of phospholipid bilayer membranes in LECC by linear solvation energy relationship (LSER) models. The correlation coefficients between MECC, LECC, and log Po=w values were determined for a group of analytes of different hydrophobicity. Two different liposome systems were evaluated: DPPG/DPPC/cholesterol (24/46/30 mol%); and, DPPG/DPPC (30/70 mol%). The studies were carried out at 36
C, which is close to the main phase-transition temperature of pure DPPC. For DPPC MLVs, the Tm is about 41.3
C; LUVs melt at approximately the same temperature, but it has been reported [100] that the Tm of SUVs is about 37
C. Furthermore, cholesterol induces an intermediate state in phospholipid molecules with which it interacts, increasing the fluidity of the hydrocarbon chains below the main phasetransition temperature and decreasing the fluidity above it. In these studies [87,88] on solvation properties, no attention was paid to the Tm of the SUV liposomes that were used. However, the authors mentioned that the

Figure 7. Chiral separation of D - and L -tryptophan in M1C4-phospholipid–lysozyme coated capillary. (a) Coating with M1C4-PC-lysozyme and (b) coating with M1C4-PC/PS (80:20 mol%)-lysozyme. Running conditions: 50 lm i.d. capillary; total length, 48.5 cm; length to the detection window, 40 cm; capillary temperature, 25
C; applied voltage, 20 kV; sample injection, 10 s at 50 mbar; UV detection, 214 nm; running buffer, 20 mM (ionic strength) Tris at pH 7.4 [101].

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final product was a relatively clear homogeneous solution, suggesting the presence of liposomes in a liquidcrystalline phase. A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was recently developed for the chiral CE separation of D - and L -tryptophan [101]. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. However, when 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the EOF was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. Coating with 4 mM M1C4 and then 1 mM PC/PS (80/ 20, mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution (Fig. 7). Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D - and L -tryptophan was achieved without lysozyme in the running buffer.

4. Conclusions There are several ways in which phospholipids and liposomes can be immobilized onto columns in LC. Immobilized artificial membranes are composed of silicapropylamino particles onto which phospholipids are covalently bound. Stationary phases are typically endcapped with C-10 or C-3 groups. IAM phases are difficult to prepare, but fortunately they are commercially available. Stationary phases containing immobilized liposomes can be prepared sterically or dynamically or by using hydrophobic ligands, covalent binding, or the avidin–biotin technique. A popular method has been steric immobilization by freeze-thawing. A considerable challenge at the moment is the development of commercial ILC phases with good characteristics (repeatability, reproducibility, stability). Another important area of interest is the development of gels with larger pore sizes to allow the immobilization of larger liposomes; the larger the external surface area of the liposome, the better it mimics natural membranes. Although only liposomes of simple phospholipid composition have been immobilized so far, future studies can be expected to turn to the immobilization of liposomes of complex composition and even real phospholipid membranes. The use of liposomes in capillary electromigration techniques is still uncommon. In most studies, the liposomes have been employed in the background electrolyte solution, but there is increasing interest in their use as capillary coating material. 580

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In general, separation techniques utilizing phospholipids and liposomes have been rather infrequently applied and the focus has instead been on developing the techniques and characterizing the phases employed. Future studies can be expected to deal more specifically with the use of immobilized phases (phospholipids, liposomes, and proteoliposomes) to solve bioanalytical and biomedical problems. Together with the exploitation of nanotechnology, the use of biomimetic membranes for affinity studies and the separation of biomolecules will surely find its own niche in analytical separation science.

Acknowledgements Financial support from the Academy of Finland under Grants SA 2022176 (SKW), 78785 (SKW), and SA 206296 (MLR) is acknowledged.

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