Physical and dynamically coating of stationary phases to the

In CE(C), adsorption of analytes to the capillary wall is deleterious for the separation efficiency. However, this adsorption effect can be used as driving force to prepare a stationary phase layer in a controlled and structured manner. The preparation of such coatings is more facile and inexpensive compared to chemically bonded stationary phases. However, the lifetime and the stability of such adsorbed layers is limited.

The adsorbed coatings are categorized in physical and dynamic coatings, according to the strength of adsorption. Physical coatings are strongly adsorbed on the capillary wall and will shield the negatively charged silanol groups. Dynamic coatings are only weakly attached to the wall and the adsorbed modifier is actually added to the mobile phase [50].

The first applications of adsorbed stationary phases have already been reported in CE analyses, where the coating suppressed or inverted the EOF [87-92]. In electrochromatography, however, the coated stationary phase should introduce a chromatographic mode, while avoiding the time consuming steps to anchor the coating chemically to the wall.

Liu and co-workers investigated and reviewed in 1999 the adsorption effects and the chromatographic behavior of several cationic surfactants, basic proteins, peptides and amino acids [93-95]. More recently, new developments in physically and dynamically adsorbed coatings have extensively been reviewed by Doherty et al., Guihen et al., Cheong et al. and Xu et al. [20,50,51,53].

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69 More recently, the application of graphene coated capillaries attracted some interest for the separation in RP-CEC mode [96-100]. In a first step the capillary wall is functionalized with amino-groups. Consequently, the epoxy groups of the graphene-oxide nano sheets will react with the amino-moieties via a SN2 nucleophilic displacement. The successful separation of

some non steroidal anti-inflammatory drugs and a mixture of endocrine-disrupting chemicals has been reported on this type of column. However, the separations are characterized by low retentions due to the finite thickness of the coating. Therefore, the thickness of the layers should be optimized without any loss of efficiency.

4 Monolithic columns in capillary electrochromatography

Initially, monolithic columns were developed as an alternative for the packed bed columns in HPLC. The application of monolithic columns in liquid chromatography gained a lot of interest as the permeability of these columns are higher compared to packed columns. The higher permeability results in longer columns as column lengths are restricted by the generated back pressure. Furthermore, monolithic columns demonstrate a slow decrease of efficiency with high mobile-phase velocities and are therefore applicable for fast, efficient analyses [101-104]. Monolithic columns for CEC were already developed in the early stages of electrochromatography as they exhibit some specific advantages such as the easiness of preparation, the easy modification of the functional groups and the compatibility with small inner diameter capillaries (20-100 µm) [105-108]. Moreover, the absence of retaining frits combined with the high sample capacity explains the high interest in monolithic CEC.

Monolithic CEC will not be a subject in this work and therefore the further literature study will be concise. However, CEC with monoliths and all their aspects have been extensively researched during the last 15 years and numerous reviews have been published [83,109- 118].

Monolithic CEC can be divided in two main categories: silica-based monoliths and organic polymer monoliths.

The organic monoliths are synthesized in the capillary by the in-situ polymerization of various copolymers. The reaction is instigated by free radical, thermal or UV-initiation. The organic porous monoliths in CEC are typically based on acrylamide, methacrylate and

70 styrene polymers and the pore size can be controlled by selecting the monomer concentration, the type of crosslinker and by adjusting the porogen solvent. Generally, the hydroxyl functions of the silica wall are no longer accessible, therefore an EOF generating moiety has to be copolymerized or grafted on the monolith to instigate the mobile-phase flow in CEC. However, the organic polymers are prone to swelling and shrinkage effects in organic solvents, limiting their repeatability and life time [119-121].

Silica based monoliths can be prepared by three different approaches: the fusion of silica particles by sintering, the entrapping of silica particles by a sol-gel process and the polymerization of silicon alkoxide precursors by sol-gel processes. Actually, the last approach is the most genuine one and therefore the most popular one to create a monolithic column. The two other approaches are developed to immobilize a packed bed without the necessity of frits. Silica monoliths do not exhibit the swelling process during analyses but are more complicated to synthesize as they need post-modification [102,115]. Moreover, the silica monoliths are only applicable in a limited pH range (3-9). Furthermore, silica monoliths are prone to shrinkage during the curing step of their preparation. As a consequence, a small gap will exist between the wall and the monolith, inducing wall effects and band broadening effects [103,122].

Figure III.3. SEM image of a silica-based monolith with through pore sizes of 1-2 µm [122]. 25 µm

(a)

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71

5 Separation modes applied in CEC

The described column formats and their varieties can be applied in different separation modes. Comparable to HPLC, CEC separations can be divided into four major categories. The majority of the separations performed in CEC are based on the differences in polarity or hydrophobicity of the solute molecules. Also, a significant amount of research in CEC is focused at the separation of enantiomers. To a lesser extent the selective separations of charged ions via an ion-exchange type of method and the separations of solutes based on their sizes is also described in literature.