Preparation of chiral monoliths with new modulation of the monolith surface chemistry for the enantioseparation of chiral drugs by nano-liquid chromatography.

Here, we report the preparation and application of two new chiral monoliths for the enantioseparation of chiral drugs in nano-LC. Using 3­chloro-2-hydroxypropylmethacrylate (HPMA-Cl, 2) as a precursor monomer, two different chiral monomers namely, Nα-Boc-Lys-HPMA (3A) and Nα-Fmoc-Lys-HPMA (3B) were synthesized and used for the preparation of chiral polymer monoliths. The first monolithic column (referred to as monolith I) was prepared by an in-situ polymerization of Nα-Boc-Lys-HPMA as the chiral monomer and ethylene dimethacrylate while the second monolithic column (referred to as monolith II) was prepared by an in-situ polymerization of Nα-Fmoc-Lys-HPMA as the chiral monomer and ethylene dimethacrylate as the crosslinker. Methanol and 1-propanol were used as the porogenic solvents. The prepared chiral monoliths were investigated for the enantioseparation of chiral drugs, including ß-blockers (e.g., atenolol, propranolol, metoprolol) and anti-inflammatory drugs (e.g., ketoprofen, ibuprofen, flurbiprofen, naproxen, etodolac). The enantioseparation could be achieved via the formation of π-π interactions on the aromate-rich and aromate-poor chiral molecules while enantioseparation mechanism of chiral drugs included mostly π-π interactions and hydrogen bonding. Monolith II showed better enantioselectivity than Monolith I and the resolution values up to 2.12 were successfully achieved.

Nano-Liquid Chromatography with a New Monolithic Column for the Analysis of Coenzyme Q10 in Pistachio Samples.

Coenzyme Q10 (CoQ10) is a vital substance found throughout body. It helps convert food into energy and is eaten small amounts in foods. CoQ10 has gained great interest in recent years as a potential candidate for the treatment of various diseases. The content of CoQ10 in food samples is a crucial quality index for foods. Therefore, the development of sensitive separation and quantification method for determining the amount of CoQ10 in various samples, especially in foods, is an important issue, especially for food nutrition. In this study, a new, miniaturized monolithic column was developed and applied for the determination of CoQ10 in pistachio samples by nano-liquid chromatography (nano-LC). The monolithic column with a 50 µm i.d. was prepared by in situ polymerization using laurylmethacrylate (LMA) as the main monomer and ethylene dimethacrylate (EDMA) as the crosslinker. Methanol (MeOH) and polyethyleneglycol (PEG) were used as porogenic solvents. The final monolithic column was characterized by using scanning electron microscopy (SEM) and chromatographic analyses. The monolithic column with a 50 µm i.d. was applied to the analysis of CoQ10 in pistachio samples in nano-LC. This analytical method was validated by means of sensitivity, linearity, precision, recovery, and repeatability. The LOD and LOQ values were 0.05 and 0.48 µg/kg, respectively. The developed method using the monolithic column was optimized to achieve very sensitive analyses of CoQ10 content in the food samples. The applicability of the method was successfully demonstrated by the analysis of CoQ10 in pistachio samples.

Nano-liquid chromatography with monolithic stationary phase based on naphthyl monomer for proteomics analysis.

Monolithic poly(2-vinylnaphthalene-co-divinylbenzene) columns were introduced, for the first time, and were evaluated as the separation media for nano-liquid chromatography (nano-LC). These columns were prepared by in-situ polymerization of 2-vinylnaphthalene (2-VNA) as the functional monomer and divinylbenzene (DVB) as the crosslinker in a fused silica capillary column of 50 µm i.d. Various porogenic solvents, including tetrahydrofuran (THF), dodecanol and toluene were used for morphology optimization. Final monolithic column (referred to as VNA column) was characterized by using scanning electron microscopy (SEM) and chromatographic analyses. Alkylbenzenes (ABs), and polyaromatic hydrocarbons (PAHs) were separated using the VNA column while the column offered excellent hydrophobic and π-π interactions under reversed-phase conditions. Theoretical plates number up to 41,200 plates/m in isocratic mode for ethylbenzene could be achieved. The potential of the final VNA column was demonstrated with a gradient elution in the  separation of six intact proteins, including ribonuclease A (RNase A), cytochrome C (Cyt C), lysozyme (Lys), ß-lactoglobulin (ß-lac), myoglobin (My) and α-chymotrypsinogen (α-chym) in nano LC system. The column was then applied to the peptide analysis of trypsin digested cytochrome C, allowing a high peak capacity up to 1440 and the further proteomics analysis of COS-7 cell line was attempted applying the final monolithic column in nano-LC UV system.

Determination of the geographic origin of 52 honey samples based on the assessment of anionic content profiling with a new algorithm using monolithic column-based micellar nano-liquid chromatography.

In the present study, a new micellar nano LC-UV was, for the first time, reported for the separation and determination of five anions (chloride, nitrite, bromide, sulfate and nitrate) in 52 honey samples. Based on this approach, a graphene oxide-based monolithic column was prepared and applied for the samples. Various amounts of hexadecyltrimethyl-ammonium bromide (HTAB) in the mobile phase were used in order to optimize the separation conditions. The baseline separation was achieved using mobile phase with 25/75% (v/v) ACN/10 mM phosphate buffer at pH 3.4, while the amount of HTAB was optimized as 0.22 mM in the mobile phase. The whole method was validated and it leads to high sensitivity. The LOD values were found in the range of 0.02-0.22 µg/kg, while LOQ values were found in the range of 0.06-0.18 µg/kg. The method allowed to achieve sensitivity analyses of anionic content in 52 honey samples. All data were evaluated using a new algorithm for geographic origin discrimination. K-nearest neighbor algorithm (K-NN), cubic support vector classifier (K-DVS), and K-Mean cluster analysis were used for geographic origin discrimination of honeys. The accuracy of the whole model was calculated as 94.4% with the K-DVS method. The samples from five provinces were classified 100% correctly, while two of them were classified with one misclassification, with an accuracy of 89.9% and 83.3%, respectively. PRACTICAL APPLICATION: The new platforms and advanced technologies are crucial for advanced food analysis. In this article, a novel methodology was attempted for the determination of geographic origin of 52 honey samples. In this sense, micellar nano LC technique with a homemade monolithic nano-column was, for the first time, applied for the anion analysis using a new algorithm.

Hydrophobic AEROSIL®R972 Fumed Silica Nanoparticles Incorporated Monolithic Nano-Columns for Small Molecule and Protein Separation by Nano-Liquid Chromatography.

A new feature of hydrophobic fumed silica nanoparticles (HFSNPs) when they apply to the preparation of monolithic nano-columns using narrow monolithic fused silica capillary columns (e.g., 50-µm inner diameter) was presented. The monolithic nano-columns were synthesized by an in-situ polymerization using butyl methacrylate (BMA) and ethylene dimethacrylate (EDMA) at various concentrations of AEROSIL®R972, called HFSNPs. Dimethyl formamide (DMF) and water were used as the porogenic solvents. These columns (referred to as HFSNP monoliths) were successfully characterized by using scanning electron microscopy (SEM) and reversed-phase nano-LC using alkylbenzenes and polyaromatic hydrocarbons as solute probes. The reproducibility values based on run-to-run, column-to-column and batch-to-batch were found as 2.3%, 2.48% and 2.99% (n = 3), respectively. The optimized column also indicated promising hydrophobic interactions under reversed-phase conditions, while the feasibility of the column allowed high efficiency and high throughput nano-LC separations. The potential of the final HFSNP monolith in relation to intact protein separation was successfully demonstrated using six intact proteins, including ribonuclease A, cytochrome C, carbonic anhydrase isozyme II, lysozyme, myoglobin, and α-chymotrypsinogen A in nano-LC. The results showed that HFSNP-based monolithic nanocolumns are promising materials and are powerful tools for sensitive separations.

ProFlow nano-liquid chromatography with a graphene oxide-functionalized monolithic nano-column for the simultaneous determination of chloramphenicol and chloramphenicol glucuronide in foods.

Chloramphenicol (CAP) is an effective antibiotic with broad spectrum against gram-positive and gram-negative bacteria, while it is used to treat various infections in animals. Although CAP is banned for usage in the livestock products including, milk, honey, seafood, and royal jelly, CAP is still often detected in foods of animal origin, posing a threat to consumer health. The use of CAP is restricted in many countries due to its side effect in human metabolic process according to the Expert Committee on Food Additives (ECFA) recommendation. Chloramphenicol glucuronide (CAPG) is also a metabolic product of CAP, which may be a hazardous chemical for human health. Therefore, the development of sensitive separation and quantification method is an important issue, especially for food safety. Herein, we reported the preparation and application of a monolithic nano-column for CAP and CAPG analyses in foods by ProFlow Nano liquid chromatography (ProFlow Nano LC). The monolithic nano-column was prepared by an in situ polymerization using 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA) and followed graphene oxide (GO) modification. After characterization, the monolithic nano-column was used for the analysis of CAP and CAPG in honey and milk samples by ProFlow Nano LC. The whole method was validated in terms of linearity, sensitivity, precision, recovery, and repeatability, while it led to obtain high sensitivity with limit of quantification was found as 0.02 µg/kg for CAP. Limit of quantification for CAPG was found as 0.08 µg/kg. The developed method with monolithic nano-column was optimized to achieve very sensitive analyses of CAP and CAPG in the food samples. The applicability of the nano-column was successfully demonstrated by the analysis of CAP and CAPG in milk and honey samples. PRACTICAL APPLICATION: This article describes the preparation and application of a monolithic nano-column for the separation and determination of chloramphenicol and chloramphenicol glucuronide in food samples by ProFlow Nano LC. The use of new and advanced techniques is a crucial issue in the food science and technology. In this sense, this study demonstrated a new food analysis method using advanced instrumental technique with a homemade monolithic nano-column.

Open tubular nano-liquid chromatography with a new polylysine grafted on graphene oxide stationary phase for the separation and determination of casein protein variants in milk.

In this study, a novel narrow capillary (i.e., 20 µm i.d.) coating procedure was reported for the preparation and application of open-tubular (OT) column for the separation of casein (CN) protein variants in milk. Based on this approach, a new poly-L-lysine (PLL) grafted on graphene oxide stationary phase was developed for OT nano-liquid chromatography (OT-nano LC). In this preparation, methacryloyl graphene oxide nanoparticles (MGONPs) were synthesized and used as reactive monomer. Fused silica capillaries with 20 µm i.d. were used for the preparation of OT column. After silanization of the capillary column, the stationary phase was prepared by MGONPs graft polymerization and PLL modification. It was shown that the use of MGONPs amount was important for the capillary coating. A column without PLL modification was also prepared for comparing the effect of PLL. SEM images of the column showed that MGONPs were bound to the surface of capillary in a dense monolayer. Alkylbenzenes (ABs) were used for the chromatographic characterization of the OT column. The results showed that the modification was successfully performed and allowed satisfactory separation of ABs. The developed OT column in the ProFlow nano-LC was applied to the separation of casein (CN) protein variants, including αs1-CN, αs2-CN, ß-CN and Ƙ-CN. Results indicated that the column provided a promising separation of casein protein variants due to both hydrophobic interactions and repulsive electrostatic interactions. Run-to-run and column-to-column reproducibilities were satisfactory (i.e., ≤3.12%). Real sample application of the OT column was demonstrated using a milk sample. Several isoforms of αs1-CN could be detected. All results demonstrated that the developed OT column with a novel coating procedure was promising for advanced nano-liquid chromatographic separations.

Nano-liquid chromatography with a new nano-structured monolithic nanocolumn for proteomics analysis.

Herein, we report the preparation and application of a new nano-structured monolithic nanocolumn based on modified graphene oxide using narrow fused silica capillary column (e.g., 50 µm internal diameter). The nanocolumn was prepared by an in situ polymerization using butyl methacrylate, ethylene dimethacrylate, and methacryloyl graphene oxide nanoparticles. Dimethyl formamide and water were used as the porogenic solvent. After polymerization, the obtained nanocolumn was coated with dimethyloctadecylchlorosilane in order to enhance the hydrophobicity. Both isocratic and gradient nano-liquid chromatographic separations for small molecules (e.g., alkylbenzenes) and macromolecules (e.g., intact proteins) were performed. Theoretical plates number up to 3600 plates/m in isocratic mode for propylbenzene were achieved. It was demonstrated that the feasibility of graphene oxide modified monolithic nanocolumn for high-efficiency and high-throughput nanoscale proteomics analysis. The high resolving power of monolithic nanocolumn yielded sensitive protein separation with narrower peak width while a high-resolution analysis of peptides from trypsin-digested cytochrome C could be obtained. Graphene oxide based monolithic nanocolumns are promising and can allow to powerful tools for trace proteom sample analysis.

Graphene oxide-octadecylsilane incorporated monolithic nano-columns with 50 µm id and 100 µm id for small molecule and protein separation by nano-liquid chromatography.

In this study, graphene oxide-octadecylsilane incorporated monolithic nano-columns were developed for protein analysis by nano liquid chromatography (nano LC). The monolithic column with 100 µm id was first prepared by an in situ polymerization using ethylene dimethacrylate (EDMA), 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl), and methacryloyl graphene oxide nanoparticles (MGONPs). MGONPs were synthesized by the treatment of 3-(trimethoxysilyl)propylmethacrylate (TMSPM) and GO. Tetrahydrofuran (THF) and dodecanol were used as the porogenic solvent. The resulting column was functionalized by dimethyloctadecylch lorosilane (DODCS) for the enhancement of hydrophobicity. The functionalization greatly improved the baseline separation of hydrophobic compounds such as polyaromatic hydrocarbons (PAHs). The optimized monolith with respect to total polymerization mixture was characterized by using Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) X-ray diffraction (XRD) and chromatographic analyses. The blank monoliths without functionalization exhibited poor separation while a good separation performance of MGONPs functionalized monoliths was achieved. The monolith with 100 µm id was evaluated in protein separation in nano LC using RNase A, Cytochrome C, Lysozyme, Trypsin, and Ca isozyme II as the test proteins. It was shown that protein separation mechanism was based on large π-system of GO and hydrophobicity of the monolithic structure. Theoretical plates number up to 57 600 plates were achieved. The nano-column with 50 µm id was also prepared using the same polymerization mixture under the same chemical conditions. These nano-columns were employed for protein separation by nano LC, and the dependence of both nano-column performance on the internal diameter was also discussed.

Miniaturized LC in Molecular Omics.

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

Recent advances and applications in LC-HRMS for food and plant natural products: a critical review.

In the last decade, interest in food and plant natural products (FPNPs) has been growing due to their medical applications. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is the key to obtaining pure natural products for structure elucidation as well as for development into therapeutic agents. In this review, we will provide a summary of recent advances and applications related to the analysis of the most common FPNPs in studied matrices, in particular, polyphenols, peptides, carotenoids, alkaloids, terpenoids and glucosinolates by LC-HRMS. This paper also reviews the critical revision of this topic covering the works published in the last 4 years (early 2016-mid 2019). In addition, it gives an overview of the current state of various screening strategies (e.g. targeted, suspected, untargeted or retrospective) with discussion on future directions and perspectives of this technique. Graphical abstract.

Organic polymer-based monolithic capillary columns and their applications in food analysis.

In recent years, the use of organic polymer monolithic capillary columns in separation science has gained popularity due to the fact that they are easy to fabricate and do not require retaining frits. These materials have been applied in different fields including foods, proteomics, and pharmaceuticals. The interest in food analysis still needs to develop in order to increase the sensitivity towards micro/nano-scale food applications for food samples of < 5 µg (e.g., foodomics). In this regard, polymer monolithic capillary columns offer great separation capability in the food analytical separation science. We review the most important applications in food analysis using polymer monolithic capillary columns. In addition, several examples of the use of capillary separation methods combined with mass spectrometry detection in food analysis are summarized.

Chiral separation and determination of amino acid enantiomers in fruit juice by open-tubular nano liquid chromatography.

A novel chiral porous-layer stationary phase was developed for use in open-tubular nano liquid chromatography. The stationary phase was prepared by an in-situ polymerization of 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA). The reactive chloro groups at the surface of the porous stationary phase were reacted with ß-Cyclodextrin (ß-CD). The resulting morphology was characterized by using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FT-IR). The chromatographic performance of the column was evaluated by hydrophilic interaction chromatography (HILIC). Amino acids were used as test solutes. The running buffer conditions for the enantioseparation were found to be 85% acetonitrile (ACN):10%MeOH: 5% H2 O at 0.1% v/v trifluoro acetic acid (TFA) (flow rate: 800 nL/min). The enantioseparation provided high theoretical plate numbers up to 26 000 platesm-1 . A good retention capacity within satisfactory retention times was also achieved. Real sample applicability of this column to the separation of amino acid enantiomers in fruit juice sample was demonstrated.

Boronic acid-fumed silica nanoparticles incorporated large surface area monoliths for protein separation by nano-liquid chromatography.

In this study, a novel boronic acid-fumed silica nanoparticles incorporated hybrid monolithic stationary phase for glycoprotein separation by nano-liquid chromatography was developed. The stationary phase was prepared in 100 µm capillary by an in situ copolymerization of methacryloyl-fumed silica nanoparticle (MFSNP), 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), and ethylene dimethacrylate (EDMA) in a binary porogenic solvent composed of cyclohexanol and dodecanol. The preparation of the monolith was optimized by investigating the ratio of functional monomer to crosslinker and the effect of MFSNP content. The resulting monolithic column was functionalized with 3-aminophenylboronic acid (3-APBA). The column morphology, pore size, and specific surface area of the fabricated monolith were characterized by scanning electron microscopy, X-ray photo electron spectroscopy, and BET equation by means of the nitrogen adsorption-desorption isotherm, respectively. Good permeability stability and column efficiency were observed on the monolith with nano-flow. The results also indicated that the MFSNP content was very important for final preparation of the monolithic stationary phase. The monolith with MFSNP could achieve better separation than one without MFSNP. The chromatographic performance of the monolith with respect to hydrophobic/affinity interactions was evaluated by the separation of alkylbenzene derivatives, proteins, and glycoprotein, respectively. The column efficiencies for alkylbenzenes on the hybrid monolithic column reached to 15,600-25,000 plates/m at the velocity of 1.2 mm/s in nano-liquid chromatography.

Monolithic stationary phases with incorporated fumed silica nanoparticles. Part II. Polymethacrylate-based monolithic column with "covalently" incorporated modified octadecyl fumed silica nanoparticles for reversed-phase chromatography.

This study is concerned with the incorporation of surface modified fumed silica nanoparticles (FSNPs) into polymethacrylate based monolithic columns for use in reversed phase chromatography (RPC) of small solutes and proteins. First, FSNPs were modified with 3-(trimethoxysilyl)propylmethacrylate (TMSPM) to yield the "hybrid" methacryloyl fumed silica nanoparticle (MFSNP) monomer. The resulting MFSNP was then mixed with glyceryl monomethacrylate (GMM) and ethylene dimethacrylate (EDMA) in a binary porogenic solvent composed of cyclohexanol and dodecanol, and the in situ copolymerization of MFSNP, GMM and EDMA was performed in a stainless steel column of 4.6 mm i.d. The silanol groups of the hybrid monolith thus obtained were grafted with octadecyl ligands by perfusing the hybrid monolithic column with a solution of 4% w/v of dimethyloctadecylchlorosilane (DODCS) in toluene while the column was maintained at 110°C for 6h (in a heated HPLC oven). One of the originalities of this study was to demonstrate MFSNP as a novel derivatized "hybrid monomer" in making RPC monolithic columns with surface bound octadecyl ligands. In this respect, the RPC behavior of the monolithic column with "covalently" incorporated FNSPs having surface grafted octadecyl ligands was evaluated with alkylbenzenes, aniline derivatives and phenolic compounds. The results showed that the hybrid poly(GMA-EDMA-MFSNP) having surface bound octadecyl ligands exhibited hydrophobic interactions under reversed phase elution conditions. Furthermore, six standard proteins were baseline separated on the column using a 10min linear gradient elution at increasing ACN concentration in the mobile phase at a flow rate of 1.0mL/min using a 10 cm×4.6mm i.d. column. The relative standard deviations (RSDs) for the retention times of the tested solutes were lower than 2.1% and 2.4% under isocratic elution and gradient elution conditions, respectively.

Monolithic stationary phases with incorporated fumed silica nanoparticles. Part I. Polymethacrylate-based monolithic column with incorporated bare fumed silica nanoparticles for hydrophilic interaction liquid chromatography.

Fumed silica nanoparticles (FSNPs), were incorporated for the first time into a polymethacrylate monolithic column containing glyceryl monomethacrylate (GMM) and ethylene dimethacrylate (EDMA) in order to develop a new monolithic column for hydrophilic interaction high performance liquid chromatography (HILIC). When compared to poly(GMM-EDMA) monolithic column without FSNPs, the same monolithic column with incorporated FSNPs yielded important effects on HILIC separations. The effects of monomers and FSNPs content of the polymerization mixture on the performance of the monolithic column were examined in details, and the optimized stationary phase was investigated over a wide range of mobile phase composition with polar acidic, weakly basic and neutral analytes including hydroxy benzoic acids, nucleotides, nucleosides, dimethylformamide, formamide and thiourea. The retention of these analytes was mainly controlled by hydrophilic interactions with the FSNPs and electrostatic repulsion from the negatively charged silica surface in the case of hydroxy benzoic acids and nucleotides. The electrostatic repulsion was minimized by decreasing the pH of the aqueous component of the mobile phase, which in turn enhanced the retention of acidic solutes. Nucleotides were best separated using step gradient elution at decreasing pH as well as ACN concentration in the mobile phase. Improved peak shape and faster analysis of nucleosides were attained by a fast linear gradient elution with a shallow decrease in the ACN content of the ACN-rich mobile phase. The run-to-run and column-to-column reproducibility were satisfactory. The percent relative standard deviations (%RSDs) for the retention times of tested solutes were lower than 2.5% under isocratic conditions and lower than 3.5 under gradient conditions.

Chiral ligand-exchange separation and determination of malic acid enantiomers in apple juice by open-tubular capillary electrochromatography.

This study describes the application of an open tubular capillary column for chiral ligand-exchange separation and determination of malic acid enantiomers in apple juice by open-tubular capillary electrochromatography (OT-CEC). The open tubular column was prepared by in-situ grafting polymerization of 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl) and followed by L-Histidine (L-His) modification. L-His was used as a chiral ligand-exchange selector and copper (II) as a central ion. The electrochromatographic characterization of the open tubular column was performed with the use of thiourea as an electroosmotic flow (EOF) marker. Factors affecting electrochromatographic enantioseparation of malic acid were also studied. The running buffer conditions for optimum enantioseparation of malic acid were found to be ACN/5.0mM CuSO4, 20.0mM (NH4)2SO4 (60/40%, v/v) adjusted to pH 3.0. The separation and determination of the enantiomers of malic acid in the apple juice solution diluted 10- to 40-folds were successfully achieved.

A new anion-exchange/hydrophobic monolith as stationary phase for nano liquid chromatography of small organic molecules and inorganic anions.

In this study, an anion-exchange/hydrophobic polymethacrylate-based stationary phase was prepared for nano-liquid chromatography of small organic molecules and inorganic anions. The stationary phase was synthesized by in situ polymerization of 3-chloro-2-hydroxypropylmethacrylate and ethylene dimethacrylate inside silanized 100 µm i.d. fused silica capillary. The porogen mixture consisted of toluene and dodecanol. The pore size distrubution profiles of the resulting monolith were determined by mercury intrusion porosimetry and the morphology of the prepared monolith was investigated by scanning electron microscope. Good permeability, stability and column efficiency were observed on the monolithic column with nano flow. The produced monolithic column, which contains reactive chloro groups, was then modified by reaction with N,N-dimethyl-N-dodecylamine to obtain an anion-exchange/hydrophobic monolithic stationary phase. The functionalized monolith contained ionizable amine groups and hydrophobic groups that are useful of anion-exchange/hydrophobic mixed-mode chromatography. The final monolithic column performance with respect to anion-exchange and hydrophobic interactions was assesed by the separation of alkylbenzene derivatives, phenolic compounds and inorganic anions, respectively. Theoretical plate numbers up to 23,000 plates/m were successfully achieved in the separation of inorganic anions.

Open-tubular CEC with a new triethanolamine bonded stationary phase for biomolecule separation.

This study describes the preparation and electrochromatographic application of a new open-tubular capillary column with triethanolamine functionalized stationary phase. The stationary phase was synthesized by in situ grafting polymerization with 3-chloro-2-hydroxypropylmethacrylate based reactive monomer, and followed triethanolamine functionalization. The different 3-chloro-2-hydroxypropylmethacrylate contents on the separation efficiency were studied. The results indicated that the 3-chloro-2-hydroxypropylmethacrylate content (e.g., 15.3v/v%) on the inner surface of the capillary was very important for final preparation of the polymer stationary phase. The electrochromatographic characterization of the stationary phase was performed using alkylbenzene derivatives. The pH effect on the electroosmotic flow was also investigated. The open tubular column functionalized with triethanolamine allowed to operate in the anodic electroosmotic flow mode in the system. With anodic electroosmotic flow mode, favorable separations of the amino acids and the nucleosides were successfully achieved with high column efficiens ranging from 142000 to 257000 plates/m. Good repeatability was gained with relative standard deviation of the migration time and peak areas less than 2.2% for run to run (n=5) and less than 3.2% day to day (n=3). Furthermore, real sample applicability of this column to the separation of amino acids in white tea sample was demonstrated.

Novel tentacle-type polymer stationary phase grafted with anion exchange polymer chains for open tubular CEC of nucleosides and proteins.

A novel and simple method for preparation of a tentacle-type polymer stationary phase grafted with polyethyleneimine (PEI) anion exchanger was developed for open tubular capillary electrochromatography (OT-CEC) of nucleosides and proteins. The polymeric stationary phase was prepared using 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl)-based reactive monomer. The preparation procedure included pretreatment of the capillary inner wall, silanization, in situ graft polymerization with HPMA-Cl and PEI modification. To compare with the tentacle-type capillary column with PEI functionalization, a monolayer capillary column without PEI functionalization was also prepared. The electrochromatographic characterization of the prepared open tubular column was performed using alkylbenzenes. The electroosmotic flow (EOF) with regard to PEI concentrations and the running buffer pH was investigated. The separation conditions of the nucleosides and the proteins were optimized. The modified tentacle-type column with high anion exchange capacity has proven to afford better retention and resolution for the separation of nucleosides and proteins. The PEI functionalization column can also provide long-term stable use for biomolecule separation using a single capillary with relative standard deviation values of retention times of less than 2%. The results indicate that the present method for open tubular capillary preparation with a HPMA-Cl-based reactive monomer is promising for OT-CEC biomolecule separation.