Cemil Aydoğan

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector, N-methacryloyl-l-histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d,l‐phenylalanine, d,l‐tyrosine, and d,l‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l‐enantiomers of aromatic amino acids migrated before d‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.

Molecularly imprinted cryogel for L‐glutamic acid separation

A molecular recognition based L‐glutamic acid (L‐GLU) imprinted cryogel was prepared for L‐GLU separation via chromatographic applications. The novel functional monomer N‐methacryloyl‐(L)‐glutamic acid‐Fe3+ (MAGA‐Fe3+) was synthesized to be complex with L‐GLU. The L‐GLU imprinted cryogel was prepared by free radical polymerization under semifrozen conditions in the presence of a monomer‐template complex MAGA‐Fe3+‐L‐GLU. The binding mechanism of MAGA‐Fe3+ and L‐GLU was characterized by Fourier transform infrared (FTIR) spectroscopy in detail. FTIR analyses on the synthesized MAGA‐Fe3+‐GLU complex reveals bridging bidentate and monodentate binding modes of Fe3+ in complex with the carboxylate groups of the glutamate residues. The template L‐GLU could be reversibly detached from the cryogel to form the template cavities using a 100 mM solution of HNO3. The amount of adsorbed L‐GLU was detected using the phenyl isothiocyanate method. The L‐GLU adsorption capacity of the cryogel decreased drastically from 11.3 to 6.4 μmol g−1 as the flow rate increased from 0.5 to 4.0 mL min−1. The adsorption onto the L‐GLU imprinted cryogel was highly pH dependent due to electrostatic interaction between the L‐GLU and MAGA‐Fe3+. The PHEMAGA‐Fe3+‐GLU cryogel exhibited high selectivity to the corresponding guest amino acids (i.e., D‐GLU, L‐ASN, L‐GLN, L‐, and D‐ASP). Finally, the L‐GLU imprinted cryogel was recovered and reused many times, with no significant decrease in their adsorption capacities.

Chiral separation-based ligand exchange by open-tubular capillary electrochromatography.

Chiral ligand-exchange enantioseparation of aliphatic and aromatic amino acids was successfully performed using a new open-tubular zwitterionic column with tentacle-type polymer stationary phase. The polymeric stationary phase was prepared using 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), a new reactive monomer. The preparation procedure of the open-tubular column included silanization, in situ graft polymerization with HPMA-Cl, and L-histidine (L-His) modification. L-His was used as a chiral ligand-exchange selector and copper(II) as a central ion. Successful enantioseparation of six pairs of amino acid enantiomers was achieved with a buffer of 5 mM CuSO₄, 20 mM (NH4)₂SO₄ at pH 3.0.

Cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins by nano liquid chromatography†

In this study, vinyl phenyl boronic acid modified lauryl methacrylate-based monolithic column was successfully prepared for cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins in nano LC. The polymeric mixture consisted of lauryl methacrylate, vinyl phenyl boronic acid as cation exchanger, ethylene dimethacrylate as cross-linker, polyethylene glycol and methanol as binary porogenic solvent, and azobisisobutyronitrile as initiator. The resulting monolith showed good permeability and mechanical stability. Different ratios of monomer and porogens were used for optimizing the properties of the column. The monolithic column performance with respect to hydrophobic and cation exchange interactions was assessed by the separation a series of alkyl benzenes and anilines, respectively. cis-Diol-containing compounds such as phenols were also utilized to evaluate the retention behaviors of the vinyl phenyl boronic acid modified monolithic column. The monolithic column showed cation exchange interactions in the separation of aniline compounds. Theoretical plate number up to 52,000 plates/m was successfully achieved. The prepared monolith was further applied to the proteins with different acetonitrile content.

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.

Polymethacrylate-based monolithic capillary column with weak cation exchange functionalities for capillary electrochromatography

Polymethacrylate-based monolith with weak cation exchange functionalities was prepared in capillary column (i.d. 100 μm, o.d. 375 μm) by in situ polymerization of butyl methacrylate, ethylene dimethacrylate and N-methacryloyl-L-glutamic acid in presence of porogens. The porogen mixture included N,N-dimethyl formamide and phosphate buffer. The preparation procedure of stationary phase contained the synthesis of monomer, silanization of capillary inner wall and in situ polymerization. The use of amino acid based monomer for the monolith synthesis is one of the originalities of this novel approach. N-methacryloyl-L-glutamic acid has two carboxyl functionalities. The separation of the solutes were performed at different acetonitrile/phosphate buffer and acetonitrile/sodium hydroxide contents. The applied voltage for the alkyl benzenes was changed between +5 and +30 kV. CEC separations of alkyl benzenes, acidic, basic, phenolic and some polycylic aromatic compounds were succesfully performed under capillary-electrochromatography mode with cathodic electroosmotic flow.

Preparation of an Electrochromatographic Stationary Phase Using a New Polymethacrylate Monolith with Chloropropyl Functionality

A new reactive capillary monolith as an alternative to the commonly employed glycidyl methacrylate-based stationary phases in capillary electrochromatography (CEC) applications was synthesized and post-functionalized with charge-bearing groups. For this purpose, a hydrophilic capillary monolith with reactive 3-chloro-2-hydroxypropyl moiety was first obtained by the copolymerization of a new methacrylate-based monomer, 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), with a methacrylic crosslinking agent, ethylene glycol dimethacrylate (EDMA). The presence of 3-chloro-2-hydroxypropyl functionality in poly(HPMA-Cl-co-EDMA) monolith allowed the synthesis of monoliths carrying strongly ionizable anionic, cationic or neutral groups such as sulfonic acid, quaternary ammonium or octadecyl, respectively, via simple and single-stage reactions. In the present study, a reactive poly(HPMA-Cl-co-EDMA) capillary monolith was functionalized with sodium bisulfite (NaHSO3) to have a strongly ionizable sulfonic acid group on the monolith. The resulting monolith providing cathodic electroosmotic flow in CEC was successfully used for the separation of phenol derivatives. The theoretical plate numbers up to 63,000 plates/column were achieved. The results showed that a new promising, reactive support that could be functionalized with different chromatographic ligands for different chromatographic applications was obtained in the study.