Applications of polymethacrylate-based monoliths in high-performance liquid chromatography

Journal of Chromatography A, 1216 (2009) 2637–2650

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Review

Applications of polymethacrylate-based monoliths in high-performance liquid chromatography E.G. Vlakh, T.B. Tennikova ∗ Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoy pr. 31, 199004 St. Petersburg, Russia

a r t i c l e

i n f o

Article history: Available online 1 October 2008 Keywords: Monolithic stationary phase Polymer sorbents HPLC

a b s t r a c t Monolithic columns were introduced in the early 1990s and have become increasingly popular as efficient stationary phases for most of the important chromatographic separation modes. Monoliths are functionally distinct from porous particle-based media in their reliance on convective mass transport. This makes resolution and capacity independent of flow rate. Monoliths also lack a void volume. This eliminates eddy dispersion and permits high-resolution separations with extremely short flow paths. The analytical value of these features is the subject of recent reviews. Nowadays, among other types of rigid macroporous monoliths, the polymethacrylate-based materials are the largest and most examined class of these sorbents. In this review, the applications of polymethacrylate-based monolithic columns are summarized for the separation, purification and analysis of low and high molecular mass compounds in the different HPLC formats, including micro- and large-scale HPLC modes. © 2008 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatographic applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ion-exchange chromatography (IEC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Application for low molecular mass compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Application for peptides and oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Application for proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Application for nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.5. Application for viral particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Reversed-phase chromatography (RPLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Hydrophobic interaction chromatography (HIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Hydrophilic interaction chromatography (HILIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2638 2638 2638 2639 2639 2642 2643 2644 2644 2647 2648 2649 2649 2649

Abbreviations: AMPS, 2-acrylamido-2-methyl-1-propanesulfonic acid; BDDMA, 1,4-butanediol dimethacrylate; BSA, bovine serum albumin; BuMA, butyl methacrylate; CIM, convective interaction media; DVB, divinylbenzene; EDMA, ethylene dimethacrylate; EMA, ethyl methacrylate; GMA, glycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; HETP, height equivalent to a theoretical plate; HIC, hydrophobic interaction chromatography; HILIC, hydrophilic chromatography; HMA, hexyl methacrylate; HPLC, high-performance liquid chromatography; IEC, ion-exchange chromatography; LMA, lauryl methacrylate; ODMA, octadecyl methacrylate; OST, octylstyrene; PBS, phosphate-buffered saline; RPLC, reversed-phase liquid chromatography; RSD, relative standard deviation; SMA, stearyl methacrylate; SPE, N,N-dimethyl-Nmethacryloxyethyl-N-(3-sulphopropyl)ammonium betaine; SEM, scanning electron microscopy; TFA, trifluoroacetic acid. ∗ Corresponding author. Tel.: +7 812 323 04 61; fax: +7 812 323 68 69. E-mail address: [email protected] (T.B. Tennikova). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.09.090

2638

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

1. Introduction Nowadays, high-performance liquid chromatography (HPLC) is a powerful tool for separation, isolation and purification of different types of natural or synthetic compounds. Considerable improvement of mass transfer characteristics was achieved by the introduction of micropellicular [1], superporous [2], non-porous [3], gigaporous, namely perfusion [4], and some other (for example the so-called “gel in shell” [5]) supports. However, mass transfer improvements of dispersed sorbents enabled to solve only partially the problem of fast and efficient separations. The problems of interparticular volume and slow interphase mass exchange have been overcome with the development of new original class of separation media called as macroporous monoliths. In this case, all liquid phase flows through the pores and mass exchange occurs dramatically faster in comparison with that for conventional packings. As it was noticed, the monoliths represent a relatively new class of stationary phases, sometimes called as continuous beds (phases). Historically, the first polymethacrylate monoliths were suggested ˇ by Tennikova and Svec [6,7] who realized the idea of a combination of advantages of convective mass transport in continuous bed support with a short length column (membrane) [8]. From the middle of the 1990s, the interest to the monoliths has been arisen dramatically. Today these phases are produced from synthetic (polymethacrylate, polyacrylamide and polystyrene) [9–14] and natural (agarose and cellulose) polymers [15], or from inorganic (silica) [16,17] base. Monoliths have the properties that distinguish them fundamentally from particle-based columns. Columns of packed particles have a void of roughly 40% of the bed volume. Monoliths represent a single body and have no void. This leads to differences in hydrodynamics. While diffusion limits mass transport in particle columns and the pores are utilized only partially, interphase mass transfer in monoliths is governed by convection, and the total pore volume is utilized. This allows a dramatic reduction in the time required for mass exchange between the mobile and stationary phases. Separation times are consequently at least an order of magnitude faster on monoliths. These, and other similarities and differences between monoliths and packed columns, have been thoroughly documented [7,21–24] and the advantages of monoliths described in recent reviews [18–20]. Most recently, Guiochon and Siouffi have published excellent reviews devoted to the theory of HPLC on monolithic supports [25,26]. Monolithic columns of different geometry (disks, rods and capillary columns) with axial flow are characterized by a linear dependency of a back pressure on flow rate within a wide range [21,27–29]. First of all, this phenomenon relates to the rigid structure of highly cross-linked polymethacrylate copolymers that, contrary to the bead-based columns, is not deformed or compressed even at high flow rates because of low back pressure characterizing such a kind of morphology. For instance, a linear relation between a pressure drop and flow rate (1–8 mL) and the length of monolithic column (from 3 to 21 mm) was proved by Miheliˇc et al. [30]. Hahn et al. [31] showed that poly(GMA-co-EDMA) short monolithic columns could be operated up to 2100 cm/h without any loss of efficiency. In radial flow operation mode, the mobile phase penetrates from the outer wall of monoliths designed as a tube toward the inner one, through it and flows out to the central hole. A decrease of the area through the mobile phase flowing from outer to inner side of tube leads to an increase of a linear flow velocity that can be more than one order of magnitude [32]. Thus, when the tube diameter is large and its thickness (separation layer) small, the change in linear flow velocity is also small and can be neglected. However, if the inner radius is small, but the thickness of the tube is signifi-

cant, the changes of linear velocity of the flow through the column are also significant. For example, Podgornik et al. [33] determined the changes in linear flow velocity for monolithic tube that had a hole with a diameter of 3 mm, a wall thickness of 16 mm and a sorbent volume of 80 mL. At 250 mL/min flow rate (maximum allowed value), the linear flow velocity at the outer surface was 255 cm/h, while in the inner part it reached the value 2972 cm/h, that corresponded to 12-fold increase. It is known that, according to the van Deemter equation, the column efficiency depends on linear flow velocity, e.g. the efficiency of column linearly decreases with increasing flow. On the contrary, many studies for polymethacrylate monolithic columns, for both axial [27,31,34] and radial [32] ones, reliably proved that the column efficiency and the dynamic binding capacity were not affected by flow. An explanation might be that some of the small pores, where the liquid is stagnant at low flow rates, become accessible at higher flow rates meaning the appearance of a convective flow. A careful comparison of macroscopic fluid dynamic behavior, hydrodynamic permeability and hydrodynamic dispersion of monolithic and particulate stationary phases is given in the paper [35]. The present review is devoted to the most widely investigated and extensively described macroporous polymethacrylate monoliths regarding their applications for different HPLC formats including ␮HPLC and large-scale HPLC modes. Macroporous polymer monoliths are produced by polymerization of organic monomers, including cross-linkers. The macroporosity of such materials is achieved by addition of porogenic solvents or pore-forming reagents, for example, polymer solutions, to polymerization mixture. Currently, a wide range of monomers are used for the synthesis of polymer monoliths. However, most of them still represent polymethacrylate and polyacrylate monoliths. Numerous excellent reviews devoted to the preparation of monolithic media have been published during the last decade. Among them, we can adduce two most recent papers [36,37]. The avalanche of interest in monoliths over the last decade can be explained by their performance advantages over particle-based media. One significant advantage is their simple synthesis. Polymer monoliths can be formed in situ into any shape as large as 8 L [38] to as small as a few nL in the channel of a microfluidic chip [39]. This feature is particularly important for micro and nanoscale devices where the packing of particulate sorbents is difficult and may be followed by poor reproducibility [40]. Despite the many advantages of polymer monoliths, they present lower surface area per volume than porous particle media and this may limit their capacity per cycle in some applications. However, their high flow rates compensate, so that productivity per unit time is as good or often better than particle-based media [24]. In this review, we have focused on HPLC applications of polymethacrylate-based monoliths. The literature and discussions were grouped according to the modes of chromatographic separations. 2. Chromatographic applications 2.1. Ion-exchange chromatography (IEC) IEC is one of the most frequently used techniques for separation of charged molecules [41]. This chromatographic mode is based on a displacement of counter-ions (usually simple anions or cations, such as chloride or sodium ions) which are associated with exchanger groups of insoluble matrix by solute molecules carrying the appropriate charge and binding reversibly to the solid support. The retention and separation of charged molecules on ion exchang-

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

ers depend on the nature of molecules, mobile phase composition, type of ion exchanger and matrix properties. One of the important characteristics of ion-exchange stationary phases is their capacity. Fast, simple and non-destructive methods for the determination of sorbent capacity are described in [42]. The recent developments in IEC using monolithic materials can also be found in the review [43]. Today, the commercially offered monolithic materials for IEC include disks, columns and tubes. The disk-shaped polymethacrylate-based separation devices were in fact the first successful example of monolithic ion exchanger. These so-called “short columns” are currently produced and distributed worldwide by BIA Separations, Ljubljana, Slovenia, under the trade name CIM disks. Standard CIM disks have a diameter of 12 mm and a thickness (bed length) of 3 mm. The company also offers Mini-CIM disks of 6 mm diameter and 2 mm thickness. Other polymethacrylate-based monolithic columns are produced by Dionex (Sunnyvale, CA, USA) under the trade name ProSwift columns. This product line comprises three ion-exchange 50 mm long columns with a diameter of 4.6 mm optimized for the separation of large biomolecules. The columns allow a flow rate of up to 8 mL/min without loss in peak resolution and have an excellent long-term stability confirmed by a large number of injections. The latest format of macro-scale monoliths is a tube where the radial flow mode is applied. These tubular columns of up to 8 L volume operating at maximum flow rate of 10 L/min are also produced by BIA Separations under the trade name CIM Tube monolithic columns. The presence on the World market of different types of commercially available IE monolithic columns as well as the suitability of this separation mode for a wide range of charged small and large molecules makes IEC the most popular techniques. The applications of polymethacrylate-based monoliths for IEC are summarized in Table 1 [44–103]. However, the most scientifically interesting or practically useful examples have to be discussed more in details. 2.1.1. Application for low molecular mass compounds Small molecules such as organic acids are routinely separated by isocratic elution mode using conventional long columns packed with porous particles. Podgornik et al. [45] reported that all attempts to separate organic acids on 3 mm length CIM QA (strong ion exchanger) monolithic disks were unsuccessful. The reason for such negative result lies in the value of the so-called Z parameter [104] that is very important characteristics regarding the separation efficiency. In IEC, the Z parameter is a constant characteristic of a molecule interaction with a separation surface and considering the amount of a displacer (dissolved in a mobile phase ions) it is needed for molecules solvation and participation in their adsorption. The general rule of efficient separation is the larger difference in Z factor between molecules to be separated, the larger differences in their retention time and, consequently, the better separation quality is achieved. Due to the small difference in Z factors for small organic acids, the successful separation has to be provided by larger number of solute–sorbent interaction steps. In other words, to perform the efficient separation of this class of compounds the longer monolithic column should be used. As it was shown by the authors a good isocratic separation of mixture consisting of four organic acids (pyruvic, malic, citric and ␣-ketoglutaric) can be fulfilled on a stack of four 3 mm length CIM QA short monolithic columns (total column length 12 mm, HETP about 50 ␮m) in less than 4 min. Thus, the monolithic column length can be a real crucial point for the cases of isocratic IE separation of small molecules with similar Z factor values. Thus, the disk format looks much more convenient for protein or DNA separations using a gradient elution mode, whereas rod columns are preferable for isocratic chromatographic mode of low molecular mass objects.

2639

An original approach to ion-exchange column preparation was described in papers [48–50]. The authors applied latex-coated polymethacrylate-based monolith for ion-exchange separation of small molecules. An excellent HPLC example was demonstrated by Zakaria et al. [49]. In this case, a 30 cm × 250 ␮m I.D. monolithic capillary column was obtained by in situ copolymerization of butyl methacrylate, ethylene dimethacrylate and 2-acrylamido2-methyl-1-propanesulfonic acid following coating by QA-latex particles. It is well known that the resolution for macroporous monolithic stationary phases practically does not depend on flow rate, whereas the separation time is reduced several folds. The authors of the paper [49] examined the effect of flow rate on a separation of seven anions in ␮HPLC format. The increase of flow rate in the range of 17–31 ␮L/min at the separation of mixture consisting of iodate, bromate, nitrite, nitrate, benzoate, toluenesulfonate and benzenesulfonate allowed the baseline separation of all analytes (Fig. 1). An interesting work dealing with the determination of trace drugs in human plasma was recently published by Yang et al. [52,53]. The weak ion-exchange polymethacrylate-based 10 mm × 4.6 mm I.D. monolithic columns were applied for the determination of five postsynaptic ␣-1 adrenoreceptor antagonists and two antibiotics. The authors demonstrated that these drugs can be easily detected and enriched using native human plasma without tedious sample pretreatment. One important finding was the stability of the column over a large number of runs. The intraday reproducibility characterized with relative standard deviation (RSD) varied between 0.8 and 1.9%. For inter-day reproducibility this parameter was found to be equal to 2.6%. No obvious changes in column efficiency and back pressure were observed within 2 months. 2.1.2. Application for peptides and oligonucleotides Peptides represent a very important class of small molecules. One of the main features of peptide structure is an irregularity of charge distribution and, consequently, an irregularity of adsorption sites. Podgornik et al. [54] applied cation-exchange chromatography on short monolithic columns for the separation of peptide mixture. Three peptides differed by their molecular mass and consisting, respectively, of 9, 11 and 15 amino acids (EYIKWEEFK, KSGDWKSKCFY and QISTKSGDWKSKCFY) were separated on CIM SO3 disks. Both gradient and isocratic elution modes were successfully used for peptide separation. In the case of gradient elution, a direct dependence of retention time on peptide length was not observed due to the difference in the amino acid composition which, in its turn, defines the interaction of peptide molecule with the charged surface. Another example of successful application of IEC on short monolithic columns for the separation of peptide mixture was demonstrated by our group [55]. Three linear lysine homologues consisting of 4, 8 and 12 lysyl residues were separated within 5 min using CIM SO3 disk and complex-shape gradient of a displacer. Fig. 2 shows that the separated peptides significantly differed by their residence time that is sufficient for fast and efficient separation of such similar compounds. As it was expected, the maximum retention corresponded to dodecalysine, whereas the minimum one related to tetralysine. The separation of four oligodeoxynucleotides differing by 2 base units only (8, 10, 12 and 14 units, respectively) was carried out by Podgornik et al. [56]. For this purpose, the commercially available CIM DEAE disks were used. Both isocratic and complex-shape gradient elution modes were used. The results obtained seemed to be very similar. The variation of monolithic disk thickness from 0.75 to 1.5 mm demonstrated a satisfactory separation of all oligonucleotides; however, the chromatographic picture, similar to the

2640

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

Table 1 Application of methacrylate monoliths for ion-exchange chromatography. Monolithic material

Application

Ref.

CIM DEAE disk

Study of temperature effect on dynamic binding capacity of proteins and amino acids to monolithic ion-exchange sorbent Separation of organic acids mixture

[44]

Separation of hydrated Zn2+ from Zn-citrate, Zn-oxalate and Zn-EDTA Separation of a model common cations (Na+ , K+ , NH4 + , Ca2+ , Mg2+ ) Separation of seven model sugars, analysis and separation of sugars derived from the enzymatically processed corn starch Separation of seven inorganic and organic anions

[46]

Separation of seven inorganic and organic anions

[50] [51]

Laboratory-made COOH-containing poly(GMA-co-EDMA) capillary column Laboratory-made poly(GMA-co-MAA-co-EDMA) capillary column modified with ethylenediamine CIM SO3 disk

Isolation of tartrate Mn(III) from Phanerochaete chrysosporium fungus culture medium Determination of trace drugs in human serum albumin and human plasma Screening of antibiotics (oxacillin and cloxacillin) in human urine and plasma Separation of three heteropeptides

CIM SO3 disk

Separation of three homolysine peptides

[55]

CIM DEAE disk

Separation of different oligonucleotides

[56]

Laboratory-made poly(GMA-co-EDMA) column modified with diethylamine CIM DEAE disk

Separation of different oligonucleotides

[57]

Investigation of the retention mechanism of various size oligonucleotides. Separation of protein mixture Separation of standard protein mixture

[58] [59–61]

Separation of standard protein mixture

[62] [63]

Quicka disk DEAE

Separation of three annexins from plasma membranes of rat Morris hepatoma 7777 Separation of protein mixture

CIM DEAE disk

Separation of plasma membrane proteins

[65]

CIM QA 4-disk stack-column CIM DEAE disk Laboratory-made poly(GMA-co-EDMA) capillary column functionalized with SO3 groups Laboratory-made QA-latex-coated poly(BuMA-co-EDMA-co-AMPS) capillary columns Laboratory-made QA-latex-coated poly(BuMA-co-EDMA-co-AMPS) capillary columns Laboratory-made QA-latex-coated poly(GMA-co-EDMA) capillary columns CIM QA disk

Laboratory-made poly(GMA-co-EDMA) column modified with diethylamine Laboratory-made poly(GMA-co-EDMA) column grafted with poly(2-acrylamido-2-methyl-1-propanesulfonic acid) Quicka disk DEAE

[45]

[47] [48] [49]

[52] [53] [54]

[64]

CIM QA disk

Separation of lignin and manganese peroxidase isoenzymes

[66–68]

CIM DEAE and CIM ethanol amine

Separation of lignin peroxidase isoenzymes

[69] [70]

CIM QA tubes

Isolation of clotting factor VIII

CIM QA and CIM DEAE disks

Isolation of clotting factor IX

[71,72]

CIM DEAE disks and tubes

[73]

[76]

CIM DEAE and CIM QA disks

Separation of standard protein mixture as well as lignin peroxidase isoenzymes. Transfer of gradient plot from axial to radial columns. Comparison of efficiency of 0.34, 0.68, 1.02, 1.36, 8, 80 and 800 mL columns Separation of standard protein mixture. Comparison of efficiency of 8, 80, 800 and 8000 mL columns Separation of IgG, HSA and IgM. Comparison of efficiency of 8, 80 and 800 mL columns Determination of chemical and chromatographic stability of methacrylate-based column. Separation of standard protein mixture Isolation of pectin methylesterase isoenzymes from tomato fruits Purification of d-lactate dehydrogenase, d-alcohol dehydrogenase and alcohol oxidase from different microorganisms Separation of IgM from IgG of human blood serum

CIM DEAE disk

Isolation of the integral membrane protein CEACAM 1

[81]

CIM QA and CIM SO3 disks

Preparative purification of pegylated myelopoietin from native myelopoietin Purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-␥) in the extract solution Separation of mixture of three model proteins

[82]

CIM DEAE tube columns CIM DEAE, CIM QA and CIM EDA tube columns CIM DEAE and CIM QA tube columns

CIM SO3 disk CIM DEAE, CIM QA and CIM ethanol diamine disks

Laboratory-made poly(GMA-co-EDMA) column firstly modified with ethylenediamine, and then reacting with chloroacetic acid Laboratory-made poly(GMA-co-EDMA) column modified with polyethyleneimine CIM DEAE disc Laboratory-made poly(GMA-co-EDMA) column modified with diethylamine CIM QA and CIM DEAE tube columns

Separation of low molecular mass Al species from high molecular mass Al compounds in spiked human serum Characterization of column efficiency for protein chromatography Purification of recombinant monoclonal IgM

[74] [75]

[77,78] [79]

[80]

[83] [84] [85] [86] [87]

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

2641

Table 1 (Continued ) Monolithic material

Application

Ref.

CIM DEAE disk

Separation of plasmid DNA

[88,89]

CIM DEAE tube

Separation of plasmid DNA

[90–92]

CIM DEAE and CIM QA disks

Separation of plasmid and genomic DNA

[93,94]

CIM DEAE disk

Separation of plasmid DNA

[95]

CIM DEAE disk

Isolation of DNA from raw foodstuff and from thermally treated samples Separation of double-stranded RNA from its satellite RNAs of Cucumber mosaic virus Determination of plasmid DNA encapsulating efficiency into liposomes Separation of plasmid DNA from Escherichia coli lysate

[96]

[100]

CIM DEAE and CIM QA disks

Investigation of the dynamic adsorption behavior of virus-mimicking nanoparticles Concentration of measles and mumps viruses

CIM QA disk

Concentration of rod-shaped tomato mosaic virus

[102,103]

CIM DEAE disk CIM DEAE disk Laboratory-made DEAE-functionalized poly(GMA-co-EDMA) column CIM DEAE disk

a

[97] [98] [99]

[101]

Poly(GMA-co-EDMA) monolithic disks produced in the early 1990s by Säulentechnik Knauer GmbH, Berlin, Germany; dimensions 25 mm × 2 mm.

experiments with organic acids, was considerably improved with increasing column length [45]. The reduction of the monolith thickness to 0.3 mm, approaching the thickness of a membrane, still provided the separation. Furthermore, it remained almost unaf-

Fig. 1. Effect of high flow rates on the separation of inorganic anions and organic acids using monolithic column coated with quaternary ammonium latex AS10 (Dionex). Conditions: column 250 ␮m I.D. × 30 cm; eluent 40 mM NaF; sample loop 50 ␮m × 5.0 cm; concentration of analytes 0.5 mmol/L; detection 195 nm. Separated peaks: 1, iodate; 2, bromate; 3, nitrite; 4, benzoate; 5, nitrate; 6, benzenesulfonate; 7, toluenesulfonate. Reprinted with permission of American Chemical Society from Ref. [49].

fected when the flow rate was doubled from 4 to 8 mL/min. From the performed experiments it is possible to conclude that the mechanism of isocratic separation of small molecules on thin polymethacrylate layer appeared to be similar to that with the use of conventional packed columns where the isocratic separation was a result of multistep adsorption/desorption process. It means that even so “thin” support has enough of theoretical plates to fulfill the rules of isocratic elution. The ability of short columns with a relatively small number of theoretical plates to achieve such separations can be explained by the lack of zone spreading due to eddy dispersion; monoliths have no void volume.

Fig. 2. Separation of linear lysyl homologues K4 , K8 , and K12 obtained by solid phase peptide synthesis on CIM SO3 monolithic disk. Conditions: 0–1 min gradient from 0 to 50% B, 1–2 min from 50 to 60% B, 2–4 min from 60 to 80% B; flow rate 5 mL/min; eluent A: 5 mM PBS buffer, pH 7; eluent B: 5 mmol/L PBS, pH 7, containing 0.5 mol/L NaCl. Reprinted with permission of Elsevier from Ref. [55].

2642

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

Similar results were shown by Sykora et al. [57], where the separation of two oligonucleotide mixtures consisting, respectively, of 7 oligodeoxyadenylic acids with 12–18 repeated units, and 13 oligodeoxythymydic acids with 12–24 units was carried out using weak anion-exchange HPLC. Several laboratory-made 50 mm × 8 mm I.D. poly(GMA-co-EDMA) columns modified with diethylamine to obtain 1-N,N-diethylamine-2-hydroxypropyl functionalities were used as monolithic stationary phases. It was shown that the increase of flow rate from 1 to 4 mL/min at maintained constant gradient volume led to the reduction of separation time from 90 to 22.5 min with equivalent resolution. Thus, both these results clearly confirmed the independence of separation on flow conditions for discussed formats of monolithic media. Recently, Yamamoto et al. [58] reported the investigation of the retention mechanism of various size oligonucleotides (4–50-mers) at linear gradient conditions using anion-exchange chromatography on monolithic disks. They have determined two parameters, namely the value of binding sites and salt concentration corresponding to the peak elution. The authors established a linear dependence of both parameters on a number of charges when molecular mass of the solutes to be separated was less than ca. 3600 (12-mer) for the first parameter, and ca. 6000 (20-mer) for the second one. However, the dependence did not stay the same for both parameters when separated oligonucleotide had a larger molecular mass (>20-mer). 2.1.3. Application for proteins The number of currently published papers on IEC of proteins with the use of polymethacrylate-based monoliths is still the dominance among other applications of monolithic IEC. Due to this fact, the main attention in this section will be paid to the most interesting researches done in this field. The separations of manganese peroxidase (MnP) and lignin peroxidase (LiP) izoenzymes with the use of ion-exchange CIM monolithic disks were performed by Podgornik et al. [66,68]. LiP and MnP are extracellular enzymatic isoforms excreted by Phanerochaete chrysosporium and involved in lignin degradation. The authors showed that four main LiPs fractions can be easily separated from a crude culture filtrate on CIM QA disk with satisfactory resolution within less than 4 min instead of conventional HPLC separation on packed column (125 mm × 4 mm) that normally took about 30 min [66,67]. It should be noted another positive feature of short monolith comparatively to the separation on a packed column. Due to a lower bed volume of a disk, the higher peak height of the same sample was observed. It means that the sensitivity of analysis is also higher; this recommends such devices for protein separations from strongly diluted biological media. Branovic et al. [71,72] have described the use of monolithic DEAE and QA short columns for the separation of clotting factor IX. The dynamic adsorption experiments revealed that the binding capacity of factor IX on QA disk was about 20% higher than that obtained for DEAE disk. At the same time, DEAE disk demonstrated better chromatographic properties for the separation of impurities in comparison with QA sorbent. The chromatographic protocol developed in paper [71] allowed the separation of vitronectin from factor IX. This finding is very important because these two proteins are not separated in some commercially available factor IX concentrates purified on conventional ion exchangers. The result obtained has to be counted as a serious advantage. Another work [72] has demonstrated the results of scaled-up experiments carried out with 8, 80 and 500 mL DEAE poly(GMAco-EDMA) tubular columns. The use of monolithic columns for the isolation of factor IX from human plasma allowed not only the reduction of separation time but also the increase of specific activity of a product by almost one order of magnitude compared to

the still practically used DEAE-Sephadex column. This result is not surprising. Obviously the longer residence time of enzyme inside a separation medium will lead to a higher probability of losing its specific activity. This process can be accelerated by being unfriendly to the labile protein chromatographic conditions. Therefore, the high speed of chromatographic process is a crucial point for enzyme purification and analysis. Among the important results of the work [72], it can also be mentioned that the peak resolution demonstrated for 8 mL column was practically identical to that obtained with 80 mL device. To reach the similar resolution on different sized monolithic columns, a gradient slope should be carefully adjusted. The simple transferring of gradient slop from smaller column to larger one normally leads to the poorer resolution on larger column. The wiser way is the recalculation of a gradient slope for larger column keeping constant resolution. For this purpose, the dimensionless parameter O was introduced by Yamamoto [105,106]: O=

L Ia G HETPLGE

(1)

where Ia is the constant equal to 1, G is the gradient slope normalized with respect to column void volume and HETPLGE is the plate height in a linear gradient elution mode. To obtain equal resolution for two columns, the dimensionless parameter O should also be equal for both devices. Due to the equality of HETP for the same stationary phases, this criterion is simplified to L1 G1 = L2 G2

(2)

Knowing that G can be written as G = BVv =

CVv CVv = VG FtG

(3)

where C is the concentration difference between the beginning and the end of the gradient, Vv is the void volume, F is the flow rate and tG is the gradient time. Since the concentration difference is equal the final expression for gradient time is as follows: tG2 = tG1

Vv2 F1 L1 Vv1 F2 L2

(4)

ˇ Strancar’s group has applied this approach to compare the separations of standard test proteins on different polymethacrylatebased monolithic columns [73]. They used axial CIM DEAE disks making a stack from 1 to 4 disks (3–12 mm column length, 0.34–1.36 bed volume) as well as CIM DEAE tubes of 8, 80 and 800 mL. The results obtained for the separation of myoglobin, conalbumin and soybean trypsin inhibitor confirmed that Eq. (4) can be practically used to transfer the gradient conditions from axial to radial chromatographic monolithic columns, as well as among radial monolithic columns of different volume. The same result was also proved in the papers [74,75] where CIM DEAE and QA monolithic tubes of 8, 80, 800 and 8000 mL volume were used for protein separation (Fig. 3). As it is seen from the presented figures, 800 mL column performs a high-resolution separation identical to 8 mL column that is a result of the correct application of Yamamoto’s method for the transition of gradient conditions between different sized columns. The monolithic column with bed volume of 8000 mL allows the isolation of about 500 g of protein per hour by utilizing 1 L per minute flow rate. This amazing result of novel monolithic technology is a serious gap for the industrial production of proteins. Additionally, the work of Vidiˇc et al. [76] has to be mentioned here. In this paper, chemical and mechanical stability of QA and

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

2643

Fig. 4. Gradient separation of protein mixture on 800 mL tube monolithic column. Conditions: mobile phases: buffer A: 20 mM Tris–HCl buffer, pH 7.4; buffer B: 20 mM Tris–HCl buffer + 1 M NaCl, pH 7.4; stationary phase: CIM DEAE 800 mL tube monolithic column. Flow rate: 800 mL/min. Gradient: 0–70% buffer B in 6 min. Sample: myoglobin (peak 1), conalbumin (peak 2) and soybean trypsin inhibitor (peak 3) with concentrations of 5, 20 and 20 mg/mL, respectively. Detection: UV at 280 nm. Reprinted with permission of Elsevier from Ref. [76].

Fig. 3. Comparison of chromatograms obtained on an 8-mL (a) and 800-mL (b) tube monolithic columns. Conditions: mobile phase: buffer A: 20 mM Tris–HCl buffer, pH 7.4; buffer B: 20 mM Tris–HCl buffer + 0.7 M NaCl, pH 7.4; stationary phase: CIM DEAE columns of various volumes. Sample: proteins: myoglobin (peak 1), conalbumin (peak 2) and soybean trypsin inhibitor (peak 3) with concentrations of 3, 9 and 12 mg/mL, respectively. Flow rate: 8 mL column, 16 mL/min; 800 mL column, 800 mL/min. Detection: UV at 280 nm. Reprinted with permission of Elsevier from Ref. [74].

DEAE polymethacrylate monoliths was investigated. For this purpose, 8 mL QA and 800 mL DEAE columns were exposed in 0.1 M and 1.0 M sodium hydroxide solutions for up to 3 and 13 months. The degradation of DEAE column owing to its 13-month exposure in 1.0 M sodium hydroxide solution was equal only to 3.7%, while the value of the same parameter determined for 3-month exposure of QA column in 0.1 M sodium hydroxide solution was determined to be around 12%. The chromatographic stability of 800 mL DEAE column was examined during repeated 50 cleaning-in-place cycles including 1000 min (50 × 20 min) [76] of exposure in 1.0 M solution of NaOH and no changes in chromatographic properties, neither protein separation, nor binding capacity, were observed (Fig. 4). Regarding these results, it can be concluded that both mechanical stability and chemical stability of polymethacrylate monoliths are high to withstand conditions required for their implementation not only at laboratory, but also at industrial scales. The study of preparative purification of pegylated myelopoietin from native myelopoietin by means of QA and SO3 CIM disks in comparison with the same process carried out on Sepharose columns was done by Hall [82]. It was established that this separation with baseline peak resolution and high peak symmetry for both QA and SO3 CIM disks appeared to be 5–10-fold faster than that registered with conventional columns. Moreover, the protein purity of pegylated myelopoietin fractions exceeded 98%. In the paper of Du et al. [86] the laboratory-made 50 mm × 4.6 mm polymethacrylate-based monolithic columns modified with diethylamine were tested regarding their efficiency in protein chromatography. It should be noticed that an interesting and novel approach for pore structure formation was suggested by

the authors. To increase the permeability of monolithic sorbent, solid granules of Na2 SO4 were introduced into the system of pore-forming agents also containing traditionally used cyclohexanol and dodecanol. It was shown that the obtained monoliths demonstrated decreased back pressure compared to those prepared without a salt. Moreover, the back pressure changed a little with the increase of dodecanol, indicating that the pores formed by Na2 SO4 granules contributed much more than those formed by the organic solvents. The values of HETP for all monoliths decreased with increasing flow rate in a range of 500–1500 cm/h and were nearly constant at flow rate higher than 1500 cm/h. This fact is in agreement with the conception of mass transport inside macroporous polymer monoliths: increasing of flow rate leads to the occurrence of convective flow in more small-sized channels. Thus, the column efficiency increases with increasing flow rate. Gagnon et al. demonstrated the purification of recombinant monoclonal IgM for human clinical trials with cation- and anionexchange monoliths [87]. Cycle time for the cation-exchange capture step was only 98 min despite the low concentration of product in the feed stream. Purity after the capture step was about 90%. Cycle time for the anion-exchange intermediate purification step was only 48 min whereas the purity was equal to 95%. Compared to the methods developed for monoliths, the HPLC using hydroxyapatite as a sorbent required more time, i.e. 195 min, even though the sample volume was one third the sample volume applied to the cation exchanger. This demonstrates a serious process time advantage for monoliths in comparison with particle-based media. 2.1.4. Application for nucleic acids The high throughput capacity of monoliths has also proven valuable for increasing manufacturing efficiency and capacity for DNA plasmids. The chromatographic behavior of nucleic acids is defined by their negative molecular charge and, therefore, they can be easily bound to the positively charged chromatographic supports like DEAE and QA anion exchangers. At first time, the separation of plasmid DNA has been described by Giovannini et al. [88]. At this pioneering work the authors found that plasmid DNA can be successfully separated under isocratic conditions on QA short monolithic column (disk). The results obtained were compared to those observed on conventional packed QA-functionalized columns, as well as on polyacrylamide monolith produced in a rod shape (UNO QA column, Bio-Rad, Hercules, CA, USA). The

2644

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

result of this comparison demonstrated the significantly higher performance of polymethacrylate-based monolithic sorbent. Further, ˇ analogous experiments were done by Strancar et al. and Urthaler et al. [90–92]. Particularly, Urthaler et al. showed the production of up to 15 g highly purified plasmid DNA per chromatographic run using 800 mL CIM DEAE tubular monolithic column. It should be mentioned that if the adsorption capacity was more or less similar to that obtained on packed column (Fractogel EMD DEAE, Merck, Darmstadt, Germany), the process speed and, consequently, productivity of monoliths was found to be 5–10 times higher. At present, the developed method of plasmid DNA purification is successfully used by Boehringer Ingelheim (Austria). In fact, this is the first industrial process based on the use of monolithic stationary phases. The feasibility of separation and analysis of DNA molecules by means of a chromatography on short monolithic columns was studied by Benˇcina et al. [93]. They used this method for the separation of 5 kilobase pair (kbp) plasmid DNA, 50 kbp genomic DNA and genomic DNA of 200 kbp. Dynamic binding capacity on CIM DEAE was unaffected by flow rate; 20 mg/mL with genomic DNA and greater than 10 mg/mL with plasmid DNA. The recovery of target compound was up to 80% under optimized conditions. The fresh and interesting work dealing with the separation of replicative double-stranded RNA from its satellite RNAs of Cucumber mosaic virus using CIM DEAE monoliths can be found in the literature [94]. The developed approach gives a possibility to reveal the pathogens presence in total nucleic acids extract originating from the infected plant tissue in only 15 min avoiding nucleic acid precipitation and electrophoretic analysis. Brgles et al. [98] applied HPLC method using CIM DEAE disk for the determination of plasmid DNA encapsulating efficiency into positively charged liposomes. The offered procedure appeared to be fast, simple, precise and, in contrast to the mostly used for this purpose methods, avoiding any kind of DNA labeling. The influence of different properties of a mobile phase and stationary phase on the purification of plasmid DNA using a 120 mm × 15 mm laboratory-made DEAE polymethacrylate monolithic column was studied by Danquah and Forde [99]. The most important result obtained by the authors was the exploration of thermal stability of plasmid DNA as a strategy to improve the purity. Exposure of cell lysate of Escherichia coli at 60–80 ◦ C for 1 min allowed the diminishing of protein and RNA contaminants which were less stable at such conditions. Toxicological analysis of plasmid samples showed decreasing endotoxin’s level with increasing NaCl concentration in the binding buffer. As to the effect of stationary phase on the plasmid DNA isolation, two parameters, such as pore size and ligand density, were examined. In the first case three monolithic materials having pore size of 400, 600 and 800 nm were used for investigation. A significant trend was observed for plasmid DNA binding. Total binding capacity of plasmid DNA increased twice with the decrease of monolith pore size from 800 to 400 nm. Unfortunately, the authors did not give any information about the total surface area of the used materials, although it was known that it could be dramatically increased with decreasing pore size. It is impossible unambiguously to assert what was the real reason of such a result, but it can be related to the mentioned tendency. The testing of three monolithic columns with DEAE group density equal to 1.25, 1.85 and 2.25 mmol/g sorbent allowed conclusion that DNA recovery increased significantly and reached values from 78% up to 88% with decreasing ligand density. Such an observation can be related to a decrease of interaction energy between large DNA molecules and surface with decreasing matrix charge density. When charge surface concentration is high, the increase of contact number of this molecule with solid phase occurs that, in its turn, may lead to the appearance of irreversible-like adsorption.

However, this problem can be easily solved by the addition of a displacer to a binding buffer, or by increasing its concentration in a desorption solvent. 2.1.5. Application for viral particles The intact virus particles consist of nucleic acid molecule incorporated into protein capsid, which, in its turn, is surrounded by a lipid bilayer and glycoproteins. Thus, virus particles are charged and, consequently, can be separated by IEC. The total charge of virus is defined by proteins envelope and depends on their pI and pH of solution. The original paper devoted to the modeling of virus-mimicking nanoparticles bearing different proteins on their outer surface was recently published [100]. The goal of this research was an investigation of special properties of dynamic adsorption behavior of such big objects. Two chromatographic modes, namely ion-exchange and affinity liquid chromatography on CIM monolithic disks, were used for comparison with the dynamic behavior of individual proteins. Adsorption for both mechanisms was dominated by a single surface protein. Branovic et al. [101] used CIM DEAE disks for the concentration of measles and mumps viruses from virus vaccines containing attenuated viruses and some preservatives. The authors showed that the suggested approach can be very useful for the improved detection of particles discussed. The ability of monolithic columns to concentrate highly diluted plant viruses was tested with a model plant virus, namely rodshaped tomato mosaic virus (ToMV), by Kramberger et al. [102,103]. The virus was concentrated using strong anion exchanger, CIM QA disk, and it has been demonstrated that ToMV could be concentrated by several orders of magnitude in one-step procedure. The developed method can be applied to concentrate diluted virus samples for the following quantitative analysis. 2.2. Reversed-phase chromatography (RPLC) Reversed-phase liquid chromatography (RPLC) is based on the interactions between non-polar groups (hydrophobic patches) of separated molecules and hydrophobic ligands (alkyl or aryl groups) belonging to the solid matrix. Surface concentration of hydrophobic adsorption sites (for example, C4–C18 alkyl ligands) usually is situated in a range from several hundreds ␮mol/mL sorbent to several mmol/mL sorbent. Initially, experimental conditions are designed for a favor transfer of a solute from a liquid to a stationary phase. It means that the adsorption step takes place in water or in some aqueous solution. In this case, from the point of view of energy of hydrophobic binding of a solute and corresponding ligand, it is advantageous to form a complex. Bound solutes are then desorbed from a reversed-phase medium by adjusting the polarity of a mobile phase. Monolithic applications of RPLC are presently dominated by modified silica [16,107–109] and polystyrene [13,14]. The dominance of silica monoliths is closely connected with their pore structure. Contrary to most polymer materials, silica-based monoliths have small-sized skeletons and a bimodal pore size distribution with micrometer-sized throughpores and nm-sized mesopores, which in turn leads to the higher surface area. These specialties of silica monoliths provide favorable chromatographic properties, namely fast separations and high efficiency. The first one, provided by low-pressure drop across the column and fast mass transfer kinetics, is typical not only for silica, but also for all monoliths as a class of chromatographic sorbents, whereas high efficiency characterized by a number of theoretical plates per unit pressure drop and binding capacity value is much higher in silica than in polymer monoliths. Indeed, the efficiency of silica mono-

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

lithic columns in RPLC for small molecules is often exceeded those for polymer materials. However, most of the surface area of silica monoliths is found within the networks of mesopores which are restricted enough to limit the sorption of slow diffusing macromolecules such as proteins. In high-performance chromatography this is observed as a decrease in plate height that worsens with increased linear flow velocity. Polymethacrylate-based monoliths are offered only by Dionex under the trade name ProSwift RP columns. However, by no clear reasons, the papers describing their applications are still absent in current literature. Examples of RPLC on polymethacrylate monoliths mainly address micro-RPLC applications (Table 2) [54,110–127]. Lee et al. [110] reported on the use of poly(butyl methacrylateco-ethylene dimethacrylate) (BuMA-co-EDMA) capillary column prepared by in situ photo- and thermo-initiated polymerization and intended for protein separation by RPLC. In this work, a mixture of four proteins (ribonuclease A, cytochrome c, myoglobin and ovalbumin) was separated using gradient elution method at flow rates of 4.2, 34 and even 100 ␮L/min (sorbent pore size was equal to 2.24 ␮m). No effect of flow rate on peak resolution was observed. It has to be noted that the highest flow rate, 100 ␮L/min, corresponded to a very high linear flow velocity equal to 85 mm/s. The use of such a high flow rate would not be possible for conventional packed columns due to a prohibitively high back pressure. The separation of the same proteins using poly(butyl methacrylate-coglycerol dimethacrylate) column was similar to that obtained on the monolith cross-linked with EDMA. This fact confirms that internal hydrophilic bridges do not significantly change the retention behavior since the hydrophobicity of a sorbent is defined by both butyl chains of monovinyl monomer and hydrocarbon polymer back bone [110]. Moravcova et al. [112] have compared the isocratic separation of nine benzene derivatives on laboratory-made poly(BuMA-coEDMA) column and column packed with Biosphere C18 beads. Both columns have demonstrated similar retention behavior, but separation on monolith appeared to be two times faster. An excellent separation of four test proteins was achieved on monolithic microcolumn based on poly(butyl methacrylateco-glycerol dimethacrylate) [113] (Fig. 5). Initially, the authors designed this sorbent for hydrophobic interaction chromatography (HIC) mode, but the column did not work properly that made them to test it for RPLC. Actually, this result was expected because the ratio monomer/cross-linker used for polymerization was 30/70 and it meant that the material with too high for HIC amount of butyl groups has been produced. Indeed, when the authors reduced the content of butyl methacrylate in polymerization mixture up to 10% (w/w) the separation by HIC mechanism was easily performed. Recently, Umemura et al. [115] prepared poly(hexyl methacrylate-co-ethylene dimethacrylate) monolithic 200 mm length × 1.02 mm I.D. column that was applied to fast efficient separation of ribonuclease A, cytochrome c, transferrin and ovalbumin. The produced 20 cm long column exhibited 3000 theoretical plates at a flow rate of 50 ␮L/min and a pressure drop less than 1 MPa. The monolithic column was stable to at least 15 MPa and allowed the operation at 15–20 times higher flow rates. For example, the authors managed the separation of four mentioned proteins at a flow rate of 1000 ␮L/min within 20 s using gradient elution. Several alkyl methacrylate-based monoliths, namely ethyl-, butyl-, hexyl-, lauryl- and octadecyl-bearing stationary phases, were prepared by Ueki et al. using in situ polymerization inside 250 ␮m I.D. capillary column [116]. The comparison of chromatographic performance was done via the separation of five alkylbenzenes. All tested columns exhibited adequate chromatographic efficiency, but the best parameter reaching 6000

2645

Fig. 5. RP-HPLC separation of four proteins on poly(butyl methacrylate-co-glycerol dimethacrylate) column. Conditions: eluent A: 90% H2 O/10% acetonitrile (v/v), 0.1% TFA; eluent B: 10% H2 O/90% (v/v) acetonitrile, 0.1% TFA; linear gradient A–B: 0% (0 min) to 100% (5 min), protein concentration 0.25 mg/mL of each; flow rate 7.4 ␮L/mL. Reprinted with permission of Wiley-VCH from Ref. [113].

plates/20 cm was observed for lauryl-bearing monolith, while the material with octadecyl hydrophobic groups demonstrated quite low flow resistance (maximum operative pressure drop was less than 0.5 MPa). The effect of separation efficiency on linear flow velocity was examined in the range 1–100 mm/s for C18 column. As expected, the separation time was reduced in inverse proportion to the flow rate, while the pressure drop linearly increased with increasing flow rate. However, in contrast to the flat van Deemter profile of analogous silica-based monoliths, the authors revealed the considerable efficiency reduction, e.g. theoretical plates number declined to one-fiftieth when the linear flow velocity was raised from 1 to 100 mm/s. As it was proved by many contributors for different HPLC modes realized on rigid polymethacrylate monoliths [8,9,21,34,49,54], normally, the monolithic column efficiency is flow rate unaffected that was also approved by developed theory [23,128]. The deviation observed by authors from the rule may be generated by inhomogeneous structure of polymer obtained that inescapably leads to the low flow resistance and, consequently, is responsible for the deterioration of column efficiency at high flow rate. Jiang et al. [117] have reported on in situ preparation of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monoliths inside a 100 ␮m I.D. capillary. The optimized monolithic column was applied to the separation of several mixtures using RP chromatographic conditions. A baseline separation of a mixture consisting of thiourea, dimethyl phthalate, anisole and naphthalene, as well as a mixture of six phenols was easily performed. The separation of a standard mixture of 13 polycyclic aromatic hydrocarbons was a baseline only for 10 compounds, whereas in the case of seven weakly basic anilines mixture, six compounds were baseline separated. In the paper [120] the authors made an attempt to compare the chromatographic characteristics of two kinds of monolithic columns, namely poly(octylstyrene-co-divinylbenzene) and poly(lauryl methacrylate-co-ethylene dimethacrylate). To reach this goal, 100 ␮m I.D. capillary columns were prepared and applied

2646

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

Table 2 Application of polymethacrylate-based monoliths for reversed-phase and hydrophobic interaction chromatography. Monolithic material

Application

Ref.

(a) Separation of benzene, toluene combined with the mixture of four homologous of 4-hydroxybenzoate (b) Separation of three steroids

[54]

Laboratory-made poly(BuMA-co-EDMA) capillary column

Separation of standard protein mixture

[110,111]

Laboratory-made poly(BuMA-co-EDMA) capillary column

Separation of nine benzene derivatives

[112]

Laboratory-made poly(BuMA-co-GMA) capillary column

Separation of standard protein mixture

[113]

Laboratory-made C8 and C18 modified poly(GMA-co-EDMA) capillary columns Laboratory-made poly(HMA-co-EDMA) monolithic semi-microcolumns Laboratory-made poly(HMA-co-EDMA) capillary column

Separation of standard protein mixture

[114]

Separation of standard protein mixture

[115]

Separation of mixture of five alkylbenzenes

[116]

Laboratory-made poly(EMA-co-EDMA) capillary column

Separation of mixture of five alkylbenzenes

[116]

Laboratory-made poly(BuMA-co-EDMA) capillary column

Separation of mixture of five alkylbenzenes

[116]

Laboratory-made poly(ODMA-co-EDMA) capillary column

Separation of mixture of five alkylbenzenes

[116]

Laboratory-made poly(LMA-co-EDMA) capillary column

Separation of mixture of five alkylbenzenes

[116]

Laboratory-made poly(SMA-co-EDMA) capillary column

Separation of six phenols

[117]

Laboratory-made poly(BuMA-co-EDMA) capillary column

Separation of mixtures consisting of three, five and seven aromatic compounds Separation of mixtures consisting of seven test aromatic compounds Comparison of the chromatographic efficiency of polystyrene and polymethacrylate-based columns. Separation of 18 BSA digested peptides Separation of mixtures consisting of four aromatic compounds

[118]

Reversed-phase chromatography CIM C18 disk

Laboratory-made poly(BuMA-co-EDMA) capillary column Laboratory-made poly(LMA-co-EDMA) capillary column

Laboratory-made poly(BuMA-co-EDMA) capillary column

[119] [120]

[121]

Laboratory-made poly(OMA-co-EDMA) capillary column

Separation of mixture of four aromatic compounds

[122]

Laboratory-made poly(BuMA-co-EDMA) capillary column

[123]

Laboratory-made poly(BuMA-co-EDMA) capillary column

Fast gradient separation of mixture of eight alkylbenzenes and four proteins Separation of several benzene derivatives

Laboratory-made poly(BuMA-co-EDMA) capillary columns

Separation of alkylbenzenes from toluene to heptylbenzene

[125]

Laboratory-made poly(LMA-co-EDMA) capillary columns

Separation of alkylbenzenes from toluene to heptylbenzene

[125]

Laboratory-made poly(BuMA-co-EDMA) capillary columns with different polymer morphology Laboratory-made poly(BuMA-co-EDMA) capillary columns

Study of effect of monolith morphology on chromatographic efficiency. Separation of alkylbenzenes Determination of stability and repeatability. Separation of standard protein mixture

[126]

Hydrophobic interaction chromatography CIM C4, C8 disks

Separation of standard protein mixture

[124]

[127]

[8,21]

CIM C3 disk

Separation of standard protein mixture

[9]

CIM C4 disk

Purification of recombinant tumor necrosis factor ␣

[34]

Laboratory-made poly(BuMA-co-HEMA-co-BDDMA) capillary column CIM C12 disk

Separation of standard protein mixture

[113]

Separation of standard protein mixture

[128]

Laboratory-made poly(GMA-co-MMA-co-EDMA) capillary column CIM C4 tube

Separation of mixture of three peptides: angiotensin I, angiotensin II and methionone enkephaline Purification of alcohol oxidase from A. ochraceu

[129] [130]

CIM Isobutyl disk

Purification of alcohol oxidase from A. ochraceu

[130]

for the separation of peptide mixture obtained as a result of BSA digestion using 10 ␮g of trypsin. Despite the differences of columns obtained, namely the larger surface area and smaller pore size of poly(OST-co-DVB), the authors made a conclusion that this column offered better chromatographic performance and higher capacity. Unfortunately, the precise column characteristics are absent in the paper, but from the presented SEM images obviously the polymer materials are significantly differing from each other. In this case, it seems a little bit strange to compare these two columns, because the same difference in efficiency and capacity may be observed for one column type (polystyrene or polymethacrylate) if the porous

characteristics will be so distinctive. However, it is worth noticing that the idea was very interesting and both types of columns can be perfectly applied for the solution of a problem discussed. Jandera et al. [123] designed the poly(BuMA-co-EDMA) monolithic capillary column that allowed gradient separations of low molecular mass alkylbenzenes (Fig. 6) as well as proteins (insulin, trypsin, BSA and lactoferrin) within 2–4 min. Among the other results achieved in the paper, one seems to be more important: in spite of the same elution order of the tested proteins on the polymethacrylate- and silica-based C18 monolithic columns, the polymer sorbent provided the significantly better separa-

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

2647

It was turned out that low-density monolith demonstrated better efficiency, minimum plate height of 15 ␮m and much higher flow permeability than that found for high-density material. In the paper [127] such important parameters as column stability and reproducibility were investigated. For this purpose, 30 poly(BuMA-co-EDMA) columns of 20 cm long and 100 ␮m I.D. were prepared by both thermo- and photo-initiated polymerization. The reproducibility was examined at two levels, namely intra-batch (column-to-column) and inter-batch (batch-to-batch), with evaluation of two parameters, e.g. retention time and back pressure across the column. According to the data of 15 successive injections and separations of the protein mixture on each column, the RSD values were calculated. The values of RSD regarding a retention time were found to be ranging from 0.8 to 1.4% for thermo-initiated polymerization, and from 0.5 to 0.7% for photo-polymerized columns. Moreover, the retention did not change even after more than 2200 protein separations monitored over 1 month. The variations in back pressure for all series of column prepared were characterized by RSD values ranging from 4 to 10% for thermo-initiated polymer, and from 10 to 12% for the columns prepared by photoinitiated polymerization. Thus, the established values for both parameters are very similar to those found for standard packed chromatographic column. It should be noted that in the case of microcolumn preparation, the in situ polymerization seems to be more simple and easy process than the packing of column with particular sorbent that are often accompanied by the problems of homogeneity. 2.3. Hydrophobic interaction chromatography (HIC)

Fig. 6. Fast gradient separation of alkylbenzenes on a poly(BuMA-co-EDMA) monolithic capillary column. Conditions: column: 170 mm × 0.32 mm I.D.; mobile phase: (A) 40% ACN in water; (B) 100% CAN; gradient 0–100% B in 10 min; flow rate 53 ␮L/min; UV detection at 254 nm; analytes: (1) benzylalcohol, (2) benzaldehyde, (3) benzene, (4) toluene, (5) ethylbenzene, (6) propylbenzene, (7) butylbenzene and (8) amylbenzene. Reprinted with permission of Elsevier from Ref. [123].

tion of BSA and lactoferrin. This result once more underlined the special place of polymer monoliths in the separation of large biomolecules. The effect of initiator type used in polymerization process on separation efficiency and selectivity was investigated by Holdˇsvendová et al. [124]. This group of co-authors synthesized poly(BuMA-co-EDMA) monolithic columns via polymerization initiated by a redox system comprising ammonium peroxodisulfate and N,N,N ,N -tetramethylethylenediamine. The most known AIBN was also used with comparative purpose. The isocratic separation of several benzene derivatives approved that column efficiency and selectivity do not depend on the type of initiation. A series of works devoted to capillary monolithic column characterization, including the determination of such important characteristics as column stability, efficiency and reproducibility, were carried out by Eeltink et al. [126,127]. First of all, the authors studied the influence of polymer morphology on the efficiency of polymethacrylate-based monoliths [126]. Poly(BuMA-co-EDMA) monolithic phases were synthesized inside fused-silica capillaries by varying the polymerization mixture content. High-density porous monoliths with unimodal pore-size distribution were prepared from 40% (w/w) monomers and 60% (w/w) porogens mixture. A low-density polymeric material, prepared using 20:80 ratio of monomers versus porogens, showed bimodal pore-size distribution and much finer structure than the monolith mentioned above.

HIC is a mild separation method mainly used for the separation of proteins in their natural status. In HIC mode, the total hydrophobicity of separation surface can be adjusted via ligands density and the number of C-atoms in a ligand’s chain. In contrast to RPLC, the surface concentration of hydrophobic ligands has to be one order of magnitude lower. Methyl, ethyl, propyl, isopropyl, butyl, octyl and dodecyl ligands have been successfully used for the separations carried out according to HIC mechanism. Nowadays, polymethacrylate-based monoliths for HIC are produced only by BIA Separations, under the trade names CIM Disks C2 and C4, as well as CIM Tubes of the same functionalities. HIC applications of polymethacrylate-based monoliths are much less widespread as, for example, RPLC or IEC. Accordingly, it would be reasonable to ask “Why?”. Most probably, the cause could be related to the specific mechanism of discussed method based on the effect of protein “desalination” followed by their “precipitation” onto hydrophobic surface. Obviously HIC is appropriate for proteins only; moreover, the proteins to be separated must have their own significant hydrophobicity. All these reasons seriously limit practical use of this method and can be applied only to very special practical needs. The examples of polymethacrylate-based monoliths for HIC are summarized in Table 2 [8,9,21,34,113,128–130]. For the first time, C8 and C12 disks were applied for the separation of standard protein mixture (myoglobin, conalbumin, soybean trypsin inhibitor and ovalbumin) at different chromatographic conditions by Tenˇ ˇ nikova and Svec [8,21,128]. Ongoing work of Strancar et al. [9] was devoted to the separation of standard protein mixture by HIC on propyl-modified poly(GMA-co-EDMA) short monolithic column. It was demonstrated that the separation of three proteins has been completed at flow rate 10 mL/min within 30 s without any reduction in separation performance. Recombinant tumor factor necrosis ␣ from E. coli lysates was purified by anion-exchange chromatography followed by HIC on C4 disk [34]. Hydrophobic interaction chromatography was performed

2648

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

2.4. Hydrophilic interaction chromatography (HILIC)

Fig. 7. Separation of protein mixture by HIC mode using poly(BuMA-co-HEMAco-BDDMA) monolithic column. Conditions: sample volume 0.5 ␮L; sample composition: a solution containing 0.2 mg/mL each of myoglobin, ribonuclease A, and lysozyme. Eluent A: 2 mol/L (NH4 )2 SO4 in 10 mmol/L sodium phosphate buffer, pH 7. Eluent B: 10 mmol/L sodium phosphate buffer, pH 7. Linear gradient A–B: 0% (0 min) to 100% (20 min) at a flow rate of 2.5 ␮L/min. Reprinted with permission of Wiley-VCH from Ref. [113].

using monolithic disk of 2 mm thick and 25 mm diameter (Quick Disk, Säulentechnik Knauer, Berlin, Germany). The comparison with Sepharose C4 column demonstrated considerable decrease in separation time, from 120 min with gel low-pressure column to about 60 min for the case of monolith. In spite of the chromatographic picture obtained on Sepharose packed column showing superior peak resolution, the purity and specific activity of tumor factor necrosis were comparable. A novel triple polymethacrylate-based copolymer for weakly HIC was suggested by Hemström et al. [113]. The laboratory-made poly(butyl methacrylate-co-2-hydroxyethyl methacrylate-co-1,4butanediol dimethacrylate) monolithic microcolumn of 250 ␮m I.D. was used for the separation of protein mixture consisting of myoglobin, ribonuclease A and lysozyme at linear gradient conditions and at a flow rate of 2.5 ␮L/min (Fig. 7). The authors found that contrary to the mechanical stability which is strongly affected by the choice of the porogenic solvents, retention in this mode was not affected by the polarity of porogens used for monoliths preparation. Recently, Isobe and Kawakami [130] reported on the application of 8 mL CIM C4 tube and CIM Isobutyl disk for HIC purification of alcohol oxidase from A. ochraceu. Both columns were efficient for the separation and demonstrated high recovery of enzyme activity.

HILIC is an alternative technique to RPLC for the separation of polar compounds. Usually, this chromatographic mode is used for the separation of carbohydrates, peptides, proteins, natural product extracts, polar pharmaceuticals, etc. Normally, HILIC is based on the use of polar stationary phases and mobile phases with elevated content of organic solvents [commonly, acetonitrile (ACN) >60%] mixed with water. The application of polyacrylamide monolithic columns can be generally met for this kind of separation [131,132] whereas the use of polymethacrylate-based columns is still rare and only two examples were found in current literature. Jiang et al. [133] demonstrated the suitability of porous zwitter-ionic monolithic column for HILIC. Such a column was prepared by thermo-initiated copolymerization of N,N-dimethyl-Nmethacryloxyethyl-N-(3-sulphopropyl)ammonium betaine (SPE) with EDMA inside a 100 ␮m I.D. capillary. To control the separation properties of prepared monolith, toluene, thiourea and acrylamide were used as a test mixture. Thiourea, which is normally used as a dead volume marker in RP chromatography, was eluted after toluene and acrylamide when the ACN concentration in a mobile phase increased from 50 to 95%. The mixture of four low molecular mass neutral amides, usually weakly retained and separated by RPLC, was easily fractionated using HILIC mechanism. To demonstrate the possibility of application of the developed monolithic column for chromatography of charged molecules, seven derivatives of benzoic acid as well as a mixture of several pyrimidines/purines and neutral compounds were excellently separated (Fig. 8). It was found that the retention time of all seven acids decreased dramatically with decreasing ACN concentration from 92 to 70%. Moreover, the authors proved that the ion-exchange mechanism could contribute significantly to the overall retention if pH of a mobile phase was above pKa of separated charged compounds. Another example is the separation of three 15-, 19- and 20-mer oligonucleotides at gradient HILIC mode (100 mM triethylamine acetate in ACN/water was used as eluents) on hybrid polyacrylamide–polymethacrylate monolithic column. Holdˇsvendová et al. [134] prepared a capillary monolithic column of 320 ␮m I.D. using N-(hydroxymethyl)methacrylamide and EDMA as co-monomers. Almost baseline separation of all tested analytes

Fig. 8. Separation of basic and neutral compounds on poly(SPE-co-EDMA) monolithic column. Conditions: column dimensions 100 ␮m I.D. × 285 mm; mobile phase 5 mM ammonium formate pH 3.0 in ACN/H2 O (95/5, v/v); UV detection wavelength 214 nm; flow rate 800 nL/min; injection volume 100 nL. Samples: (1) toluene; (2) methacrylamide; (3) acrylamide; (4) thymine; (5) uracil; (6) adenine; (7) thiourea; (8) cytosine. Reprinted with permission of the American Chemical Society from Ref. [133].

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

was achieved within 35 min at flow rate 6 ␮L/min and accompanied by highly symmetrical peak profiles and excellent baseline stability. Column-to-column reproducibility was comparable to the values found for polymethacrylate esters based monolithic columns and came to not more than 6.5%. These examples allow the conclusion of future potential of monolithic polymethacrylate-based columns for such interesting kind of applications. 3. Conclusions The foundations of monoliths go back nearly 20 years. In that time, extensive theoretical and engineering evaluations have documented and explained how their unique architecture creates equally unique fractionation characteristics. Since such stationary phases contain only flow-through pores, the usual mass transfer restriction is not observed and extremely fast separations become possible. The changing flow profile and column radius do not affect the resolution, as the mass transfer is not affected by flow velocity. The open pore structure of discussed polymer monoliths provides the high accessibility for low and high molecular mass compounds. The extensive experimental work of both academic and industrial laboratories confirms the great potential of these separation media. Since monoliths are rather novel format of stationary phases for HPLC their applications still remain more modest compared to the applications of packed columns. Undoubtedly, this is only a question of time. In this short review we were concerned with only one type of polymer monoliths, but the range of monolithic media grows dramatically every year. We also avoided the description of very modern and important applications of the same materials, namely bioaffinity separation processes. The reason is that the reader can find very spread and detailed information on this subject in several recent reviews [10,135,136]. Acknowledgment This work was supported by a personal grant to E.G.V. from the Russian Science Support Foundation (RSSF, 2008, RFBR # 08-0800876-a). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

T. Hashimoto, J. Chromatogr. 544 (1991) 257. P.E. Gustavsson, P.O. Larsson, J. Chromatogr. A 734 (1996) 231. L.F. Colwell, R.A. Hartwick, J. Liq. Chromatogr. 10 (1987) 2721. N.B. Afeyan, N.F. Gordon, I. Mazsaroff, L. Varady, S.P. Fulton, Y.B. Yang, F.E. Regnier, J. Chromatogr. 519 (1990) 1. S. Brandt, R.A. Goffe, S.B. Kessler, J.L. O’Connor, S.E. Zale, J. Biotechnol. 6 (1998) 779. T. Tennikova, B. Belenekii, F. Svec, M. Bleha, US Pat. 4.923.610, 1990; US Pat. 4.952.349, 1990. ˇ T.B. Tennikova, B.G. Belenkii, F. Svec, J. Liq. Chromatogr. 13 (1990) 63. ˇ F. Svec, T.B. Tennikova, J. Bioact. Compat. Polym. 6 (1991) 393. ˇ A. Strancar, P. Koselj, H. Schwinn, D. Josic, Anal. Chem. 68 (1996) 3483. A. Jungbauer, R. Hahn, J. Chromatogr. A 1184 (2008) 62. C. Ericsson, S. Hjerten, Anal. Chem. 71 (1999) 1621. F. Plieva, B. Bober, M. Dainiak, I.Y. Galaev, B. Mattiasson, J. Mol. Recognit. 19 (2006) 305. S. Xie, R.W. Allington, F. Svec, J.M.J. Frechet, J. Chromatogr. A 865 (1999) 169. Z. Kuˇcerova, M. Szumski, B. Buszewski, P. Jandera, J. Sep. Sci. 30 (2007) 3018. P.E. Gustavsson, P.O. Larsson, J. Chromatogr. A 925 (2001) 69. K. Unger, R. Skudas, M.M. Schulte, J. Chromatogr. A 1184 (2008) 393. O. Nunez, K. Nakanishi, N. Tanaka, J. Chromatogr. A 1191 (2008) 231. ˇ E.F. Hilder, F. Svec, J.M.J. Frechet, J. Chromatogr. A 1044 (2004) 3. ˇ F. Svec, J. Sep. Sci. 28 (2005) 729. ˇ K. Stulik, V. Pacáková, J. Suchánková, P. Coufal, J. Chromatogr. B 841 (2006) 79. ˇ T.B. Tennikova, M. Bleha, F. Svec, T.V. Almazova, B.G. Belenkii, J. Chromatogr. 555 (1991) 97. B.G. Belenkii, A.M. Podkladenko, O.I. Kurenbin, V.G. Maltsev, D.G. Nasledov, S.A. Trushin, J. Chromatogr. 646 (1993) 1.

2649

[23] N.I. Dubinina, O.I. Kurenbin, T.B. Tennikova, J. Chromatogr. A 753 (1996) 217. ˇ [24] F. Svec, T.B. Tennikova, Z. Deyl (Eds.), Monolithic Materials: Preparation, Properties and Applications (Journal of Chromatography Library), vol. 67, Elsevier, Amsterdam, 2003. [25] G. Guiochon, J. Chromatogr. A 1168 (2007) 101. [26] A.-M. Siouffi, J. Chromatogr. A 1126 (2006) 86. [27] N.D. Ostryanina, O.V. Il’ina, T.B. Tennikova, J. Chromatogr. B 770 (2002) 35. ˇ ˇ [28] M. Barut, A. Podgornik, M. Merhar, A. Strancar, in: F. Svec, T. Tennikova, Z. Deyl (Eds.), Monolithic Materials: Preparation, Properties and Applications (Journal of Chromatography Library), vol. 67, Elsevier, Amsterdam, 2003, p. 66. ˇ [29] A. Nordborg, F. Svec, J.M.J. Frechet, K. Irgum, J. Sep. Sci. 28 (2005) 2401. [30] I. Miheliˇc, D. Nemec, A. Podgornik, T. Koloini, J. Chromatogr. A 1065 (2005) 59. [31] R. Hahn, M. Panzer, E. Hansen, J. Mollerup, A. Jungbauer, Sep. Sci. Technol. 37 (2002) 1. ˇ [32] A. Podgornik, M. Barut, A. Strancar, D. Josic, T. Koloni, Anal. Chem. 72 (2000) 5693. ˇ ˇ [33] A. Podgornik, M. Barut, I. Miheliˇc, A. Strancar, in: F. Svec, T.B. Tennikova, Z. Deyl (Eds.), Monolithic Materials: Preparation, Properties and Applications (Journal of Chromatography Library), vol. 67, Elsevier, Amsterdam, 2003, p. 82. [34] J. Lukˇsa, V. Menart, S. Miliciˇc, B. Kus, V. Gaberc-Porekar, D. Josic, J. Chromatogr. A 661 (1994) 161. [35] F. Leinweber, U. Tallarek, J. Chromatogr. A 1006 (2003) 207. [36] E. Vlakh, T. Tennikova, J. Sep. Sci. 30 (2007) 2801. [37] M.R. Buchmeiser, Polymer 48 (2007) 2187. [38] A. Jungbauer, R. Hahn, J. Sep. Sci. 27 (2004) 767. ˇ [39] C. Yu, M.H. Davey, F. Svec, J.M.J. Frechet, Anal. Chem. 73 (2001) 5088. [40] R. Oleschuk, L. Shultz-Lockyear, Y. Ning, D.J. Harrison, Anal. Chem. 72 (2000) 585. [41] S. Yamamoto, K. Nakanishi, R. Matsuno, Ion-exchange Chromatography of Proteins (Chromatographic Science Series), vol. 43, Marcel Dekker, New York, 1988, p. 306. ˇ [42] N. Lendero, J. Vidiˇc, P. Brne, A. Podgornik, A. Strancar, J. Chromatogr. A 1065 (2005) 29. [43] S.D. Chambers, K.M. Glenn, C.A. Lucy, J. Sep. Sci. 30 (2007) 1628. [44] I. Miheliˇc, A. Podgornik, T. Koloini, J. Chromatogr. A 987 (2003) 159. ˇ [45] A. Podgornik, M. Barut, S. Jakˇsa, J. Janˇcar, A. Strancar, J. Liq. Chromatogr. 25 (2002) 3099. [46] P. Svete, R. Milacic, B. Mitrovic, B. Pihlar, Analyst 126 (2001) 1346. [47] Y. Ueki, T. Umemura, J. Li, T. Odake, K. Tsunoda, Anal. Chem. 76 (2004) 7007. ˇ [48] E.F. Hilder, F. Svec, J.M.J. Frechet, J. Chromatogr. A 1053 (2004) 101. [49] P. Zakaria, J.P. Hutchinson, N. Advalovic, Y. Liu, P.R. Haddad, Anal. Chem. 77 (2005) 417. [50] J.P. Hutchinson, E.F. Hilder, R.A. Shellie, J.A. Smith, P.R. Haddad, Analyst 131 (2006) 215. [51] H. Podgornik, M. Stegu, A. Podgornik, A. Perdih, FEMS Microbiol. Lett. 201 (2001) 265. [52] G. Yang, H. Liu, Y. Zhang, S. Wang, J. Yin, B. Yin, Y. Chen, J. Chromatogr. A 1129 (2006) 231. [53] G. Yang, S. Feng, H. Liu, J. Yin, L. Zhang, L. Cai, J. Chromatogr. B 854 (2007) 85. ˇ [54] A. Podgornik, M. Barut, J. Janˇcar, A. Strancar, T.B. Tennikova, Anal. Chem. 71 (1999) 2986. [55] E.G. Vlakh, G.A. Platonova, G.P. Vlasov, C. Kasper, A. Tappe, G. Kretzmer, T.B. Tennikova, J. Chromatogr. A 992 (2003) 109. ˇ [56] A. Podgronik, M. Barut, J. Janˇcar, A. Strancar, J. Chromatogr. A 848 (1999) 51. ˇ [57] D. Sykora, F. Svec, J.M.J. Fréchet, J. Chromatogr. A 852 (1999) 297. [58] S. Yamamoto, M. Nakamura, C. Tarmann, A. Jungbauer, J. Chromatogr. A 1144 (2007) 155. ˇ [59] F. Svec, J.M.J. Fréchet, Biotechnol. Bioeng. 48 (1995) 476. ˇ [60] F. Svec, J.M.J. Fréchet, Anal. Chem. 64 (1992) 820. ˇ [61] F. Svec, J.M.J. Fréchet, J. Chromatogr. A 702 (1995) 89. ˇ [62] C. Viklund, F. Svec, J.M.J. Fréchet, Biotechnol. Prog. 13 (1997) 597. ˇ [63] D. Josic, Y.P. Lim, A. Strancar, W. Reutter, J. Chromatogr. B 662 (1994) 217. [64] D. Josic, J. Reusch, K. Loster, O. Baum, W. Reutter, J. Chromatogr. 590 (1992) 59. [65] D. Josic, M.K. Brown, F. Huang, H. Callanan, M. Rucevic, A. Nicoletti, J. Clifton, D.C. Hixson, Electrophoresis 26 (2005) 2809. [66] H. Podgornik, A. Podgornik, A. Perdih, Anal. Biochem. 272 (1999) 43. ˇ [67] H. Podgornik, A. Perdih, A. Podgornik, M. Barut, A. Strancar, Am. Biotechnol. Lab. 19 (2001) 32. [68] H. Podgornik, A. Podgornik, P. Milavec, A. Perdih, J. Biotechnol. 88 (2001) 173. [69] H. Podgornik, A. Podgornik, J. Chromatogr. A 799 (2004) 343. ˇ [70] A. Strancar, M. Barut, A. Podgornik, P. Koselj, H. Schwinn, P. Raspor, D. Josic, J. Chromatogr. A 760 (1997) 117. ˇ [71] K. Branovic, A. Buchacher, M. Barut, A. Strancar, D. Josic, J. Chromatogr. A 903 (2000) 21. ˇ [72] K. Branovic, A. Buchacher, M. Barut, A. Strancar, D. Josic, J. Chromatogr. B 790 (2003) 175. ˇ [73] P. Milavec Zˇ mac, H. Podgornik, J. Janˇcar, A. Podgornik, A. Strancar, J. Chromatogr. A 1006 (2003) 195. ˇ cek, M. Barut, [74] A. Podgornik, J. Janˇcar, M. Merhar, S. Kozamernik, D. Glover, K. Cuˇ ˇ A. Strancar, J. Biochem. Biophys. Methods 60 (2004) 179. ˇ [75] M. Barut, A. Podgornik, L. Urbas, B. Gabor, P. Brne, J. Vidiˇc, S. Plevˇcak, A. Strancar, J. Sep. Sci. 31 (2008) 1867.

2650

E.G. Vlakh, T.B. Tennikova / J. Chromatogr. A 1216 (2009) 2637–2650

ˇ cek, M. [76] J. Vidiˇc, A. Podgornik, J. Janˇcar, V. Frankoviˇc, B. Koˇsir, N. Lendero, K. Cuˇ ˇ Krajnc, A. Strancar, J. Chromatogr. A 1144 (2007) 63. [77] I. Vovk, B. Simonovska, M. Benˇcina, J. Chromatogr. A 1065 (2005) 121. [78] I. Vovk, B. Simonovska, J. Chromatogr. A 1144 (2007) 90. [79] K. Isobe, Y. Kawakami, J. Chromatogr. A 1065 (2005) 129. ˇ [80] P. Brne, A. Podgornik, K. Benˇcina, B. Gabor, A. Strancar, M. Peterka, J. Chromatogr. A 1144 (2007) 120. [81] M. Rucevic, J.G. Clifton, F. Huang, X. Li, H. Callanan, D.G. Hixson, D. Josic, J. Chromatogr. A 1123 (2006) 199. [82] T. Hall, D.C. Wood, C.E. Smith, J. Chromatogr. A 1041 (2004) 87. [83] Y. Wei, X. Huang, R. Liu, Y. Shen, X. Geng, J. Sep. Sci. 29 (2006) 5. [84] M. Wang, J. Xu, X. Zhou, T. Tan, J. Chromatogr. A 1147 (2007) 24. ˇ canˇcar, J. Inorg. Biochem. 101 (2007) 1234. [85] S. Murko, R. Milaˇciˇc, J. Sˇ [86] K.-F. Du, D. Yang, Y. Sun, J. Chromatogr. A 1163 (2007) 212. [87] P. Gagnon, F. Hensel, R. Richieri, BoPharm March (Suppl.) (2008) 26. [88] R. Giovannini, R. Freitag, T.B. Tennikova, Anal. Chem. 70 (1998) 3348. ˇ [89] K. Branovic, D. Forciˇc, J. Ivanciˇc, A. Strancar, M. Barut, T. Gulija, R. Zgorelec, R. Mazuran, J. Chromatogr. B 801 (2004) 331. ˇ [90] A. Strancar, A. Podgornik, M. Barut, R. Necina, in: T. Scheper (Ed.), Modern Advances in Chromatography (Advances in Biochemical Engineering and Biotechnology), vol. 76, Springer, Berlin/Heidelberg, 2002, p. 49. ˇ [91] J. Urthaler, R. Schlegel, A. Podgornik, A. Strancar, A. Jungbauer, R. Necina, J. Chromatogr. A 1065 (2005) 93. [92] J. Urthaler, W. Buchinger, R. Necina, Acta Biochim. Polon. 52 (2005) 703. ˇ [93] M. Benˇcina, A. Podgornik, A. Strancar, J. Sep. Sci. 27 (2004) 801. [94] S. Jerman, A. Podgornik, K. Cankar, N. Cadez, M. Skrt, J. Zel, P. Raspor, J. Chromatogr. A 1065 (2005) 107. [95] A. Zöchling, R. Hahn, K. Ahrer, J. Urthaler, A. Jungbauer, J. Sep. Sci. 27 (2004) 819. ˇ [96] D. Forcic, K. Branoviˇc-Cakaniˇc, J. Ivanciˇc, R. Jug, M. Barut, A. Strancar, J. Chromatogr. A 1065 (2005) 115. [97] M. Krajacic, J. Ivancic-Jelecki, D. Forcic, A. Vrdoljak, D. Skoric, J. Chromatogr. A 1144 (2007) 111. ˇ [98] M. Brgles, B. Halassy, J. Tomasic, M. Santak, D. Forcic, M. Barut, A. Strancar, J. Chromatogr. A 1144 (2007) 150. [99] M.K. Danquah, G.M. Forde, Biotechnol. Appl. Biochem. 48 (2007) 85. [100] I.V. Kalashnikova, N.D. Ivanova, T.G. Evseeva, A.Yu. Menshikova, E.G. Vlakh, T.B. Tennikova, J. Chromatogr. A. 1144 (2007) 40. ˇ [101] K. Branovic, D. Forciˇc, J. Ivanciˇc, A. Strancar, M. Barut, T. Kosutic-Gulija, R. Zgorelec, R. Mazuran, J. Virol. Methods 110 (2003) 163. ˇ [102] P. Kramberger, N. Petrovic, A. Strancar, M. Ravnikar, J. Virol. Methods 120 (2004) 51. ˇ [103] P. Kramberger, M. Peterka, J. Boben, M. Ravnikar, A. Strancar, J. Chromatogr. A 1144 (2007) 143. [104] W. Kopaciewicz, M.A. Rounds, J. Fausnaugh, F.E. Regnier, J. Chromatogr. 266 (1983) 3.

[105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] [135] [136]

S. Yamamoto, Biotechnol. Bioeng. 48 (1995) 444. S. Yamamoto, A. Kita, J. Chromatogr. A 1065 (2005) 45. S. Altmaier, K. Cabrera, J. Sep. Sci. 31 (2008) 2551. M. Thommes, R. Skudas, K. Unger, D. Lubda, J. Chromatogr. A 1191 (2008) 57. M. Bayer, C. Hänsel, A. Mosandl, J. Sep. Sci. 29 (2006) 1561. D. Lee, F. Svec, J.M.J. Fréchet, J. Chromatogr. A 1051 (2004) 53. A. Nordborg, F. Svec, J.M.J. Fréchet, K. Irgum, J. Sep. Sci. 28 (2005) 2401. D. Moravcova, P. Jandera, J. Urban, J. Planeta, J. Sep. Sci. 26 (2003) 1005. P. Hemström, A. Nordborg, K. Irgum, F. Svec, J.M.J. Frechet, J. Sep. Sci. 29 (2006) 25. Y. Li, J. Zhang, R. Xiang, Y. Yang, C. Horvath, J. Sep. Sci. 27 (2004) 17. T. Umemura, Y. Ueki, K. Tsunoda, A. Katakai, M. Tamada, H. Haraguchi, Anal. Bional. Chem. 386 (2006) 566. Y. Ueki, T. Umemura, Y. Iwashita, T. Odake, H. Haraguchi, K. Tsunoda, J. Chromatogr. A 1106 (2006) 106. Z. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, J. Biochem. Biophys. Methods 70 (2007) 39. ˇ ˇ P. Coufal, M. Cihak, J. Suchánková, E. Tesaˇrova, Z. Bosáková, K. Stulık, J. Chromatogr. A 946 (2002) 99. ˇ J. Grafnetter, P. Coufal, E. Tesaˇrova, J. Suchánková, Z. Bosáková, J. Sevˇcık, J. Chromatogr. A 1049 (2004) 43. K.W. Ro, J. Liu, M. Busman, D.R. Knapp, J. Chromatogr. A 1047 (2004) 49. X. Shu, L. Chen, B. Yang, Y. Guan, J. Chromatogr. A 1052 (2004) 205. X. Huang, Q. Wang, H. Yan, Y. Huang, B. Huang, J. Chromatogr. A 1062 (2005) 183. P. Jandera, J. Urban, D. Moravcová, J. Chromatogr. A 1109 (2006) 60. P. Holdˇsvendová, P. Coufal, J. Suchánková, E. Tesaˇrova, Z. Bosákova, J. Sep. Sci. 26 (2003) 1623. Y. Hou, P.J. Schoenmakers, W.T. Kok, J. Chromatogr. A 1175 (2007) 81. S. Eeltink, J.M. Herrero-Martinez, G.P. Rozing, P.J. Schoenmakers, W.T. Kok, Anal. Chem. 77 (2005) 7342. ˇ L. Geiser, S. Eeltink, F. Svec, J.M.J. Fréchet, J. Chromatogr. A 1140 (2007) 140. ˇ T.B. Tennikova, F. Svec, J. Chromatogr. 646 (1993) 279. V. Szucs, R. Freitag, J. Chromatogr. A 1044 (2004) 201. K. Isobe, Y. Kawakami, J. Chromatogr. A 1144 (2007) 85. T.J. Tegeler, Y. Mechref, K. Boraas, J.P. Reilly, M.V. Novotny, Anal. Chem. 76 (2004) 6698. R. Freitag, J. Chromatogr. A 1033 (2004) 267. Z. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Anal. Chem. 79 (2007) 1243. P. Holdˇsvendová, J. Suchánková, M. Bunˇcek, V. Baˇckovská, P. Coufal, J. Biochem. Biophys. Methods 70 (2007) 23. G.A. Platonova, T.B. Tennikova, J. Chromatogr. A 1065 (2005) 19. D. Josic, J.G. Clifton, J. Chromatogr. A 1184 (2007) 2.