Electrochromatographic enantioseparation of amino acids using polybutylmethacrylate-based chiral monolithic column by capillary electrochromatography

This article describes the development of a polybutylmethacrylate‐based monolithic capillary column as a chiral stationary phase. The chiral monolithic column was prepared by polymerization of butyl methacrylate (BMA), ethylene dimethacrylate (EDMA), and N‐methacryloyl‐l ‐glutamic acid (MAGA) in the presence of porogens. The porogen mixture included N,N‐dimethyl formamide and phosphate buffer. MAGA was used as a chiral selector. The effect of MAGA content was investigated on electrochromatographic enantioseparation of d,l ‐histidine, d,l ‐tyrosine, d,l ‐phenyl alanine, and d,l ‐glutamic acid. The effect of acetonitrile (ACN) content in mobile phase on electro‐osmotic flow was also investigated. It was demonstrated that the poly(BMA‐EDMA‐MAGA) monolithic chiral column can be used for the electrochromatographic enantioseparation of amino acids by capillary electrochromatography (CEC). The mobile phase was ACN/10 mM phosphate buffer (45:55%) adjusted to pH 2.7. It was observed that l ‐enantiomers of the amino acids migrated before d ‐enantiomers. The separation mechanism of electrochromatographic enantioseparation of amino acids in CEC is discussed. Chirality 24:606–609, 2012. © 2012 Wiley Periodicals, Inc.     

Preparation and characterization of a new open‐tubular capillary column for enantioseparation by capillary electrochromatography

In order to use the enantioseparation capability of cationic cyclodextrin and to combine the advantages of capillary electrochromatography (CEC) with open‐tubular (OT) column, in this study, a new OT‐CEC, coated with cationic cyclodextrin (1‐allylimidazolium‐β‐cyclodextrin [AI‐β‐CD]) as chiral stationary phase (CSP), was prepared and applied for enantioseparation. Synthesized AI‐β‐CD was characterized by infrared (IR) spectrometry and mass spectrometry (MS). The preparation conditions for the AI‐β‐CD‐coated column were optimized with the orthogonal experiment design L₉(3⁴). The column prepared was characterized by scanning electron microscopy (SEM) and elemental analysis (EA). The results showed that the thickness of stationary phase in the inner surface of the AI‐β‐CD‐coated columns was about 0.2 to 0.5 μm. The AI‐β‐CD content in stationary phase based on the EA was approximately 2.77 mmol·m^?2. The AI‐β‐CD‐coated columns could separate all 14 chiral compounds (histidine, lysine, arginine, glutamate, aspartic acid, cysteine, serine, valine, isoleucine, phenylalanine, salbutamol, atenolol, ibuprofen, and napropamide) successfully in the study and exhibit excellent reproducibility and stability. We propose that the column, coated with AI‐β‐CD, has a great potential for enantioseparation in OT‐CEC.     

Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Cellulose tris(3-chloro-4-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSPs) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the chiral selector loading onto silica, mobile phase composition and pH, as well as separation variables on separation of enantiomers was studied. It was found that CSPs based on cellulose tris(3-chloro-4-methylphenylcarbamate) can be used for preparation of very stable capillary columns useful for enantioseparations in nano-LC and CEC in combination with polar organic mobile phases.     

HPLC enantioseparation on cellulose tris(3,5-dimethylphenylcarbamate) as a chiral stationary phase: Influences of pore size of silica gel,coating amount,coating solvent,and column temperature on chiral discrimination

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) was coated on large-pore silica gels and used as a chiral stationary phase (CSP) for high-performance liquid chromatographic separation of enantiomers. The influences of pore size of silica gel, coating amount of CDMPC, coating solvent, and column temperature on chiral discrimination were investigated. CSPs prepared with a large-pore silica gel having a small surface area showed higher chiral recognition. The amount of CDMPC adsorbed on the silica gel influenced the chiral recognition of some racemates. Loading capacity of racemates increased with an increase of the amount of CDMPC supported on the silica gel, and a CSP coated with 45% CDMPC by weight can be used for both analytical and semi-preparative scale separations. The CDMPC, coated using acetone as the coating solvent, exhibited, in many cases, higher enantioselectivity than that obtained with tetrahydrofuran F as the coating solvent. © 1996 Wiley-Liss, Inc.     

Enhanced sequence coverage in tryptic fragment analysis by two-dimensional HPLC/MS using a monolithic silica capillary column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Enantioseparation of glycyl-dipeptides by CEC using particle-loaded monoliths prepared by ring-opening metathesis polymerization (ROMP)

Novel particle-loaded monolithic capillary electrochromatography (CEC) phases for chiral separations were prepared via ring-opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 microm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8,exo,endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens, RuCl2(PCy3)2(CHPh) as initiator and silica-based particles containing the chiral selector. By suspending silica particles bearing the chiral selector in the polymerization mixture, particle-based monoliths are easily prepared. This approach has several advantages compared to particle-based separation media: (i) the concept of particle-based monoliths is broadly applicable, as any silica-based chiral phase can be used; (ii) they are inexpensive to prepare; and (iii) the manufacturing process is very simple, no sophisticated packing procedures or the preparation of end frits are required. To show the usefulness of this concept for chiral CEC, the chiral separation performance of particle-loaded CEC monoliths bearing teicoplanin aglycone, chemically bonded to 3 microm silica gel, was investigated for a set of glycyl-dipeptides. Particle-loaded ROMP CEC monoliths showed good separation performance for glycyl-dipeptides.     

Vancomycin as chiral selector for enantioselective separation of selected profen nonsteroidal anti-inflammatory drugs in capillary liquid chromatography

The chiral selector vancomycin was used either as mobile phase additive or bound as a chiral stationary phase (CSP) for the stereoselective separation of seven racemic nonsteroidal anti-inflammatory drugs (NSAIDs), fenoprofen, carprofen, flurbiprofen, indoprofen, flobufen, ketoprofen, and suprofen, by capillary liquid chromatography. The effect of the type of stationary phase, the chiral column Chirobiotic V or the achiral stationary phases Nucleosil 100 C8 HD and Nucleosil 100 C18 HD, and the concentration of vancomycin in the mobile phase on separation of the drug enantiomers were evaluated. All the drugs, except flobufen, were successfully enantioseparated on Nucleosil 100 C8 HD with 4 mM vancomycin present in the mobile phase (composed of methanol and buffer) in the reversed phase mode. On the vancomycin-bonded chiral stationary phase, it was difficult to get enantioseparations of the profen NSAIDs. However, flobufen gave better enantioseparation on the vancomycin CSP. The better enantioresolution of the majority of profen derivatives on the achiral columns with vancomycin added to the mobile phase can be attributed in particular to the higher separation efficiency of this capillary chromatographic system. In addition, vancomycin dimers, formed in the mobile phase, seem to offer a better steric arrangement for stereoselective interaction to these analytes than the vancomycin bonded on the CSP. These substantial differences in the CS structure significantly influence the chiral discrimination mechanism.     

Enantioseparation by ligand-exchange using particle-loaded monoliths: capillary-LC versus capillary electrochromatography

Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 microm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of alpha-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with alpha values up to 5.     

Enantioseparation by ligand-exchange using particle-loaded monoliths: Capillary-LC versus capillary electrochromatography

Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 μm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of α-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with α values up to 5.     

Enhanced Sequence Coverage in Tryptic Fragment Analysis by Two-Dimensional HPLC/MS Using a Monolithic Silica Capillary Column

The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.     

Enantioseparation of tetrahydropalmatine and Tröger's base by molecularly imprinted monolith in capillary electrochromatography

Two chiral compounds, Tröger's base and tetrahydropalmatine, were enantioseparated on the (5S, 11S)-(-)-Tröger's base and l-tetrahydropalmatine imprinted monolithic capillary columns with CEC, respectively. The monoliths were prepared by in situ thermal-initiated copolymerization of methacrylic acid (MAA) and ethylene dimethacrylate (EDMA). After optimizing the ratio of porogens (toluene and dodecanol), the obtained monolithic capillary columns show good flow-through property and enantioselectivity. The influences of CEC parameters such as pH of the buffer, organic solvent and salt concentration on the electroosmotic flow (EOF) and recognition selectivity were systematically investigated. Under the optimal conditions, baseline resolutions of two chiral compounds were achieved. In addition, the fast separation was obtained within 4 min by applying higher voltage and assisting pressure of 6 bar.     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

A rapid enantioseparation system of capillary electrochromatography modified by electrostatic adsorption with transfersomes

Transfersomes were a special kind of nanomaterials with higher deformability and flexibility. A rapid method for coated-column preparation using anionic transfersomes as a coating material by electrostatic adsorption was developed. With carboxymethyl-β-cyclodextrin added in running buffer as the chiral selector, the capillary electrochromatography enantioseparation system based on the transfersomes-coated column modified by electrostatic adsorption was established for the first time. Propranolol and metoprolol acted as model drugs to evaluate the enantioseparation performance, these two basic drugs achieved baseline separation with satisfactory resolution and selection factor in this transfersomes-electrochromatography system but only partial separation in bare column system. In order to get the optimal separation condition, concentration of chiral selector, buffer pH, and applied voltage were systematically investigated. A rapid and efficient enantioseparation electrochromatography system was established and showed that transfersomes as the stationary phase could efficiently improve chiral separation effect.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u=4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u=8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

Effect of the mobile phase on the retention behavior of optical isomers of the mandelic acid derivative methyl 2-phenyl-2-(tetrahydropyranyloxy) acetate by chiral HPLC

Conditions for separation of enantiomers of a mandelic acid derivative, methyl 2-phenyl-2-(tetrahydropyranyloxy) acetate (the analyte) were studied. Because of the presence of two chiral carbons, the analyte consists of four stereoisomers stable at ambient temperature. Chiral HPLC of the analyte resulted in four peaks, using an (S,S)-Whelk-O1 column with the mobile phase consisting of hexane and the t-butyl methyl ether (TBME). It was found that TBME dramatically changed the retention of the isomers, though it produced the best enantioseparation on (S,S)-Whelk-O1. The amount of TBME in the mobile phase influenced the degree of retention shift; 5% (v/v) TBME gave a bigger shift than 8% (v/v) and 10% (v/v). 2-Propanol did not produce the same results. The chiral separation was also tried on cellulose tris (3, 5-dimethyl phenylcarbamate) (CDMPC), but only three peaks were seen, indicating some but not full enantiomer resolution.     

Elucidation of the chromatographic enantiomer elution order for praziquantel: An experimental and theoretical assessment

In this work, we have studied both experimentally and theoretically the praziquantel (PZQ) chiral discrimination. According to the main results, the enantioseparation of PZQ was efficiently optimized by HPLC on the reverse phase from the Chiralpak IB column, which has cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) as a chiral selector. The thermodynamic and structural parameters obtained via density functional theory (DFT) calculations pointed out the chiral discrimination as well as the enantiomeric elution order of PZQ, thus elucidating the experimental data and validating our proposed method. Finally, the hydrogen bonds and π-π stacking interactions played a key role in the discrimination between the PZQ diastereomeric complexes formed.     

Synthesis of dendritic stationary phases with surface-bonded L-phenylalanine derivate as chiral selector and their evaluation in HPLC resolution of racemic compounds

Four dendrimers were synthesized on aminopropyl-modified silica gel using methyl acrylate and ethylene diamine as building blocks by divergent method. Four generations of chiral stationary phases (CSPs) were prepared by coupling of L-2-(p-toluenesulfonamido)-3-phenylpropionyl chloride to corresponding dendrimers. The derivatives prepared on silica gel were characterized by FT-IR, (1)H NMR, and elemental analysis. The selector loadings of these four generations of CSPs generally showed a decrease tendency with the increase of generation numbers of dendrimers. The enantioseparation properties of these CSPs were preliminarily investigated by high-performance liquid chromatography. The CSP derived from the three-generation dendrimer exhibited the best enantioseparation capability. Effects of the mobile phase composition and molecular structures of racemic mixtures on enantioseparation were further studied.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u = 4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u = 8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

Liquid chromatographic direct resolution of beta-amino acids on a doubly tethered chiral stationary phase containing N--H amide linkage based on (+)-(18-crown-6)- 2,3,11,12-tetracarboxylic acid

A doubly tethered chiral stationary phase (CSP) prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to doubly tethered primary aminoalkyl silica gel was used for the resolution of various beta-amino acids. All the beta-amino acids tested were resolved quite well, the separation (alpha) and the resolution factors (RS) being in the ranges 1.34-2.09 and 2.52-7.45, respectively, with a mobile phase of methanol-water (50:50, v/v) containing 10 mM acetic acid. The chiral recognition efficiency of the doubly-tethered CSP was found to be generally superior to that of the corresponding singly-tethered CSP in the resolution of beta-amino acids. The chiral recognition behaviors for the resolution of beta-amino acids on the doubly tethered CSP were examined by varying the type and content of organic and acidic modifiers in the aqueous mobile phase and the column temperature.     

Electrochromatographic characterization of methacrylate ester-based monolith and capillary electrochromatography separation of flavonoids

A porous polymethacrylate ester-based monolithic column for capillary electrochromatography (CEC) was designed by mean of in situ co-polymerizing lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent including cyclohexanol, 1,4-butanediol and water. After investigating the influence factors of the CEC monolithic columns, four flavonoids (i.e., Rutin, Quercetin, Kaempferol, and Quercitrin) were separated and assayed to evaluate this monolithic column with CEC method. Under optimum conditions, the CEC method exhibited high separation efficiency, with rapid separation time of 3–4 min, for the four flavonoid samples using 10 mM phosphate buffer containing 70% acetonitrile (pH 9.0). Importantly, the proposed method could provide a promising approach for rapid separation and detection in biomedicine.