KAPİLER ELEKTROKROMATOGRAFİ YÖNTEMİYLE D,L AMPİSİLİN ENANTİYOMERLERİNİN AYRIMI
Özet
Ampicillin is effective against gram-positive and gram-negative bacteria and is significantly resistant to acid. Because of these properties, it can be used in the treatment of various infections, making it a pharmacologically important antibiotic. Due to the asymmetric carbon atom in the side chain when synthesized, it is necessary to have two different enantiomeric forms and it needs to be chirally separated because only one form is useful for the body.
There are various methods for separating of chiral compounds. Within the scope of the thesis, a micelle electrokinetic capillary chromatography method, as one of sub category of capillary electrophoresis, was used. The enantiomeric separation of ampicillin was carried out by using miscelles which was used as stationary phase in the capillary column. The micelles used in the column was formed by adding an anionic surfactant (SDS) to the electrolyte solution above the critical micelle concentration.
Ligand exchange chromatography is one of the methods that separates enantiomeric molecules by using chirality. Ligand exchange is based on the formation of unstable complexes that can act as ligand and the enantiomeric separation is performed through these complexes.
In ligand exchange, the enantiomers are separated from each other by means of chiral selectors. In this study, the enantiometric separation of ampicillin was carried out by ligand exchange-micellar electrokinetic chromatography method using L-Lysine monohydrochloride as a ligand and copper (II) sulfate pentahydrate as a central ion.
For this aim, firstly L-Lys-Cu2+ amino acid-metal complex was formed to separate the ampicillin compound enantiomerically. UV-VIS spectrophotometer was used to determine the optimal molar ratios of the interacting L-Lys amino acid with the Cu2+ ion forming the L-Lys-Cu2+ complex. Characterization studies of the complex were carried out using FTIR-ATR spectrophotometer. Critical micelle concentration (CMC) was determined by current density measurement. SDS micelles obtained above the critical micelle concentration was interacted with L-Lys-Cu2+ complex. Characterization studies of the micelles and the micelles interacting with the chiral selector were made using high contrast transmission electron microscope, dynamic light scattering spectrophotometer and zetasizer analyzer.
Enantiomeric separations were performed using a micellar electrokinetic chromatography method using a capillary electrophoresis instrument. In this chromatography system, the SDS-L-Lys-Cu2+ micelle-chiral selector complex was used as a pseudostationary phase for the enantiomeric separation of ampicillin. When the electrokinetic conditions optimized, the ampicillin was enantiomerically separated. The effect of pH, SDS amount, applied electrical field, pressure, organic solvent ratio and ampicillin enantiomer ratios were investigated on the electrokinetic separation of ampicillin enantiomers. Alfasilin, sold as a drug was purchased from pharmacy and the enantiomeric purity was controlled under optimized electrokinetic conditions to check the usefulness of the prepared pseudostationary phase to control the enantiomeric purity of ampicillin, the penicillin-derived antibiotic in real samples.
As a result; the enantiomeric separation of the ampicillin compound was carried out by combining the ligand exchange mechanism with micellar electrokinetic capillary chromatography, the sub category of the capillary electrochromatography, and the optimum conditions for the enantiomeric separation were successfully determined.
Key words: Ampicillin, enantiomer, ligand exchange, capillary electrochromatography, micellar electrokinetic capillary chromatography, micelle.
