Nano-sized polymer and polymer-coated particles in electrokinetic separations

Trends in Analytical Chemistry 120 (2019) 115656

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Trends in Analytical Chemistry journal homepage: www.elsevier.com/locate/trac

Nano-sized polymer and polymer-coated particles in electrokinetic separations L.A. Kartsova a, D.V. Makeeva a, *, V.A. Davankov b a b

Saint Petersburg State University, Saint Petersburg, Russian Federation Nesmeyanov-Institute of Organoelement Compounds, Moscow, Russian Federation

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 10 September 2019

Capillary electrophoresis (CE) is a rapidly developing method of efficient and selective separation of charged and uncharged compounds. Application of various modifiers of the background electrolyte (BGE) and capillary walls provide enhancement of selectivity and reproducibility of CE analyses. Nanoparticles (NPs) are the most promising modifiers, primarily due to their unique surface chemistry, formation of stable suspensions in BGEs, compatibilities with organic solvents and MS detection. Polymer NPs combine the advantages of nano-sized particles and the ease of dense functionalization. The combination of these properties can be also achieved by joint application of polymers with inorganic-based NPs. Here we review recent application examples of polymer NPs and discuss various synergetic examples of simultaneous use of polymers along with inorganic NPs for the separation of wide classes of compounds. We also consider approaches to capillary modification with NPs and types of “polymer-NPs” combinations for the coating and pseudo-stationary phase formation. © 2019 Elsevier B.V. All rights reserved.

Keywords: Polymer nanoparticles Nanoparticles Polymers Capillary electrochromatography Micellar electrokinetic chromatography Capillary coatings Pseudo-stationary phase

1. Introduction Capillary electrophoresis (CE) is a fast, cheap, simple to perform method of separation of charged analytes [1]. Since the introduction of MEKC by Terabe in 1984 [2] the method became applied for the separation of neutral compounds as well. Micelle-forming surfactants (cationic or anionic) at the concentration exceeding the critical micelle concentration in background electrolyte (BGE) serve as the pseudo-stationary phase (PSP) for the partitioning and separation of neutral compounds. On the other hand, the insufficient concentration sensitivity of CE can be improved by application of various on-line concentration techniques which allow sufficiently decrease limits of detection as well as manipulate with matrices of analytes. All this makes CE a powerful method for the application in medicine [3], ecology [4], in the emerging fields of proteomics [5] and metabolomics [6]. In our opinion, none of the chromatographic methods have undergone so many modifications since they have been discovered as CE has. All of the modifications were proposed to overcome sufficient disadvantages of the classic CE, which are low

* Corresponding author. E-mail address: [email protected] (D.V. Makeeva). https://doi.org/10.1016/j.trac.2019.115656 0165-9936/© 2019 Elsevier B.V. All rights reserved.

concentration sensitivity, contamination of internal capillary surface, sorption of basic compounds on the fused silica capillary walls and insufficient selectivity with respect to compounds with similar electrophoretic mobilities. The problem of low sensitivity can be successfully resolved by application of various on-line concentration techniques, while the others could be eliminated by using appropriate modifiers of the system. This could be compounds of various nature which serve as coatings of capillary walls (to prevent the basic analytes sorption, variate the electroosmotic flow (EOF) mobility and its direction, or perform the chromatographic separation mode of analytes on the capillary walls) and/or compose pseudo-stationary phases for the enhanced separation of neutral and charged analytes by means of hydrophobic and electrostatic interactions, correspondingly. First generation of the modifiers was presented by low molecular weight compounds (amines, surfactants), which have been replaced by linear or branched polymers with great number of functional groups. Polymeric modifiers in a lot of cases can form stable physically adsorbed coatings of the capillary walls, while application of polymers can afford PSPs that are compatible with organic solvents which are often required for the separation of highly hydrophobic analytes. However, most perspective modifiers in the moment seem to be nanoparticles (NPs) primarily due to their unique surface

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Abbreviations AETMAC APS APTAC AuNPs BGE CE CEC COF EOF FASS DDAB DMEAPL LHRH LPA MEKC MOF NPs

[2-(acryloyloxy)ethyl] trimethylammonium chloride ammonium persulfate (3-acrylamidopropyl)trimethylammonium chloride gold nanoparticlaes background electrolyte capillary electrophoresis capillary electrochromatography covalent-organic framework electroosmotic flow field-amplified sample stacking didodecyldimethylammoniun bromide dimethylethanolamine aminated poly(chloromethyl styrene) luteinizing hormone-releasing hormone linear polyacrylamide micellar electrokinetic chromatography metal-organic framework nanoparticles

chemistry, formation of highly stable suspensions in BGEs, compatibility with organic solvents and MS detection. NPs suitably for capillary electrophoresis application include carbon, silica, metallic and metal oxide nanomaterials, and polymer NPs [7]. The first review article that summarized examples of NPs application as PSPs in capillary electrophoresis was published in 1997 [8], where authors formulated main requirements for particles which could serve as PSPs in electrokinetic chromatography, discussed peak broadening problems in particle-containing systems and factors influencing the peak shape, as well as recent application examples. It is of interest that it was polymer NPs which were first applied as PSPs [9] for the separation of catechols. In spite of the fact that the utilizing of PNPs started in 1989, there were only a few research articles devoted to this topic by 2006 [10]. However, the high resolution ability, efficiency and MS compatibility were quite inspiring. The use of gold, silver, titanium oxide NPs, fullerenes, carbon nanotubes together with polymer NPs as PSPs and coating materials in CEC, including information on nanoparticles implementation in microchip electrochromatography was discussed a year later in Refs. [11,12]. However, more recent reviews [4,7,13e15] did not actually cover the latest information on successful application of polymer NPs in electrokinetic methods. Polymer NPs (PNPs) are usually based on amphiphilic polymers (10e250 nm) which form stable suspensions in water or BGE and generally consist of hydrophobic core and charged functional groups on the surface. Unlike inorganic NPs, polymer-based NPs have intramolecular cavities which are accessible to analytes. They also form more stable suspensions which could be stored unchangeable for years. Thus, polymer NPs combine the advantages of nano-sized particles and polymers in terms of high surface area-tovolume ratio and variability of numerous functional groups. However, combination of these properties can be also achieved by joint application of polymers with inorganic-based NPs. Polymers can efficiently cover the surface of NPs. They could provide better attachment of modifiers to the capillary surface, stabilization of inorganic NPs suspensions and variation of functionality at the same time. In this article we generalize the achievements of application of polymer NPs and synergetic examples of “polymer-inorganic NPs” as capillary coatings and pseudo-stationary phases in electrokinetic

NSAE ODMQC

nano-sized anion-exchangers quaternized cellulose modified with octadecyl fragments PAHs polyaromatic hydrocarbons PDA poly(dopamine) PDDA poly(diallyldimethylammonium chloride) PDMAEMA poly(2-(dimethylamino) ethyl methacrylate) PDMA poly(N,N-dimethylacrylamide) PEO poly(ethylene oxide) PGMA poly(glycidyl methacrylate) PDMS poly (dimethylsiloxane) PLL poly(L-lysine) PNPs polymer nanoparticles PS polystyrene PSPs pseudo-stationary phase TMAPL trimethylamine aminated poly(chloromethyl styrene) RAFT radical addition-fragmentation chain transfer QC quaternized aminocellulose ZIF-8 zeoliticimidazolate framework-8

separations of wide classes of analytes. To the best of our knowledge, it is the first review article on this topic. 2. Capillary coatings In the review article [16] authors formulated the following criteria that NPs should meet to be applied as a dynamic or permanent coating of fused silica capillary: 1) stability of suspensions in a variety of CE background electrolytes; 2) desired selectivity in the interaction with various analyte molecules; 3) electric charge providing different mobility than that of the electroosmotic flow; 4) matched mobility to the co-ions of the background electrolyte to alleviate peak broadening; 5) small mass-transfer resistance; 6) no effect on detection efficiency; 7) high surface area coverage. Due to exceptionally high surface-to-volume ratios as well as wide possibility to variate the chemistry of surface functional groups NPs can sufficiently increase separation efficiency, selectivity and reproducibility of CE analyses. Unlike inorganic, polymeric NPs (PNPs) form stable suspensions in water (as well as in a lot of BGEs), not liable to particle agglomeration, while displaying high adhesion to the fused silica capillary surface due to electrostatic as well as nonspecific interactions. Variety of exposed functional groups which can be positively and/or negatively charged or electrically neutral imply a variety of functions of PNPs as coating materials. They can suppress or reverse the electroosmotic flow, act as an ion-exchange stationary phase for open tubular CEC, prevent sorption of positively charged and basic compounds on the capillary walls, and increase selectivity through nonspecific hydrophobic interactions with analytes. Polymers polyfunctionality can be efficiently used for the variation of sorption characteristics of inorganic NPs to improve their adhesion on the capillary surface, successful postfunctionalization of prepared coating as well as NPs stabilization. In this chapter we consider various application examples of polymeric NPs concerning different separation modes (ion-exchange, reversed-phase, affinity, chiral) for the separation and concentration of wide classes of analytes. The second part of the chapter will be devoted to the mutual application of polymers with inorganic NPs in terms of coating formation and improving separation selectivity.

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2.1. Polymer NPs as capillary coatings The wide possibilities for variation of functional groups of PNPs which are responsible for different interactions with various classes of compounds make it possible to manipulate the separation selectivity. Table 1 demonstrates examples of utilizing PNPs for the realization of various separation mechanisms CE, which are ionexchange, mixed-mode, reversed-phase, chiral, affinity and for the separation of different classes of compounds (aromatic charged and uncharged compounds, peptides, amino acids, antibiotics, catecholamines and etc.). Covalent immobilization and dynamic physical adsorption of PNPs are used for the coating of capillary walls as well as for the functionalization of the PNPs which can be modified before or after (post-functionalization) the formation of coating on the capillary walls. These examples are discussed in detail below. 2.1.1. Ion-exchange mode Ion-exchange capable latex nanoparticles display a large surface-to-volume ratio and have a plenty of ion exchange sites situated on the surface of the particles, compared to bigger polymer matrix particles. That is why they could provide a high mass transfer and separation efficiency in CE. At the same time, compared to inorganic NPs, they still have vacant cavities in the volume of the beads providing higher ion capacity. Charged PNPs also demonstrate stability and lack of aggregation for long periods of time. The first applications of PNPs in terms of exploiting their ionexchange properties were described in Ref. [11] review and included the works from the group of Haddad and coworkers [17e19], where polymeric NPs were used as an ion-exchange area for preconcentration of ionic analytes followed by CE separation of cations. In Ref. [17] a coating consisting of a double layer of oppositely charged Dionex latex particles was described. The first layer was formed by anion-exchange Dionex AS5A latex particles (60 nm), the second layer included cation-exchange CS3 latex particles (300 nm). This double layer coating provided stable pHindependent EOF and an ion-exchange capacity of 2.69 nequiv./ column. The capillary was divided in two zones, which were PNPs capillary coating as a preconcentration area and an untreated capillary. The prepared capillary was utilized for the on-line preconcentration and separation of monovalent organic bases, alkaline metal ions and alkaline earth metal ions after an elution step using a transient-isotachophoretic gradient. The method was applied for the analyses of alkaline earth metals with low mM detection limits (0.6e9.5 mM). Nevertheless, restricted ion-exchange capacity even in the case where the outer latex NPs layer of high capacity was used showed the limitations of the proposed approach for samples of high ionic strength. Authors made an assumption of possible improving the performance of the system by using a dual layer latex NPs adsorbed on a monolithic support. The proof did not take long and in the same year the authors presented a silica monolith column coated with functionalized latex particles for the separation of inorganic anions [19]. A significant increase in the retention of analytes which was explained by higher phase ratio and higher ion-exchange capacity of monolith columns was observed. Such an appealing property of PNPs ion-exchange medium for the preconcentration of analytes as high ion capacity can turn out to be a drawback concerning necessity of using high ionic strength electrolytes in order to elute the analytes from the nano-sized ionexchanger. This circumstance could result in problems with Joule heating effects and lower separation efficiencies. The influence of the monolith nature of the support should be also taken into account. For example, coatings based on anion-exchange particles immobilized onto the methacrylate monolithic beds displayed a mixed-mode separation ability on account of the methacrylate

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matrix [18]. These mixed-mode interactions contributed to the enhanced separation of organic anions with similar electrophoretic mobilities. The preconcentrated organic and inorganic anions were eluted from the first part of the column by transient isotachophoresis gradient onto the coupled unmodified capillary part and separated on the latter. Such an approach allowed decreasing the detection limits up to 10 400 times which ranged between 1.5 and 12.0 nM for the organic anions [18]. Application of nano-sized anion-exchangers (NSAE) for the realization of the mode which combines the separation principles of ion chromatography and capillary electrophoresis was described in the latest publications [20e22]. Suspensions of NSAE based on styrene and 8% divinylbenzene copolymer matrix functionalized with quaternary ammonium groups were prepared and applied for the formation of an anion-exchange capable phase on the fused silica capillary walls [20,22]. The proposed fast and simple approach to the formation of NSAE-based stationary phase on the internal fused silica surface included 15 min rinsing of the capillary with diluted water suspension of NSAE. Physically adsorbed coating turned out to be extremely stable in a wide range of pH (from 2 to 10), did not require NSAE additives to the BGE to maintain the surface coverage and showed improved separation efficiency (N ¼ 148e732 *103 t.p./m) and selectivity (Rs ¼ 1.2e5.7) of carboxylic acids. Despite the prevailing role of electrostatic interactions, the nonspecific interactions are also of great importance and make a significant contribution to the attachment of PNPs to the capillary surface. For instance, in Ref. [21] the ability of cation-exchange negatively charged PNPs to modify the capillary surface and provide anodic electroosmotic flow in the 2e8 pH range was described. Thus, the authors observed the adhesion of PNPs even to the capillary surface having the same charge. However, stability of the thus dynamically adsorbed coating was insufficient, and it was necessary to make an addition of cation-exchange NPs into the BGE to maintain the modified surface. Utility of such a coating was demonstrated on the improved separation of catecholamines [21]. The modified capillary provided additional cation-exchange interactions which positively affected the resolution of cationic analytes. An example of application of ion-exchange latex nanoparticles for the reversion of EOF direction as well as improvement of separation performance by ion-exchange mechanism was reported [22,23]. The first publication was devoted to the preparation of a trimethylamine-aminated polychloromethyl styrene (TMAPL) nanolatex and their application for the on-line preconcentration and determination of bromate in tap water by the field-amplified sample stacking (FASS) OT-CEC method with the TMAPL-coated capillary column [22]. The highly stable coating created strong reversed EOF and ensured a robust method for a sensitive detection and quantitation of trace amounts of inorganic anions. Inspired by these results, authors developed a method of preparation of a new dimethylethanolamine aminated polychloromethyl styrene nanolatex (DMEAPL), coated the latter on a capillary column walls and achieved baseline separation of tetracycline antibiotics including tetracycline, oxytetracycline, doxycycline and chlorotetracycline in pig plasma [23]. To improve the sensitivity authors applied DMEAPL-coated capillary in combination with FASS concentration and demonstrated better resolution of analytes compared to that on uncoated capillary. 2.1.2. Separations based on hydrophobic interactions. Reversedphase mode To the best of our knowledge, the reversed phase mechanism for the separation of neutral compounds on the PNPs-coated capillary walls was realized in 2012, only [24]. Authors used the PNPs based

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Table 1 Summary of polymer NPs application in CEC. Base of PNPs

Functional groups/type of functionalization

Type of immobilization on the capillary walls

Separation mode

Analytes

Ref.

Polystere (Dionex latex particles)

Dynamic physical absorption

Ion-exchange

Inorganic anions and cations

[17,19]

Dynamic physical absorption on the silica monolith column

Ion-exchange

Inorganic anions

[19]

Polystere (Dionex latex particles)

Dionex AS18 latex with quaternary ammonium groups and CS3 with sulfonate groups/e Dionex AS18 latex with quaternary ammonium groups and CS3 with sulfonate groups/e Dionex AS18 latex with quaternary ammonium groups/e

Physically absorbed on the methacrylate monolithic beds

Aromatic organic anions

[18]

Polystyrene -divinylbenzene

Quaternary ammonium groups/e

Dynamic physical absorption

Mixed-mode (ion-exchange, hydrophobic interaction) Ion-exchange

[20,21]

Dynamic physical absorption Covalent

Ion-exchange Chiral separation

Covalently attached on pretreated by (3-aminopropyl) trimethoxysilane capillary

Metal ion affinity interactions

Phosphopeptides, His-tagged proteins

[27]

Dynamic physical absorption

Ion-exchange

Tetracycline antibiotics

[23]

Nanochitosan

Sulfonic groups/e Bovine serum albumin/covalent immobilization 1.Iminodiacetic acid ligands/ covalent; 2. Metal salt (FeCl3 or NiSO4) post-functionalization/ dynamic physical absorption Aminated with dimethylethanolamine e

Inorganic anions, carboxylic acids Catecholamines DL-tryptophan

Covalent

Chiral separation

[29,30]

Poly (glycidylmethacrylate)

Lysine/covalent immobilization

Divinylbenzene and vinylbenzyl chloride copolymer

N,N-dimethyldecylamine groups/ covalent immobilization

Covalently attached on the aminosilylated FS capillary Dynamic physical absorption onto the fused silica capillary with surface sulphonation

Hydrophobic interactions Reversed-phase

Catechins, tryptophane, a-tocopherols Tryptophane, tyrosine, phenylalanine Aromatic compounds (benzene derivates, aromatic aldehydes and ketones)

Polystyrene

Polychloromethylsrtyrene

on divinylbenzene and vinylbenzyl chloride copolymer aminated with N,N-dimethyldecylamine groups. The particles exhibited both long hydrophobic C10 links and quaternary ammonium end groups. The former provided sites for the realization of reversed phase mechanism of separation while the later were responsible for the strong electrostatic interactions with sulfonated capillary walls to ensure stable capillary coating. Application of PNPs in this case allows using organic modifiers of BGE because the coating remains stable unlike that formed by detergents. Aqueous-organic BGEs could be essential for the separation of hydrophobic analytes like benzene derivatives and aromatic aldehydes and ketones which, indeed, were successfully separated by the above authors. Separation of amino acids inclinable to predominant hydrophobic interactions was performed with lysine-substituted poly(glycidyl methacrylate) (PGMA) nanoparticles immobilized on the capillary walls. First, a ring-opening reaction between epoxy groups of the PGMA NPs and an amino-silylated fused capillary surface [25] resulted in a dense polymeric monolayer on the surface that was then subjected to further modification with lysine. The post-functionalized coating provided the reversed strong anodic electroosmotic flow especially in acidic conditions and a higher retention and resolution of a model mixture of amino acids (tryptophan, tyrosine and phenylalanine), compared with uncoated as well as Lys-functionalized monolayer coated capillaries. Separation was achieved due to hydrophobic interactions between the analytes and the NP-coating. In Ref. [26] authors demonstrated the first example of a stationary phase based on a boron-containing covalent-organic framework (COF) for the CEC of both charged and neutral analytes. The COF has a rigid structure, possesses permanent porosity and low density. Authors proposed a simple poly(dopamine) (PDA)supported layer-by-layer method of growing boron-COF crystals (50 nm) on the capillary walls. The coating revealed a reversedphase retention mode of uncharged analytes (ethyl-, n-propyland n-butyl-benzenes), mainly by p-p and hydrophobic

[22] [31]

[25] [24]

interactions with the COF matrix. At the same time, the fabricated capillary columns also demonstrated good resolving ability to acidic and basic compounds (benzoic acids, anilines). 2.1.3. Separation based on metal-affinity interactions Authors [27] proposed a method of preparation of capillaries coated with modified polysterene NPs for the enrichment and selective purification of certain proteins from other proteins, which was based on metal-affinity mechanism of separation. It should be noted that the first application of polystyrene (PS) NPs as a coating for the separation of proteins was demonstrated as far back as 1998 [28], where authors proposed to use 40e140 nm polystyrene particles which have been derivatized with a-u-diamines such as ethylenediamine or 1,10-diaminodecane for the suppression of interactions of basic analytes with bare silica capillary surface. In spite of promising results any further application of NPs in this direction was not considered until 2006 [27]. PS NPs were covalently attached on the capillary walls by flushing the pretreated silanized capillary with a suspension of epoxy modified latex diluted with borate buffer pH 8.5 at room temperature. After that iminodiacetic acid ligands were immobilized on the PS NPs surface followed by flashing the capillary with FeCl3 or NiSO4 aqueous solutions to initiate selective affinity interactions with phosphopeptides and His-tagged proteins correspondingly. This kind of application of PS NPs was aimed at the formation of an open tubular capillary of high capacity and large surface area and realizing capillary electrochromatography mode of separation. Authors achieved an enrichment of phosphopeptides at a micro volume scale with recoveries ranging from 92 to 95%. This stable covalently immobilized coating demonstrated great compatibility with MALDI-MS detection. 2.1.4. Chiral separation A covalent attachment of PNPs on the capillary surface for the realization of chiral separations by CEC was described in Ref. [29].

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The authors proposed an approach to the formation of a chiral stationary phase based on nano-sized natural polymer chitosan. This approach included copolymerization of methacrylatemodified nano-chitosan with methacrylamide on the silanized capillary surface. The coating demonstrated chiral separation ability for a few classes of compounds as a-tocopherols, catechines and tryptophan. Authors moved further and proposed different immobilization procedures to increase the efficiency of chitosane attachment and alter its separation ability [30]. The chiral separation mode was also performed with chemically immobilized bovine serum albumin on polystyrene NPs followed by attaching the latter to the capillary walls, as described in Ref. [31]. Direct immobilization of proteins on the capillary walls results in monolayers with only small ligand capacity and consequently poor resolution of analytes. In terms of increasing active surface area and ligand capacity the polystyrene NPs present a perfect substrate for the formation of stationary phases on the capillary surface because they are easy for preparation and modification. Authors demonstrated a rapid enantio separation (3 min) along with higher resolution of DL-Trp on nanoparticle-coated columns, compared with the monolayer coating type. The prepared columns possessed good stability and run-to-run and column-to-column reproducibility. All the above examples demonstrate the wide prospects of application of PNPs for the improvement of separation selectivity as well as concentration sensitivity of the CE technique, PNPs demonstrate a number of advantages over the low molecular weight and polymeric modifiers in terms of coating stability and separation performance due to the higher total surface area and wider possibilities for functionalization and use of various separation modes. However, the high ion capacity and interference of the polymer matrix of PNPs could influence the separation of charged analytes in both positive and negative ways. In the latter case lower separation efficiencies of analytes could be observed. The other drawbacks of PNPs as capillary coatings are the same as those for polymers or inorganic NPs. They include the necessity of preparation of monodisperse particles or particles with the same polydispersity for the satisfactory reproducibility of analyses and irreversible sorption of NPs on the capillary walls. The summary of PNPs application with various functionalities is shown in Table 1. 2.2. Synergetic applications of polymers and NPs as capillary coatings Special attention should be paid to polymer-modified inorganic NPs. Metal and metal oxide NPs are extremely interesting materials that permit improving of surface area of coatings in capillaries for the OT CEC. However, some of the particles require stabilization to prevent their aggregation or chemical destruction and form longlasting coatings on the walls [32e35]. For instance, there are a lot of application examples of citrate- or surfactant-stabilized gold nanoparticles (AuNPs) [36,37], wherein the stabilizing agents differ from reducing compounds used during the nanoparticle synthesis. Importantly, some charged polymers can simultaneously serve both as reducing and stabilizing agents and allow one-step preparation of stable suspensions suitable for immediate formation of durable and chemical stable capillary coatings [38]. Incorporation of the high surface-to-size-ratio inorganic NPs into shells of polyfunctional polymers having great adsorption potential can provide stable coatings with developed surface area, which is especially important for the realization of CEC separation. It should be mentioned that the “polymer-NPs” systems for the coating formation in CEC could be prepared by either application of polymercoated NPs or by attachment of NPs on the polymer-modified capillary walls. We classified all the examples of coatings based on “polymer-NPs” systems in terms of the type of inorganic NPs

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which were chosen as a core. Among them are Au, SiO2 NPs and other NPs (TiO2, ZrO2, Fe3O4-COOH, etc.). The polymers serving as a shell for the above inorganic NPs are responsible for such properties as stability, direction of EOF and types of interactions with analytes. 2.2.1. “Polymer-AuNPs” systems as capillary coatings Gold nanoparticles draw particular attention for the application in CEC due to the ease of their chemical modification. At the same time, it is necessary to prevent particles agglomeration and adsorption of basic compounds on the NPs surface by their stabilization with low- and high-molecular-weight compounds. Unlike surfactants, polymers could promote NPs synthesis and be used both as reducing and stabilizing agents. Moreover, it is possible to perform the one-step simultaneous synthesis of both AuNPs and polymers directly in capillary. Not only do polymers protect NPs from the agglomeration, but they also vary the adsorption properties of NPs and types of interactions with analytes. For example, poly(ethylene oxide) (PEO) interacts with proteins by hydrophilic regions and improve separation selectivity [32]. Positively charged polymers (quaternized aminocellulose (QC) [39], poly(diallyldimethylammonium chloride) (PDDA) [38,40,41]) provide strong attachment of NPs on the capillary walls due to the electrostatic interactions and prevent proteins and basic analytes sorption. Especial attention should be paid to poly (dopamine) which could take part in electrostatic, p-p aromatic, cation-p interactions as well as hydrogen bonding and metal coordination. Examples of simultaneous application of these polymers with AuNPs are discussed below. In Ref. [32] authors proposed PEO to modify didodecyldimethylammoniun bromide (DDAB)-capped gold nanoparticles in order to improve CEC separation of both acidic and basic proteins. These surfactant-protected gold NPs (AuNPs) are more stable compared to generally used citrate-protected AuNPs which are unstable in high ionic strength solutions and at low pH. DDABcapped gold NPs prevent sorption of proteins on capillary walls as well as provide a stable coating of fused capillary. Authors showed that it was an exactly unique combination of DDAB-capped AuNPs and PEO molecules that provided the best separation of proteins. Neutral PEO molecules attach to the DDAB-capped gold NPs surface due to hydrophobic regions and interacts with protein molecules by exposed to the surface hydrophilic groups. At the same time adsorption of PEO on the NPs makes the PEO stiffer and less extended compared to the free linear polymer coils. Such PEO modified DDAB-AuNPs systems were prepared by a simple mixing of NPs directly with PEO. By using of AuNPs modified with 0.05% PEO capillaries authors performed well resolved analyses of proteins with peak efficiencies for the basic proteins, including Rchymotrypsinogen, ribonuclease A, trypsinogen, cytochrome C, and lysozyme, ranging between 619 000 and 1 007 000 plate/min. The capillaries were also appropriate for the separation of five acidic proteins with low pI values, providing high peak efficiency, excellent reproducibility (RSDs for migration time less than 0.60%), and no further dynamic coating was required. In Ref. [39] authors proposed to use positively charged QC to stabilize AuNPs and apply the polymer-modified NPs (QC-AuNPs) as a coating material to decrease protein sorption and reduce analyses time. QC-AuNPs were prepared by mixing HAuCl4 and QC  solutions. Anions AuCl 4 replaced Cl anion on the polymer chains of QC, followed by reduction and formation of AuNPs. Authors proved that AuNPs occupied only a small number of the quaternary ammonium groups of QC, as follows from the FT-IR spectra and XRD patterns of QC and QC-Au nanocomposites. Residual positively charged groups covered the negatively charged adsorption sites on the fused silica capillary surface and inhibited protein adsorption.

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Moreover, AuNPs can also directly interact with capillary walls by formation of hydrogen bonds between AuNPs and silanol groups. This may additionally decrease sorption of proteins on the capillary walls. Application of QC-AuNPs increased the anodic EOF by ca. 14% compared with the pure QC capillary. All these factors benefit the QC-AuNPs coating in terms of stability and increased separation efficiency and selectivity as well as decreased protein sorption on the capillary walls. In Ref. [38] authors proposed to apply charged polymerprotected gold nanoparticles as semipermanent capillary coating in CE-MS for the analysis of heroin and its basic impurities. Due to the use of PDDA it was proposed a simple and fast method of AuNPs preparation, where PDDA served as both reducing and stabilizing agent. The coating was prepared by rinsing the capillary with PDDAAuNPs solution for 15 min. The coating possessed tolerance to methanol, AcN and 0.1 M HCl and demonstrated the improved the run-to-run, capillary-to-capillary repeatabilities, peak shape, analysis time and peak resolution of heroin and its basic impurities, which were superior to the PDDA-coated capillary. Authors believed that the viscosity of PDDA-Au which is only 90% of that of PDDA allow to form relatively easily a homogeneous surface coating by pumping of a suspension of the modifier through the capillary. Similar AuNPs stabilized by PDDA were also successfully applied for the prevention of sorption on the capillary walls and efficient separation of opioid peptides in Ref. [40]. Authors generated a stable long-term coating, which provided positively charged surface of capillary walls and large surface-to-volume ratio due to PDDA and AuNPs respectively. The coated capillary was shown to be adapted for the analysis of complex biological samples. In Ref. [41] the charged polymers were used as suppliers of AuNPs sorption on the capillary surface for the preparation of mixed-mode coatings. Capillaries were coated sequentially by positively charged poly (diallyldimethylammonium chloride), negatively charged poly (sodium-4-styrenesulfonate) and positively charged AuNPs. The polymeric support provided robustness of protein analyses over more than 810 runs and high stability of the coating against 0.01 NaOH, HCl and BGE concentrations up to 70 mM. AuNPs increased the coating density that resulted in high performance for acidic and basic proteins. The coating also demonstrated reversed-phase retention of polyaromatic hydrocarbons (PAHs). Self-polymerization of dopamine and formation of a PDA film which is easily attached to the surface of organic and inorganic materials is an inspiring instrument for the creation of coatings [42]. PDA could serve as a supplier for further surface modification providing high adhesion, stability of the coatings and various types of interactions with analytes. Based on quite a large number of publications devoted to PDA-supported coating during the last decade as well as review articles [42,43] it could be concluded that this area is still of great interest to scientific community. More detailed information about functions of PDA in electrophoretic systems as well as in micro extraction application was discussed in Ref. [43]. Amino groups of PDA could be also efficiently used for the covalent immobilization of NPs on the coated by PDA capillary surface. Thus, the strong affinities of gold for amino groups and to AueS bond formation were implemented for the enzyme immobilization on the capillary surface [44]. AuNPs which were covalently attached to the surface of capillary PDA monolith served as immobilization centers for the a-glucosidase attachment. Prepared capillaries were applied for the selective screening of a-glucosidase inhibitors in extracts of natural products. AuNPs were responsible for the attachment and stability of a-glucosidase on the capillary walls and provided high reproducibility and separation selectivity of analyses, while the PDA monolith provided the fixation of AuNPs.

Another example of PDA application as a support for incorporation of NPs to provide high surface to volume coating and strong attachment to the fused silica capillary walls for the chiral separation was shown in Ref. [45]. Authors prepared a novel enantioselective column for OT-CEC based on thiol-substituted bcyclodextrin covalently bonded on polydopamine-gold nanoparticles. PDA layer was prepared by in-capillary self-polymerization of dopamine under alkaline conditions. After that gold nanoparticles were introduced into capillary and attached by both electrostatic and affinity interactions between gold NPs and amino groups on PDA followed by attachment of thiols-bearing b-cyclodextrine through SeAu covalent bonding. Authors separated 7 pairs of chiral drugs on the developed coating. Capillaries containing AuNPs provided sufficient increase of separation efficiency compared to b-cyclodextrine-PDA coating. It could be also explained by the increased phase ratio and surface area. The similar modification system (AuNPs coated with PDA molecules) was reported in Ref. [46] for the modification of PDMS microchip channel of a microfluidic CE system. Capillary coating was formed in situ by in-capillary polymerization, where HAuCl4 acted as an oxidant to trigger dopamine polymerization and the source of gold nanoparticles, while dopamine acted as a reductant and a monomer for polymerization process. Produced polymer was simultaneously deposited on the microchip surface due to high adhesion of PDA. Thus authors proposed an approach for the formation of a coating, as stable as covalently attached, which is as simple and rapid to prepare as the dynamic coatings. Authors especially noted that the synergic effect of PDA and AuNPs is responsible for the controllable, stable EOF and anti-fouling properties of modified PDMS microchannels. The coating was tested by separation of five amino acids (arginine, proline, histidine, valine and threonine) with high efficiency and reproducibility. Immobilization of DNA on the PDA/AuNPs coated capillary was described in a more recent work [47]. Authors prepared the PDA coating with deposited AuNPs on it by simple one-step operation. The next procedure was a simple immobilization of thiolated DNA fragments on the modified capillary with formation of a selfassembled monolayer of thiolated DNA on the AuNPs surface due to covalent bond between gold and sulfur. Prepared coating was tested in terms of chiral separation ability and demonstrated baseline separation of DL-tryptophan enantiomers. Another example of PDA/AuNPs cooperation was demonstrated by the same group in Ref. [48], where coated capillaries were applied for a sensitive analysis of glutathione in bacteria and HaCaT cells. The mechanism of PDA/AuNP immobilization on the glass microchannel and the separation and detection of reduced glutathione (GSH) and oxidized glutathione (GSSN) by the coated microchip is visualized on the Fig. 2. 2.2.2. “Polymer - SiO2 NPs” systems as capillary coatings The main advantages of SiO2 NPs over the other NPs are low toxicity and cost, stability over a wide pH range and ease of functionalization via the well-established silanization reaction. Polymers could improve adhesion properties of SiO2 NPs, vary the charge of the surface and bring novel sites for the interaction with analytes. In Ref. [49] authors prepared the polyfunctional coating based on modified SiO2 NPs, which is capable to separate neutral as well as charged analytes. Authors modified mesoporous silica nanoparticles with poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) in order to enhance adsorption of a coating based on SiO2 NPs onto the inner wall of a fused silica capillary. After that, the PDMAEMAmodified SiO2 NPs were immobilized onto the inner surface of a pretreated and functionalized with octadecylsilane capillary. The presence of the polymer on the surface of NPs was responsible for

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Fig. 1. Schematic illustration of the formation of PDA (A) and TiO2 NPs deposited on the PDA coated OT column (B).

the attachment of NPs on the capillary walls. SEM images showed a rough inner surface on the modified capillaries. Due to polyfunctionality the prepared coating demonstrated improved separation of neutral compounds (thiourea, naphatalene and biphenyl) as well as charged proteins (lysozyme, cytochrome, a-chymptrypsinogen A) at pH 6. Under these conditions, the coating acquires a positive charge which prevents sorption of basic proteins and manifests itself in improving of peak tailing and separation efficiency. Authors noticed that both the interactions with C18 coating of the walls and positive charge of adsorbed nanoparticles play important role in the protein separation. Layer-by-layer approach was proposed in Ref. [50], where SiO2 NPs were placed between several poly (L-lysine) (PLL) layers on the capillary walls. Controlling the last layer (PLL or SiO2 NPs) it was possible to change the direction of EOF and analysis time as well as adopt the system for different kinds of analytes. A PLL outer layer provides reversed EOF and is appropriate for the anionic

compounds with respect to the speed of the analysis, while a SiO2exposed layer was beneficial for the determination of biogenic amines regarding speed and sensitivity. Multilayer coatings demonstrated better resolution and reproducibility of analyses due to smaller exposed area of the bare silica surface and longer diffusion paths. An example which differs from the mentioned above works was described in Ref. [51]. A polymer was applied for the stabilization of NPs on the capillary surface after the formation of SiO2 NPs layer on the walls of a microchannel for chip proteins separation [51]. The SiO2 NPs were stabilized with a methacrylate polymer prepared in situ through photopolymerization as a post-functionalization step. Based on SEM images it was proven that methacrylate matrix links the nanoparticles at specific sphere-sphere contact points. It leads to improved stability of the structure at high electric fields, allowing fast and efficient separation of proteins with different molecular weights, which were aprotinin (6.5 kDa), trypsin

Fig. 2. The mechanism of PDA/AuNP immobilization on the glass microchannel of GSH and GSSH by the coated microchip. The sample reservoir (SR) was filled with the sample solution. Sample Waste Reservoir (SW), Buffer Reservoir (BR) and Buffer Waste Reservoir (BW) were filled with running buffer.

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inhibitor (20 kDa), ovalbumin (45 kDa), and bovine serum albumin (67 kDa). Authors reported high reproducibility of analyses. The % RSD of the protein migration times varied between 0.3 and 0.5% (n ¼ 4, in 1 day) and 0.83% over a period of 7 days (n ¼ 28 runs) in a single device, at high field strength. 2.2.3. “Polymer - TiO2, ZrO2 NPs” systems as capillary coatings TiO2 and ZrO2 NPs film could be efficiently used owing to their high chemical stability, pH resistance, amphoteric nature and ability to provide metal oxide affinity chromatography mode for the separation of proteins. In Ref. [52] authors developed a facile and effective approach to prepare OT columns including TiO2 NPs deposited onto the surface of PDA pre-modified capillary. Fig. 1 presents a schematic illustration of PDA formation followed by TiO2 NPs attachment to the modified capillary surface. TiO2 NPs deposited on the pre-modified PDA column by chelation, which also boost nucleation and growth of titanium NPs on the surface. NPs improved the surface area of the column and consequently the separation efficiency and selectivity of proteins analyses. Stability and reproducibility of the capillary was proved by the analyses of real objects including separation and identification of b-lactoglobulin and five glycoforms of ovalbumin in diluted egg white. The same approach was performed in Ref. [53], where PDAcoating was used as an adhesive for the zirconia-coated column preparation. It led to improving of surface-to-volume ratio and stability of NPs coating of the capillary walls. The described in Ref. [53] two-step approach was simple and provided preparation of a physical coating with stability similar to that of the covalent coating. The prepared zirconia coated capillaries enhanced separation performance of alkaloids and provided fast and highly efficient separation of inorganic anions. 2.2.4. Polymers with other NPs as capillary coatings There are some examples of simultaneous application of polymers with other inorganic NPs. In Ref. [54] authors used modified by poly(allyldimethylammonium chloride) capillary to provide electrostatic attachment of carboxyl modified magnetic nanoparticles (Fe3O4-COOH) on the capillary surface. NPs improved the separation resolution of amino acids, proteins and dipeptides, while the positively charged polymer attached on the capillary surface insured stability of the coating. The separation on the modified capillaries combines chromatographic and electrophoretic mechanisms. In Ref. [55] authors proposed to apply natural magnesiumalumosilicate attapulgite nanoparticles in cooperation with linear polyacrylamide (LPA) for the preparation of stable fused silica capillary coatings. This coating suppressed EOF, showed great reproducibility across 43 runs of a protein mixture and reduced protein adsorption on the capillary walls. Due to the high-surface-area nanoparticles of attapulgite the amount of linear polyacrylamide molecules on the functionalized capillary wall was higher which resulted in significantly lower electroosmotic flow than in the case of typical LPA coating. High reproducibility of analyses and stability of the doped with attapulgite NPs LPA capillary coatings allowed to perform top-down proteomics of E. coli cells by CZE-MS/MS. Some metal-organic frameworks (MOFs) based on Zn (II) ions and organic ligands deposited on PDA coatings were successfully applied in Refs. [43,56] for the separation of neutral, acidic and basic compounds. MOFs possess a permanent porosity and high stability and could provide a unique selectivity. At the same time the PDA promotes the attachment of MOFs on the capillary surface and ensures the stability of coatings. In Ref. [43] authors proposed the simple PDA-assisted method of zeolitic imidazolate framework8 (ZIF-8) immobilization on the capillary surface. ZIF-8 crystals on the modified capillaries are capable of the p-p and hydrophobic

interactions with analytes, while the metal ions in the crystals could enhance the interactions with hydroxyl groups. All these factors improved separation selectivity of dihydroxybenzene isomers compared to the simple PDA column. Here, the immobilization of ZIF-8 onto the inner surface of silica capillary was promoted by PDA-coating. In Ref. [56] authors proposed the use of macrocyclic porous three dimensional organic molecules as ligands for the formation of MOFs based on Zn (II). The similar PDA-assisted method of MOFs immobilization was proposed. Due to their permanent cavities, shape-persistence and good stability the coatings provided selective separation of neutral compounds, acidic drugs, food additives and small biomolecules. All the mentioned above works indicate the high prospects and variations of synergetic joint application of NPs and polymers. The later could sufficiently increase the stability of inorganic particles and prevent their agglomeration, enhance the stability of coating and variate the functionality of NPs-based stationary phase by interacting with analytes with multiple functional groups. Despite the modification of NPs with polymers presents one additional step in the coating preparation, in a lot of cases this step is quite simple and fast. “Polymer-NPs” associates could be prepared by direct mixing of components prior to the coating formation or could be prepared in situ, when the monomer and the seed for NPs fill the capillary and the polymerization process proceeds together with NPs growing on the capillary walls. NPs bring about an enhanced surface area of the coating, which positively influences the selectivity and separation efficiency. Discussed examples are summarized in the Table 2. 3. Pseudo-stationary phases PSPs in capillary electrophoresis allow spreading the application of electrokinetic methods for the separation of uncharged analytes as well as compounds with close electrophoretic mobilities, including enantiomers. Distinct from micelles, nanoparticles possess high stability, compatibility with organic solvents and MS detection [57]. In the first review of 1997 [8] authors formulated main requirements for NPs to be applied as PSPs, including necessity of a charge on the surface providing self-migration of NPs in an electric field and elimination of their co-elution with EOF. Authors particularly pointed out the importance of the difference between EOF and electrophoretic mobilities of nanoparticles; the difference creates a window for the analytes separation. The larger the difference, the wider the resolution window of analytes could be expected. Especial attention was paid for peak broadening in NPs systems, which could be influenced by several factors, among them are electrophoretic mobilities of the particles, surface properties, porosity and composition of BGE. Application of various types of NPs as PSPs was later also revealed in 2006 by C. Nilsson and S.Nilsson [10]. Authors described examples of application of silica, gold, molecularly imprinted and polymeric NPs as PSPs for the separation of different classes of compounds (phenol derivates [58], polyaromatic hydrocarbons [59], steroids [60], aromatic acids [61], DNAs [62], catechols [9] etc.). There were cited the articles including application examples of polymeric NPs based on polystyrene-divinylbenzene (sulfonated or not), methacrylic acid, methyl methacrylate and other for the separation of alkaline metals [63], amines [64], catechols [9] and even for improving the resolution of propranolol enantiomers [65]. At the same time authors noticed that in spite of promising results, there were just a few number of manuscripts devoted to application of polymeric NPs as PSP in MEKC. In this chapter, we present more recent application examples concerning not only polymer

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Table 2 Summary of synergetic applications of polymers and NPs as capillary coatings. NPs

Polymer/Type of incorporation with NPs

Functions of coating

Type of immobilization on the capillary walls/postfunctionalization

DDAB-capped Poly (ethylene oxide)/Physically Dynamic physical adsorption/e Prevention of sorption of basic AuNPs absorbed by mixing the AuNPs directly analytes, interactions with PEO with PEO hydrophilic groups Dynamic physical adsorption/e Prevention of sorption of basic Au Quaternized cellulose (QC)/Physically analytes absorbed by introduction of HAuCl4 into the QC solution Dynamic physical adsorption/e Prevention of sorption of basic Poly(diallyldimethylammonium analytes, combined chloride)/Physically absorbed by electrophoretic and mixing the AuNPs directly with PDDA chromatographic separation mechanisms Prevention of sorption of basic Sequential dynamic physical 1. Positively charged poly adsorption of positive, negative analytes, reversed-phase mode (diallyldimethylammonium chloride), 2. Negatively charged poly (sodium-4- polymers and AuNPs on the capillary walls/e styrenesulfonate)/Support for the AuNPs attachment on the capillary walls Affinity interactions Poly (dopamine)/Support for the Covalent immobilization of covalent AuNPs immobilization AuNPs on the PDA coated capillary/Post-functionalization with a-glucozidase Covalent immobilization of Chiral separation AuNPs on the PDA coated capillary/Post-functionalization with thiols b-cyclodextrine Combined electrophoretic and In situ in-capillary polymerization of PDA and NPs chromatographic mechanisms of separation formation/e Chiral separation In situ in-capillary polymerization of PDA and NPs formation/Postfunctionalization with thiolated DNA SiO2 Prevention of sorption of basic Physical adsorption of Poly (2-(dimethylamino) ethyl analytes, reversed-phase PDMAEMA-SiO2 on the methacrylate/In situ graft separation capillary walls/Postpolymerization functionalization with octadecylsilane Poly (L-lysine)/Support for the NPs Attachment Poly (methacrylate)/Physically adsorbed on the SiO2 NPs surface attached to the capillary walls Attapulgite

Fe3O4-COOH

TiO2

Linear polyacrylamide (LPA)/One-step polymerization of acrylamide in capillary in presence of NPs Poly (allyldimethylammonium chloride)/Support for the NPs attachment Poly (dopamine)/Support for the NPs immobilization

ZrO2

Layer-by-layer dynamic physical adsorption of PLL and SiO2/e Dynamic physical adsorption/ Post-functionalization with poly (methacrylate) prepared in capillary Physical adsorption/e

Combined electrophoretic and chromatographic mechanisms of separation Prevention of sorption of basic analytes

Sequential dynamic physical adsorption of polymer and NPs/ e Chelation NPs on the incapillary polymerized PDA coated capillary/e

Combined electrophoretic and chromatographic mechanismsof separation Combined electrophoretic and chromatographic mechanismsof separation

ZIF-8

NPs but also polymer-coated nanoparticles as PSPs in electrokinetic separations. 3.1. Polymer NPs as PSPs C. Palmer and coworkers carried out a number of works devoted to PNPs application as PSPs. At [57] authors demonstrated the usability of a diblock copolymer consisted of acrylic acid (hydrophilic) and butyl acrylate (hydrophobic) parts, it formed latex nano-sized particles with hydrophobic core and hydrophilic shell (63 nm) as

Analytes

Detection Ref.

Proteins

UV

[32]

Proteins

UV

[39]

Heroin and its basic impurities Opioid peptides

UV, MS

[38]

UV

[40]

PAHs, acidic and basic proteins

UV

[41]

a-Glucozidase inhibitors

UV

[44]

Tryptophan, phenylalanine, DAD histidine, fexofenadine, promethazine, tropicamide and terbutaline Amino acids UV

[46]

DL-tryptophan Glutathione

UV UV

[47] [48]

1. Neutral compounds (thiourea, naphatalene and biphenyl) 2. Charged proteins (lysozyme, cytochrome, achymptrypsinogen A) Biogenic amines and acids

UV

[49]

LIF

[50]

Proteins

UV

[51]

Proteins (proteomic), CZEMS/MS

MS/MS

[55]

Amino acids, proteins, dipeptides

UV-Vis

[54]

Proteins ( b-lactoglobulin DAD and five glycoforms) Inorganic anions and UV alkaloids Diphenol isomers, benzene UV derivates, PAHs

[45]

[52] [53] [43]

a PSP for the electrokinetic separation and on-line concentration with UV and MS detection. Polymerization with radical additionfragmentation chain transfer (RAFT) has made the synthesis of these PNPs simple, strictly controlled and reproducible. Unlike micelles PNPs provided stable suspension in BGEs, compatible with MS detection (do not decrease ionization intensity or cause fouling of MS interface), they could be used in a low concentration in BGE and for this reason they do not interfere with the UV detection of analytes. Authors noticed that such PNPs possess high ability to the hydrophobic interactions even for the charged basic compounds. It

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is of great interest that such PNPs could sufficiently increase efficiency of sweeping on-line concentration especially of highly retained analytes. Combination of the on-line concentration with MS detection using PSP based on PNPs provided 10e20 ppb range of detection limits. The next publication [66] includes fundamental investigation of interaction behavior of the described above PNPs. Surprisingly, these particles revealed increased hydrophobic interactions and low affinity to the cationic analytes. It allows making conclusions about their conformation. The hydrophobic block of the copolymers is highly available and forms the liquid like region, which is responsible for the reversed-phase retention of analytes, while the hydrophilic region adds only a minor contribution to the retention of analytes. Replacing of functional groups from carboxylic to sulfonic did not reveal any significant differences in separation selectivity and retention mechanism, while change of hydrophobic core blocks from butyl acrylate to ethyl acrylate resulted in significant decrease in methylene selectivity for five alkylphenyl ketones separation [67]. Authors concluded that in case of the PNPs used as PSPs the influence of the hydrophobic core on the separation selectivity is dominant and could be finely tuned for the generation of unique selectivity for the separation in MEKC mode. More detailed investigations [68] revealed that the size of these anionic diblock PNPs as well as their electrophoretic mobilities depend on the relative hydrophobicity of the core of NPs, which is capable to include water in the intermolecular cavities. The more hydrophobic core, the less water was included in the core. Consequently, these NPs provided greater electrophoretic mobilities due to dense core and greater charge density of the NPs surface. Besides, hydrophobic cores demonstrated higher selectivity for the separation of neutral compounds and their application instead of SDS micelles required smaller concentrations and could be compatible with MS-detection. Possibility of preparation and successful application of positively charged diblock core-shell NPs was described in a further publication [69]. The same RAFT polymerization was used to prepare latex NPs with two cationic monomers [2-(acryloyloxy)ethyl] trimethylammonium chloride (AETMAC), (3-acrylamidopropyl)trimethylammonium chloride (APTAC) and hydrophobic monomers (butyl-, ethyl-, and methyl-acrylates). To prevent interactions of the positively charged PNPs with fused silica capillary walls authors proposed to use the homopolymer of AETMAC. Application of diblock core-shell cationic copolymers on such a coated capillary revealed the similar to anionic PNPs trends in separation selectivity, rather than to cationic surfactants. Positively charged PNPs provided predominantly hydrophobic interactions with analytes and could be efficiently used as PSPs for the separations requiring reversed EOF. However, in the case of analyses of high molecular weight, like peptides, the electrostatic interactions have a major effect on the interactions with PNPs-based PSP [70]. Authors applied a partial filling technique to reveal the interaction of PNPs based on methacrylic acid and ethylenglycoldimethacrylate and peptides which possess different basic properties. PNPs were introduces into capillary right before the analytes introduction, followed by CE analyses. Since the PNPs under consideration were negatively charged at CE conditions, they were moving in opposite direction to positively charged protein. Thus, proteins had to run through the PNPs plug, interacting with them. Application of such an approach resulted in improved peaks resolution of oxytocin and methioninenkephaline, while the more basic bradykinin and luteinizing hormone-releasing hormone (LHRH) were not detected in presence of NPs. It indicates the strong ion-ion interactions between positive sites of peptides and negative sites of PNPs. The advantages of simultaneous usage of both hydrophobic core and hydrophilic shell of polymer-based NPs were mentioned in

Ref. [71], where authors proposed to apply nano-sized particles based on cellulose. Solubility problem of this natural polymer was resolved by using a quaternized aminopropyl-cellulose modified with octadecyl fragments (ODMQC). Synthesized ODMQC contained both hydrophobic and hydrophilic groups, which make this cellulose derivative an amphiphilic polymer. ODMQC molecules self-assemble into a stable nanoscaled micelle structures in aqueous solution. So they could be used both as additives to the BGE acting like a PSP and dynamic coatings of capillary walls. Moreover, the lack of UV active groups eliminates their interference with UV detection. The authors showed that ODMQC additives in BGE lead to the formation of reversed EOF (in the range of pH 3.0e12.0), suppression of proteins sorption on the capillary walls, allow to separate both positively and negatively charged compounds. They also can act as a PSP for the separation of neutral compounds. The cycle of described above works demonstrates the attractive sides of PNPs application in electrokinetic separations as PSPs including low concentration requirement, MS-detection compatibility and unique selectivity which is different from traditional micelles. It is of especial interest that, unlike inorganic NPs, the chemistry of the surface groups of described PNPs makes a lesser contribution to the interaction with analytes compared to the core of the particles which remained accessible to analytes and available for hydrophobic interactions. The main disadvantage, which is the necessity of reproducible and controllable synthesis of PNPs, can be resolved now by RAFT polymerization; it allows safe preparation of PNPs with a variety of terminal groups and hydrophobic core. 3.2. Polymer-coated NPs as PSPs The combination of AuNPs with PEO polymers as PSPs for capillary electrophoresis separation of double-stranded DNA fragments was reported in Ref. [72]. In this research authors proposed to use “AuNPs-PEO” associates as additives to the BGE which are capable to interact with DNA fragments and provide their separation. Application of these associates provides reduction of the BGE viscosity and permits to carry out the analyses faster compared to BGE containing PEO alone. Due to the high surface area AuNPs enhance the interactions between DNA fragments and PEO adsorbed on the NPs surface and improve the sieving effect in the separation. The resolution of analytes was significantly influenced by the size of both PEO and AuNPs. More particular investigation of PEO, AuNPs, pH and ethidium bromide additives on the separation performance was described in a further publication [73]. The developed method, called nanoparticle filled capillary electrophoresis method with PEO coated AuNPs, was extended to the separation of long chain double-stranded DNA [62] and further to the genotyping of a-thalassemia deletions using AuNPs filled capillary electrophoresis with multiplex PCR [74]. AuNPs for the separation of DNA fragments were also efficient in combination with hydroxypropyl cellulose [75], LPA and poly(N,Ndimethylacrylamide) (PDMA) [76,77] matrices. In all cases NPs improved interactions of polymers with analytes by means of high surface area. It also resulted in the lower required concentration of polymers in the BGEs and lower viscosity of the sieving medium for the separation. AuNPs were shown to be applied with other polymer coatings as PSPs for the separation of high molecular weight compounds. In Ref. [78] AuNPs coated with poly(diallyldimethylammonium chloride) were used for an efficient separation and on-line concentration of acidic and basic proteins. It was shown that increasing of AuNPs in BGE (wherein the PDDA concentration stays constant) sufficiently improves separation selectivity as well as stacking efficiency. At the same time the AuNPs additives have not influenced the EOF mobility. It makes them perfect candidates for improving

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the on-line concentration effect, followed by capillary electrophoresis resolution of both acidic and basic proteins. The authors observed 314-fold sensitivity improvement compared to normal hydrodynamic injection. PDDA-modified AuNPs demonstrated the best separation of analytes compared to PEO, CTAB and poly (vinyl alcohol) in terms of sensitivity, separation selectivity, speed and repeatability. All the described above examples prove the benefits of joint application of polymers and NPs in terms of improving surface ratio of PSP and amplification of “polymer-analytes” interactions. These “polymer-NPs” systems turned out to be very useful for the separation of high molecular weight biomolecules (DNAs, acidic and basic proteins). 4. Conclusion In this review we summarize the examples of application of polymer NPs and “polymereinorganic NPs” associates in electrokinetic separations of wide classes of compounds. Polymer NPs can form stable suspensions in BGEs, possess high surface area and adhesion to the capillary surface and can be easily functionalized. Unlike inorganic NPs, their core is accessible for the interactions with analytes and plays an important role for the improving of separation selectivity of neutral compounds. We also described several examples of synergetic action of polymers with inorganic NPs. Polymers substantially increase the sorption characteristics of NPs as well as change the functionality of the NPs surface, while the NPs being applied with polymers provide the high surface area and improve separation selectivity. In this review we demonstrated that the mentioned modifiers are extremely perspective particularly in CE applications and could be used for the improved separation of proteins, DNAs, PAHs, amino acids, catecholamines and etc. Acknowledgments This work was supported by the Russian Science Foundation (grant no. N 19-13-00370). References [1] L.A. Kartsova, Capillary Electrophoresis, Problems of Analytical Chemistry, vol. 18, Nauka, Moscow, 2014 (in Russian). [2] S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya, T. Ando, Electrokinetic separations with micellar and open tubular capillaries, Anal. Chem. 56 (1984) 111e113. https://doi.org/10.1021/ac00265a031. [3] R.L.C. Voeten, I.K. Ventouri, R. Haselberg, G.W. Somsen, Capillary electrophoresis: trends and recent advances, Anal. Chem. 90 (2018) 1464e1481. https:// doi.org/10.1021/acs.analchem.8b00015. [4] A.M. Mebert, M.V. Tuttolomondo, M.I.A. Echazú, M.L. Foglia, G.S. Alvarez, M.C. Vescina, P.L. Santo-Orihuela, M.F. Desimone, Nanoparticles and capillary electrophoresis: a marriage with environmental impact, Electrophoresis 37 (2016) 2196e2207. https://doi.org/10.1002/elps.201600132. [5] B.R. Fonslow, J.R. Yates, Capillary electrophoresis applied to proteomic analysis, J. Sep. Sci. 32 (2009) 1175e1188. https://doi.org/10.1002/jssc.200800592. [6] K. Sasaki, H. Sagawa, M. Suzuki, H. Yamamoto, M. Tomita, T. Soga, Y. Ohashi, Metabolomics platform with capillary electrophoresis coupled with highresolution mass spectrometry for plasma analysis, Anal. Chem. 91 (2019) 1295e1301. https://doi.org/10.1021/acs.analchem.8b02994. [7] W. Hu, T. Hong, X. Ga, Y. Ji, Applications of nanoparticle-modified stationary phases in capillary electrochromatography, Trends Anal. Chem. 61 (2014) 29e39. https://doi.org/10.1016/j.trac.2014.05.011. [8] B. Gottlicher, K. Bichmann, Application of particles as pseudo-stationary phases in electrokinetic chromatography, J. Chromatogr. A 780 (1997) 63e73. https://doi.org/10.1016/S0021-9673(97)00218-5. [9] R.A. Wallingford, A.G. Ewing, Capillary electrophoresis, Adv. Chromatogr. 29 (1989) 65e67. [10] C. Nilsson, S. Nilsson, Nanoparticle-based pseudostationary phases in capillary electrochromatography, Electrophoresis 27 (2006) 76e83. https://doi.org/10. 1002/elps.200500535. [11] Z. Zhang, B. Yan, Y. Liao, H. Liu, Nanoparticle: is it promising in capillary electrophoresis? Anal. Bioanal. Chem. 391 (2008) 925e927. https://doi.org/10. 1007/s00216-008-1930-2.

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