High Pressure Liquid Chromatography Instrumentation Research Articles

Isolation of Glyphosate-Resistant Bacterial Strains to Improve the Growth of Maize and Degrade Glyphosate under Axenic Condition

Glyphosate is a non-selective herbicide that is used to control perennial weeds in agriculture. However, its vast application may result in glyphosate residues in the food chain. Due to its toxicity to non-target organisms, glyphosate-contaminated soils needed to be remediated, and bioremediation is a conventional remedial method. The success of this depends on the isolation of bacteria with the ability to degrade glyphosate. The goal of this study was to isolate glyphosate-degrading bacteria from the rhizosphere of maize and wheat with a repeated application history of glyphosate for 5–10 years and test their roles in promoting the growth of maize (Zea mays) and glyphosate degradation in vitro. Eleven isolated bacteria were inoculated, and their role in plant growth was compared at different levels (100 and 200 mg/kg) of glyphosate. The results revealed that E. ludwigii improved the highest shoot length by 26% and the root length by 34% compared to the control at 100 mg/kg. The relative water contents in leaves significantly improved by 58% using P. aeruginosa at 100 mg/kg. The maximum electrolyte leakage from leaves significantly reduced by 73% using E. ludwigii at 100 mg/kg compared to the control (uninoculated). A high-pressure liquid chromatography instrument was used to assess the glyphosate concentrations. The highest degradation of glyphosate was observed in treatments inoculated with E. ludwigii (99 and 40%), P. aeruginosa (95 and 39%), K. variicola, (91 and 38%) E. cloacae (92 and 38%), and S. liquefaciens (87 and 36%), respectively, at 100 and 200 mg/kg within 28 days. These five strains demonstrated a great potential for degrading glyphosate and promoting the growth of maize in vitro, and they will be further exploited for the biodegradation of glyphosate and the growth promotion of broader crop species in situ in the near future.

Analytical method development and determination of hydrocortisone acetate and fusidic acid simultaneously in cream formulation, by reversed-phase HPLC.

In this study, an accurate, simple, economical, precise and reproducible reversed-phase HPLC method was developed for the estimation of fusidic acid and hydrocortisone acetate, according to the International Conference on Harmonization guidelines, in a cream formulation. Chromatographic separation was achieved by isocratic elution, on a Shimadzu reversed-phased high-pressure liquid chromatography instrument, equipped with a C18 column (150 × 4.6 mm, 5 μm) and UV detector at 225 nm wavelength, using acetonitrile and 0.05% trifluoroacetic acid (60:40), as a mobile phase and diluent, at flow rate 2 ml/min and an injection volume of 20 μl. The calibration curves were acquired with concentration range 80-120% and mean percentage recoveries for hydrocortisone acetate and fusidic acid were 100.14 and 100.81%, respectively. The limits of detection was obtained as 6.0667 and 6.807 μm ml-1 and the limits of quantification were 20.204 and 20.628 μm ml-1 for hydrocortisone acetate and fusidic acid, respectively. All of the validation parameters were within the acceptance criteria, as per International Conference on Harmonization requirements, for hydrocortisone acetate and fusidic acid. This method was found to be validated, simple, rapid and applicable for the simultaneous estimation of hydrocortisone acetate and fusidic acid by reversed-phased high-pressure liquid chromatography, for routine analytical testing in quality control, with a run time of 8 min.

Fast Analytical Separations with High-Pressure Liquid Chromatography

High-pressure liquid chromatography (HPLC) has established itself as a critically important analytical method for many research and commercial laboratories. Employers expect today’s chemistry graduates to have a working knowledge of liquid chromatography techniques including HPLC. In addition to HPLC, it is becoming more important to educate students about newer separation technologies such as ultra-high-pressure liquid chromatography (UHPLC). Unfortunately, these systems cost significantly more than traditional HPLC instruments, preventing their broad utilization in instrumental teaching laboratories. We have developed an ultra-fast isocratic separation method using traditional HPLC instrumentation that separates five compounds in 1 min. When utilized as a teaching activity, this ultra-fast separation allows students to develop an analytical method, generate standard calibration curves, and analyze unknown samples in a single teaching laboratory period. Furthermore, chromatograms obtained with this new method are similar to UHPLC, allowing students to experience separations similar to those obtained on newer UHPLC systems using a traditional HPLC.

Evaluation of 1.0 mm i.d. column performances on ultra high pressure liquid chromatography instrumentation

The present study focuses on the evaluation of 1.0 mm i.d. (internal diameter) columns on a commercial Ultra-High Pressure system. These systems have been developed specifically to operate columns with small volumes, typically 2.1 mm i.d., by reducing extra-column volume dispersion. The use of columns with smaller i.d. results in a reduced solvent consumption and required sample volume. The evaluation of the columns was carried out with samples containing neutral and pharmaceutical compounds. In isocratic mode, the extra-column volume produced additional band broadening leading to poor performances compared to equivalent 2.1 mm i.d. columns. By increasing the length of the column, the influence of the extra-column bandspreading could be reduced and 75,000 plates were obtained when four columns were coupled. In gradient mode, the effect of the extra-column contribution on efficiency was limited and about 80% of the performance of the 2.1 mm i.d. columns was obtained. Optimum conditions in gradient mode were further investigated by changing flow rate, gradient time and column length. A different approach of the calculation of peak capacity was also considered for the comparison of the influence of these different parameters.

Separation of haemoglobin HbE and HbA2 by the fully automated, high‐pressure liquid chromatography Tosoh HLC‐723 G7 analyzer

High-pressure liquid chromatography instruments specifically devised for separating haemoglobin (Hb) fractions have been increasingly employed by the hospital laboratories over the recent years since they allow easy and fast screening for several Hb variants. Although such instruments may be proposed as sensitive, specific and reliable alternatives to the classic electrophoretic techniques, a major drawback of this screening strategy is the almost identical retention time of several Hb variants. In particular, at least 18 Hb variants have been reported in the same retention window as HbA(2), including HbE, the second most common beta-chain variant in humans after sickle cell trait. Recently, we evaluated the performance characteristics of an improved buffer formulation originally conceived for Hb variants separation procedures on the fully automated high-pressure liquid chromatography instrument Tosoh G7. At variance with other fully automated high-pressure liquid chromatography analyzers, the elution pattern on the G7 in subjects heterozygous for HbE is characterized by the presence of four suggestive peaks (HbF, HbA, HbA(2) and HbE), confirming the effective separation of HbE from HbA(2). Because of its potential value in the diagnosis of the thalassaemia syndromes, the effective separation of HbA(2) from HbE can provide clinical laboratories with a valuable information for the diagnostic reasoning.

Comparison of glycated albumin and hemoglobin A1c levels in diabetic subjects on hemodialysis

Glycated albumin is thought to more accurately reflect glycemic control in diabetic hemodialysis patients than hemoglobin A(1c) because of shortened red cell survival. To test this, glycated hemoglobin and albumin levels were measured in blood samples collected from 307 diabetic subjects of whom 258 were on hemodialysis and 49 were without overt renal disease. In diabetic subjects with renal disease, relative to those without, the mean serum glucose and glycated albumin concentrations were significantly higher while hemoglobin A(1c) tended to be lower. The glycated albumin to hemoglobin A(1c) ratio was significantly increased in dialysis patients compared with the controls. Hemoglobin A(1c) was positively associated with hemoglobin and negatively associated with the erythropoietin dose in hemodialysis patients, whereas these factors and serum albumin did not significantly impact glycated albumin levels. Using best-fit multivariate models, dialysis status significantly impacted hemoglobin A(1c) levels without a significant effect on glycated albumin. Our results show that in diabetic hemodialysis patients, hemoglobin A(1c) levels significantly underestimate glycemic control while those of glycated albumin more accurately reflect this control.

Monolithic columns—a new concept of separation in the sequential injection technique

In this contribution, the coupling of monolithic column with sequential injection technique (SIA), as a new possibility of implementation of a separation step in SIA is described. Monolithic columns were developed based on a new sol–gel technology. Due to the presence of macropores, the monolithic columns possess a much higher porosity than conventional particulate high pressure liquid chromatography (HPLC) columns. Consequently, monoliths can be coupled to the SIA system without loss of performance or limitations due to the very low column back-pressure. The aim of our study was to develop and test the new SI separation manifold and to demonstrate its functionality in the simultaneous determination of four different compounds (methylparaben, propylparaben, sodium diclofenac and internal standard butylparaben) in standard solutions and in a topical emulgel. A Chromolith ® Flash RP-18e, 25 mm×4.6 mm (Merck, Germany) column and a FIAlab ® 3000 system (USA) with an eight-port selection valve and 10 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile:water (40:70 (v/v)) + 0.05% triethylamine, pH 2.5, gradient flow rate 8–20 μl s −1. A novel reversed-phase sequential injection chromatography (SIC) technique with UV spectrophotometric detection was optimised and validated. The validation parameters showed very good results. The analysis time was less than 8 min. The method was found to be applicable for the routine analysis of the active compound sodium diclofenac and preservative propylparaben in a pharmaceutical product, topical diclofenac emulgel 1%. The coupling of the monolithic columns with sequential injection manifold provides an excellent tool to solve the separation problems without using HPLC instrumentation and with low cost per analysis and low consumption of organic solvents (non-continuous flow).

32] The measurement of ω- and ω-1 hydroxylation of fatty acids by mixed-function oxidase systems

Summary Several methods are described in the literature 2,6,11 for the separation, identification, and quantitation of the products of fatty acid hydroxylation catalyzed by either bacterial or mammalian monooxygenases. The free fatty acid and its hydroxylated metabolites can be separated using silicic and chromatography, but the possible ω and ω-1 hydroxy fatty acids cannot be resolved using this technique. However, the methyl esters of the two major hydroxylated metabolites can be resolved by either thin-layer, gas, or high-pressure liquid chromatography. Thin-layer chromatography of ω and ω-1 hydroxy fatty acid methyl esters usually requires repeated solvent development to adequately separate the two metabolites. Although radio-gas chromatography is sometimes limited by CO 2 quenching, complete separation and quantitation of a number of hydroxy fatty acid methyl esters can also be achieved using this method. High-pressure liquid chromatography (HPLC) can be used to completely resolve the two methylated hydroxy metabolites, and this separation can be achieved easily using an isocratic HPLC instrument consisting of a solvent delivery system and a micro-particle octodecylsilane column. This system, coupled with a refractive index detector, allows the collection of the methyl esters of the metabolites and lauric acid in minimum volume.

Atomic Absorption Detector for Chromium Organometallic Compounds Separated by High Speed Liquid Chromatography

Abstract Atomic absorption spectroscopy (AAS) has been demonstrated to be a metal-specific detector for chromium organometallic compounds separated by high pressure liquid chromatography (HPLC). Column effluent from the HPLC instrument was fed directly to the aspirator of the atomic absorption instrument set to record absorption due to chromium. Well defined peaks were obtained. In addition to being specific for a given metal, the method is free of some of the constrictions placed upon conventional ultraviolet absorption and refractive index detectors commonly used with HPLC. For example, solvents which absorb strongly in the ultraviolet may be employed and there is no need to use expensive spectroquality solvents. The method promises to be particularly useful for the analysis of specific organometallic compounds found in liquefied coal and shale oil.