Use of nanoparticles in capillary and microchip electrochromatography

Chiral drugs exert pharmacological effects through strict matching with chiral biological macromolecules and chiral recognition. Each enantiomer has different pharmacological activities, metabolic processes and rates, as well as toxicity pharmacokinetic characteristics owing to the difference in its interactions with the chiral environment. Therefore, method development for the resolution of chiral drugs is of great significance for the synthesis of chiral drugs and for quality control during the production process. Molecularly imprinted polymers (MIPs) are prepared by using a target molecule as the template. MIPs demonstrate highly specific recognition properties toward the target molecule since they have specific spatial molecular structures and functional groups. Hence, MIPs are particularly suitable for the separation and purification of chiral drugs. Capillary electrochromatography (CEC) offers the advantages of high separation efficiency and high selectivity owing to the dual separation mechanisms including capillary electrophoresis and liquid chromatography. By using MIPs as the stationary phases for CEC, the advantages of the two technologies can be combined to achieve efficient separation of chiral drugs. MIPs were first applied to CEC for chiral resolution in 1994, and since then, there have been notable advances in this field. The four main chiral separation modes in CEC involve the use of MIPs as the stationary phases of open tubular, packed, and monolithic columns, and as the pseudostationary phase in the separation medium. This review summarizes the research progress of these four methods and reveals the potential of MIPs in chiral resolution by CEC. The advantages and disadvantages of these methods are commented. MIPs as the stationary phases of packed columns can allow for chiral separation. However, the preparation of packed columns in narrow capillaries is difficult. In addition, frits must be prepared at the ends of the capillaries to seal the MIPs. The frits lead to the formation of bubbles during the CEC analysis, thus resulting in poor repeatability and stability. These problems can be overcome by using MIP-based open tubular columns. Furthermore, conditioning of open tubular columns is easy and less time-consuming. However, open tubular columns have limited capacity. MIP-based monolithic columns have greater capacity than do open tubular columns, and frits are not required in this case. However, in situ preparation of MIPs monolith in narrow capillaries is still challenging. The application of MIPs to chiral CEC can also be realized by using them as pseudostationary phases (additives) in the separation medium, and this allows for ease of operation. Moreover, the amount of MIPs introduced into the capillary can be accurately controlled. Thus, the batch-to-batch reproducibility can be improved, but this has the disadvantage of increased MIP consumption. In order to further expand the potential of MIPs in chiral CEC, the following aspects must be considered. First, improvement of the preparation method. In most reported MIP-based-chiral CEC techniques, the peaks of the imprinted molecules show severe tailing, and this problem must be resolved. Improving the mass transfer rate of the prepared MIPs may be a suitable solution in this regard. Second, development of new functional monomers. A functional monomer is an indispensable component in the preparation of MIPs. New functional monomers can be prepared according to the "three-point interaction" rule. Third, selection of template molecules. A single enantiomer of chiral drugs is used as the template molecule to prepare chiral MIPs. The method is not suitable for the preparation of MIPs of chiral drugs for which a single enantiomer is difficult to obtain. Therefore, appropriate choice of the template molecules for these drugs is imperative. Fourth, discussion of chiral separation mechanism. The mechanism of interaction between the template molecules and MIPs needs to be explored further, in order to obtain theoretical guidance for the design and preparation of chiral MIPs.

In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.

CD-MONs (β-cyclodextrin-based microporous organic networks), derived from β-cyclodextrin, possess notable hydrophobic characteristics, a considerable specific surface area, and remarkable stability, rendering them highly advantageous in separation science. This research aimed to investigate the utility of CD-MONs in chromatography separation. Through a monomer-mediated technique, we fabricated an innovative CD-MON modified capillary column for application in open-tubular capillary electrochromatography (OT-CEC). The CD-MON-based stationary phase on the capillary's inner surface was analyzed using Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). We assessed the performance of the CD-MON modified capillary column for separation purposes. The microstructure and pronounced hydrophobicity of CD-MON contributed to enhanced selectivity and resolution in separating diverse hydrophobic analytes, such as alkylbenzenes, halogenated benzenes, parabens, and polycyclic aromatic hydrocarbons (PAHs). The maximum column efficiency achieved was 1.5 × 105 N/m. Additionally, the CD-MON modified capillary column demonstrated notably high column capacity, with a methylbenzene mass loading capacity of up to 197.9pmol, surpassing that of previously reported porous-material-based capillaries. Furthermore, this self-constructed column was effectively utilized for PAHs determination in actual environmental water samples, exhibiting spiked recoveries ranging from 93.2 to 107.9% in lake water samples. These findings underscore the potential of CD-MON as an effective stationary phase in separation science.

Recent applications and chiral separation development based on stationary phases in open tubular capillary electrochromatography (2019–2022)

Evaluation of a composite nanomaterial consist of gold nanoparticles and graphene-carbon nitride as capillary electrochromatography stationary phase for enantioseparation

Enantiomer separation by electrochromatography employing modified cyclodextrins as stationary phases is performed in two ways. (i) Polysiloxane-linked permethylated beta-cyclodextrin (Chirasil-Dex 1) or related selectors are coated and immobilized onto the inner surface of a capillary column. Enantiomer separation is performed in the open tube and the method is referred to as open-tubular capillary electrochromatography (o-CEC). (ii) Silica-linked native beta-cyclodextrin, permethylated beta-cyclodextrin (Chira-Dex 2) or hydroxypropyl-beta-cyclodextrin are filled into a capillary column and the bed is secured by two frits. Enantiomer separation is performed in a packed column and the method is referred to as packed capillary electrochromatography (p-CEC). In a unified instrumental approach, method (i) as well as method (ii) can be operated both in the electro- and pressure-driven modes (o-CEC vs. open-tubular liquid chromatography (o-LC) and p-CEC vs. p-LC). It is demonstrated that the electro-driven variant affords higher efficiencies at comparable elution times. Employing a single open-tubular column coated with Chirasil-Dex 1, a unified enantioselective approach can be realized in which the same selectand is separated using all existing chromatographic modes for enantiomers, i.e., gas chromatography (GC), super-critical fluid chromatography (SFC), o-LC and o-CEC. As the chiral selector is utilized as a stationary phase, an additional chiral selector may be added to the mobile phase. In the resulting dual chiral recognition systems, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) may be observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand.

Chiral covalent organic framework-based open tubular capillary electrochromatography column for enantioseparation of selected amino acids and pesticides.

N-doped graphene quantum dots are carbonaceous smart nanomaterials that play a promising role in analytical chemistry methods, especially as sensors, high-performance liquid chromatography, and gas chromatography stationary phases. However, their application in electromigration methods was limited to the role of absorbing probes for indirect detection or a pseudostationary phase, while their high surface area and functionality present broader opportunities to increase analytical capabilities of the capillary electrochromatography method. The current work explores their ability to form a stationary phase on the inner walls of a fused-silica capillary for the capillary electrochromatography alone and with the following surfactant modification. It reports stability, the working pH range, efficiency, and selectivity of the novel capillary coating obtained with its further surfactant modification using imidazolium ionic liquids. The stationary phase was applied to separate a mixture of neutral and charged analytes, namely aromatic amino acids and steroid hormones, and the coating applicability to real samples was demonstrated with the food supplement analysis.

The development of sol-gel open-tubular column technology in capillary electrochromatography (CEC) is reviewed. Sol-gel column technology offers a versatile means of creating organic-inorganic hybrid stationary phases. Sol-gel column technology provides a general approach to column fabrication for microseparation techniques including CEC, and is amenable to both open-tubular and monolithic columns. Direct chemical bonding of the stationary phase to the capillary inner walls provides enhanced thermal and solvent stability to sol-gel columns. Sol-gel stationary phases inherently possess higher surface area, and thus provide an effective one-step alternative to conventional open-tubular column technology. Sol-gel column technology is applicable to both silica-based and transition metal oxide-based hybrid stationary phases, and thus, provides a great opportunity to utilize advanced material properties of a wide range of nontraditional stationary phases to achieve enhanced selectivity in analytical microseparations. A wide variety of stationary phase ligands can be chemically immobilized on the capillary inner surface using a single-step sol-gel procedure. Sol-gel chemistry can be applied to design stationary phases with desired chromatographic characteristics, including the possibility of creating columns with either a positive or a negative charge on the stationary phase surface. This provides a new tool to control electroosmotic flow (EOF) in the column. Column efficiencies on the order of half a million theoretical plates per meter have been reported for sol-gel open-tubular CEC columns. The selectivity of sol-gel stationary phases can be easily fine-tuned by adjusting the composition of the coating sol solution. Open-tubular columns have significant advantages over their packed counterparts because of the simplicity in column making and hassle-free fritless operation. Open-tubular CEC columns possess low sample capacity and low detection sensitivity. Full utilization of the analytical potential of sol-gel open-tubular columns will require a concomitant development in the area of high-sensitivity detection technology.

Capillary electrochromatography (CEC) has been realised as a combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) allowing the separation of analytes based on their differing adsorption and migration behaviour. There is a considerable interest in developing new methodologies for CEC, which uses chromatographic beads or monoliths as stationary phases or alternatively employs open tubular (OT) capillaries relying on surface modifications. Besides the bulk flat flow profile of the mobile phase in CEC columns allowing an enhanced resolution, due to the influence of the applied electrical field there are other possibilities to provide a higher resolution with better detection. As an additional newly means for peak sharpening and sample enrichment isotachophoresis and a “sweeping mechanism” could be envisaged, which both take advantage of the inhomogeneous electric field pattern in the capillary by choosing appropriate leading and terminating background electrolytes (BGEs). Transient isotachophoresis would be another technique to provide an increased plate height in the separations, using an optimized sample matrix, e.g. containing a relatively high acetonitrile content, with the same leading and terminating BGEs. In this thesis, a new method to generate a “sharpening zone”, which is similar to the concept of transient isotachophoresis, was established using an aqueous sample matrix containing an organic solvent. Target analytes within this zone could be concentrated whilst they migrated through the capillary. A novel technique was developed that used the sharpening zone for sweeping and concentrating several unresolved analyte peaks allowing an enhanced peak resolution. As the aim was to concentrate only some part of the analyte zone containing compounds of interest, the position and width of the sharpening zone needed to be well controlled. Various techniques to tune the width and position of the sharpening zone were thus developed, involving modifying the organic modifier content in the BGE, applying pressure additionally to the electroosmotic flow (EOF) and modifying the inner surface of the capillaries. This work also critically evaluated some related existing knowledge and theoretical approaches in CEC including the concept of tuneable system eigenpeaks (noncomigrating or ghost peaks caused by waves of an electric field in the capillary). Although peak broadening caused by eigenpeaks (which often suppresses resolution in CEC) has been well described for aqueous or pure non-aqueous BGEs, a systematic study of eigenmobility in aqueous/acetonitrile BGEs had not been reported. The experiments related to the eigenmobility led to a new approach to tune the eigenpeak position in chromatograms whereby a selective sample peak sharpening mechanism was elaborated using the sharpening zone. The novel approach was demonstrated for the separation of various peptides in the CE mode and for the resolution of incompletely resolved chiral amino acids in CEC. These investigations furthermore led to the development of an appropriate stationary phase, which was produced using techniques for an in-column synthesis of a molecular imprinted polymer (MIP). Well prepared MIPs are capable of specifically enhancing the retention of target analytes, herein focusing on enantio-selective isolation of chiral molecules. Since such a MIP was rather rigid, which often caused capillary clogging when used as a conventional monolithic stationary phase, this MIP was incorporated into porous layer open tubular (PLOT) capillaries. Numerous experimental difficulties were overcome with respect to the vinylisation of long fused-silica capillaries and for the polymerisation of the MIP within using thermal- and photo-polymerisations. A method for the characterization of such MIP-PLOT capillaries was elaborated involving the Langmuir adsorption theory. The new columns were evaluated using chromatography, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and CEC analysis. All experiments were designed according to the principles of Green Chemistry. These concepts involved the use of small scale analysis, simulation studies reducing the number of performed experiments, systematic studies with low amounts of solvents and materials, and optimisation of synthesis methods to reduce time, energy and reagent consumption. The theories, methods and results presented in this thesis will, in the future, allow further targeted developments of novel analytical tools for a green, sensitive, selective and improved resolution analysis of chemical and biological compounds including chiral compounds, peptides and proteins.

The paper describes the enhanced separation of o-, m-, p-dihydroxybenzene by capillary electrochromatography (CEC) using gold nanoparticles (AuNPs) as stationary phase. The effect of the AuNPs concentration upon separation was investigated. The experimental parameters, including separation voltage, pH, and concentration of running buffer, were optimized. Under the optimum conditions, a good resolution of three dihydroxybenzene isomers was obtained within 15 min in a 50 cm effective length capillary modified with 0.02 nmol/L AuNPs at a separation voltage of 16 kV in a 50 mmol/L acetate buffer (pH 5.0). The linear ranges were from 10(-6) to 10(-4) mol/L and the detection limits were as low as 10(-7) mol/L. This method was successfully used to analysis two kinds of hair coloring agent sample with recoveries in the range of 90-105% and relative standard deviations (RSD) less than 5.0%.

Homochiral zeolite-like metal-organic framework with DNA like double-helicity structure as stationary phase for capillary electrochromatography enantioseparation

Escherichia coli adhesive coating as a chiral stationary phase for open tubular capillary electrochromatography enantioseparation

The analysis of basic pharmaceutical compounds by capillary electrochromatography using continuous bed stationary phase

Metal organic framework- modified monolithic column immobilized with pepsin for enantioseparation in capillary electrochromatography