Reversed-phase Chromatography Technique Research Articles

Development, Validation and Application of HPLC Method for Metformin in Rabbit Plasma

Objective:This study was aimed at conducting a pharmacokinetic evaluation of metformin in rabbit plasma samples using rapid and sensitive HPLC method and UV detection.Methods:Acetonitrile was used for protein precipitation in the preparation of plasma samples. Reverse phase chromatography technique with silica gel column (250 mm × 4.6 mm, 5 μm) at 30°was used for the separation purpose. Methanol and phosphate buffer (pH 3.2) mixture was used as a mobile phase with flow rate 0.8 ml/min. The wavelength of UV detector was adjusted at 240 nm.Results:The calibration curve was linear in a range of 0.1-1 µg/ml with R² = 0.9982. The precision (RSD, %) values were less than 2%, whereas, accuracy of method was higher than 92.37 %. The percentage recovery values ranged between 90.14 % and 94.97 %. LOD and LOQ values were 25 ng/ml and 60 ng/ml, respectively. Cmax and AUC0-t values were found to be 1154.67 ± 243.37 ng/ml and 7281.83 ± 210.84 ng/ml.h, respectively after treating rabbits with a formulation containing 250 mg metformin.Conclusion:Based on the above findings, it can be concluded that present method is simple, precise, rapid, accurate and specific and thus, can be efficiently used for the pharmacokinetic study of metformin.

Temperature-triggered Protein Adsorption and Desorption on Temperature-responsive PNIPAAm-grafted-silica: Molecular Dynamics Simulation and Experimental Validation

Poly(N-isopropylacrylamide) (PNIPAAm) grafted onto silica, which may be used for reverse phase chromatography (RPC), was simulated and synthesized for protein separation with temperature-triggered adsorption and desorption. Molecular dynamics simulation at an all-atom level was performed to illustrate the adsorption/desorption behavior of cytochrome c, the model protein, on PNIPAAm-grafted-silica, a temperature responsive adsorbent. At a temperature above the lower critical solution temperature (LCST), the PNIPAAm chains aggregate on the silica surface, forming a hydrophobic surface that is favorable for the hydrophobic adsorption of cytochrome c, which has a high exposure of hydrophobic patches. At temperatures below the LCST, the PNIPAAm chains stretch, forming hydrophilic surface due to hydrogen bonding between PNIPAAm and surrounding water. Desorption of cytochrome c on the PNIPAAm-grafted-silica surface occurs as a result of competition with water, which forms hydrogen bonds with the protein. The conformational transitions of both cytochrome c and PNIPAAm are monitored, providing molecular insight into this temperature-responsive RPC technique. PNIPAAm-grafted-silica beads were synthesized and used for the adsorption and desorption of cytochrome c at approximately 313 K and 290 K, respectively. The experimental results validate the molecular dynamics simulation. In comparison to conventional RPC, using temperature as a driving force for RPC reduces the risk of protein denaturation caused by exposure to chaotropic solvents. Moreover, it simplifies the separation process by avoiding the buffer exchange operations between the steps.

Purification of Lycopene by Reverse Phase Chromatography

This work investigates the potential of various hydrophobic matrices for the separation and purification of lycopene from microbial biomass obtained from fermentation using Blakeslea trispora. The lycopene purification method was developed in two steps. In the first step, carotenoids were extracted from the biomass of B. trispora with petroleum ether/acetone (1:1). In the second step, this partially purified carotenoid extract was further purified by reverse phase chromatography technique. Various binding conditions were studied to achieve maximum amount of lycopene bound onto the chromatographic matrices. Column studies on the elution conditions of lycopene using linear and step gradient method were investigated. Purification of lycopene using packed bed column by reverse phase chromatography matrix HP20 was carried out using step gradient of 55% isopropyl alcohol in acetone followed by 65% isopropyl alcohol in acetone. The purity and recovery of lycopene was checked using Knauer high-performance liquid chromatography (HPLC) system. A 89% recovery of lycopene of HPLC purity 95% was achieved with substantial success. Proton magnetic resonance of the purified sample showed close resemblance with the chemical shifts (δ values) of the standard lycopene.

Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymic DNA synthesis

The reversed-phase chromatography technique was employed in the measurement of DNA synthesis at the primers d(pT)n, r(pU)n, d(pA)n, and r(pA)n (n = 1-16) in the presence of template poly(dA) or poly(dT). DNA synthesis was catalyzed by Escherichia coli DNA polymerase I Klenow fragment, Physarum polycephalum DNA polymerase beta-like, P. polycephalum DNA polymerase alpha, and human placenta DNA polymerase alpha. Values of Km and Vmax were measured as functions of the primer chain lengths. It was found that all mononucleotides and small oligonucleotides served as primers of DNA synthesis. Values of the logarithm of both Km and Vmax increased linearly until primers had attained a chain length of 9-12 nucleotides, where a break was observed. The incremental as well as the absolute values of Km were interpreted in terms of free binding energies. These together with other data indicate that the 3'-ultimate nucleotide of the primer contributes a decisive amount of free energy of binding to DNA polymerase both from the nucleoside and from the phosphate moiety. The incremental increase is due to a complementary interaction between bases of primer and template buried in the binding cleft of the polymerase. It is also the ultimate nucleotide that determines whether the ribonucleotide or the deoxyribonucleotide is an efficient primer. It is of interest that the major results seem preserved for all four DNA polymerases. An energetic model for the binding of the template-primer was proposed and compared with available crystallographic data.