Two fluoroalcohols as components of basic buffers for liquid chromatography electrospray ionization mass spectrometric determination of antibiotic residues

Two fluoroalcohols--1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFTB)--were evaluated for the first time as volatile buffer acids in the basic mobile phase for reversed-phase chromatography with electrospray ionization-mass spectrometric (LC-ESI-MS) detection of five antibiotics. Chromatographic separation as well as positive and negative ion ESI-MS intensities using these novel buffer components were compared to traditional buffer systems. Overall, the highest signal intensities and best chromatographic separation for the five antibiotics (ciprofloxacin, norfloxacin, ofloxacin, sulfadimethoxine and sulfamethoxazole) were achieved using 5 mM HFIP as the buffer acid to methanol : water mobile phase (pH of the aqueous component adjusted to 9.0 with ammonium hydroxide). Comparable results were achieved using 5 mM HFTB (pH adjusted to 9.0 with ammonium hydroxide). The suitability of HFIP for analysis of antibiotic residues in lettuce is demonstrated.     

Characterization of poly(butylene terephthalate) by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Poly(butylene terephthalate) (PBT) samples have been analyzed with size-exclusion chromatography (SEC) using a mixed solvent of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and chloroform as the mobile phase. Several matrices and different sample deposition methods have been investigated to analyze PBT with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Optimum results have been acquired by depositing PBT on top of a 2,4,6-trihydroxyacetophenone matrix. The found MALDI-TOF-MS method can be used to analyze the end group functionalities of PBT, as demonstrated with the samples at hand. By combining SEC (off-line) with MALDI-TOF-MS, absolute molecular masses of PBT can be measured, and these have been found to be considerably lower than those determined with SEC using polystyrene standards.     

Synthesis of Poly(Styrene)-block-poly(methacrylate)-block-poly(styrene) via Site Transformation Reaction

Abstract

Triblock copolymers with polystyrene outer blocks and an inner polymethacrylate block were synthesized by a site transformation reaction using anionic and cationic polymerization techniques. In order to obtain such ABA block copolymers, two synthetic routes have been applied. In the first case, different methacrylates (methyl methacrylate, 2-ethylhexyl methacrylate) were polymerized anionically with a bifunctional initiator to get poly(methacrylate) dianions later forming the inner block whereas in the second case poly(styrene)-block-poly(methacrylate) anions were synthesized by monofunctional initiation via sequential monomer addition. In a subsequent step, the living chain ends of the methacrylate dianions on one side, and the diblock copolymer anions on the other side, were functionalized with 1,4-bis(l-bromoethyl)benzene in order to obtain a potential bifunctional or monofunctional macroinitiator for the cationic polymerization of styrene. Then, styrene was polymerized cationically with the macroinitiator in the presence of SnCl₄ as coinitiator and _n Bu₄NBr as a common ion salt in CH₂Cl₂ at -15°C. Block formation was proven by SEC measurements, preparative SEC and NMR characterization.     

Fluoroalcohols as novel buffer components for basic buffer solutions for liquid chromatography electrospray ionization mass spectrometry: retention mechanisms

Two fluoroalcohols--1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFTB)--were evaluated as volatile buffer acids in basic mobile phases for LC-ESI-MS determination of acidic and basic compounds. HFIP and HFTB as acidic buffer components offer interesting possibilities to adjust retention behavior of different analytes and expand the currently rather limited range of ESI-compatible buffer systems for basic mobile phases. Comparing with commonly used basic buffer components the fluoroalcohols did not suppress the ionization of the analytes, for several analytes ionization enhancement was observed. RP chromatographic retention mechanisms were evaluated and compared to traditional buffer system. All trends in retention of the acidic and basic analytes can be interpreted by the following model: the neutral fluoroalcohols are quite strongly retained by the stationary phase whereas their anions are less retained, thus their amount on the stationary phase is dependent on mobile phase pH; the anions of the fluoroalcohols form ion pairs in the mobile phase with the basic analytes; the fluoroalcohols on the stationary phase surface compete with acidic analytes thereby hindering their retention; the fluoroalcohols on the stationary phase bind basic analytes thereby favoring their retention.     

Development of a reversed-phase high-performance liquid chromatography method for the analysis of components from a closed-loop insulin delivery system

A reversed-phase HPLC method has been developed which enables separation of the three components of a closed-loop insulin delivery system, namely concanavalin A methacrylamide (Con A-MA), dextran methacrylate (Dex-MA) and bovine insulin. The analysis of Con A-MA represents a significant challenge due to the formation of multiple conformations on contact with the chromatographic surface and the mobile phase. The extent of conformational change is shown to be dependent on a number of parameters: column temperature, mobile phase pH, contact time with the chromatographic surface, salt type and concentration and the organic modifier. By manipulation of these variables, protein denaturation can be minimised and recovery improved.     

Effect of multi-walled carbon nanotubes on non-isothermal crystallization kinetics of polyamide 6

The non-isothermal crystallization behaviors of multi-walled carbon nanotubes (MWNTs)/polyamide 6 (PA6) composites were investigated by differential scanning calorimetry (DSC). Three methods, namely, Avrami, Ozawa and Mo, were carried out to analyze the non-isothermal crystallization data. The results showed that the MWNTs in PA6 acted as effective nucleation agents. However the crystallization rate of composites obtained was lower than that of the neat PA6. It is indicated that the presence of MWNTs influenced the mechanism of nucleation and the growth of PA6 crystallites.     

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCI) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disul-fide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with re-versed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).     

Retention behaviour of beta-blockers in HPLC using a monolithic column

The chromatographic behaviour of a new HPLC-stationary phase (Chromolith RP-18e) is described for separation of beta-blockers. The effects of the different chromatographic conditions (buffer system, pH value, content of organic modifier, injection volume and flow rate) on the separation behaviour were studied. At higher flow rates the peaks seemed to be more symmetrical than at lower flow rates. The use of buffer or salt in the mobile phase for this separation is found to be very essential and the counter-anion type of this buffer or salt significantly affected the retention behaviour of beta-blockers while the cation type did not play the same important role.     

On-column amperometric detection in capillary electrophoresis with an improved high-voltage electric field decoupler

The analysis of polyamide-6 oligomers and polymer is usually performed with expensive fluorinated alcohols like 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Formic acid is well known as a mobile phase additive to adjust pH in reversed-phase high-performance liquid chromatography. However, formic acid is seldom used as a modifier to perform gradient elution chromatography on octadecyl-modified silica-based columns. Here we demonstrate the determination of cyclic and linear polyamide-6 oligomers using formic acid as a modifier on an octadecyl-modified silica-based column. This column was shown to be stable for more than 5000 column volumes, even when a mobile phase of 65-95% formic acid in water at a flow of 1 ml/min is applied. With formic acid under the conditions used (65-95% formic acid in water) the oligomers are retained on the column, while the polymer does not precipitate. In comparison, during adsorption and separation with a HFIP gradient, precipitation of the polymer occurs. The implications of the different separation mechanisms, i.e., adsorption vs. precipitation chromatography are discussed. Loadability is shown to be much better with the formic acid system. However, with formic acid as a modifier UV detection below 250 nm is not feasible. The less sensitive evaporative light scattering detector is used to detect the polyamide oligomers in the formic acid phase. In addition it is shown that capillary zone electrophoresis (CZE) with UV-absorbance detection using HFIP is an attractive combination as HFIP is UV-transparent and CZE allows low modifier consumption.     

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol L^?1 guanidine hydrochloride (GuHCI) or 8.0 mol L^?1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol L^t-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disulfide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).     

New Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers

Most synthetic polymers are distributed in more than one parameter of molecular heterogeneity. For hydrophobic copolymers there are different chromatographic techniques available to analyse these distributions. As a result of the increasing interest in hydrophilic polymers and copolymers new chromatographic techniques are developed for the characterization of these polymers as well. However, very frequently these polymers contain highly polar or charged functional groups making them soluble only in aqueous mobile phases. There are several problems related to the use of aqueous mobile phases in polymer chromatography. Even the SEC analysis of such copolymers is not straightforward. As for HPLC in aqueous mobile phases, there are only a few applications in the literature so far. In addition to the fact that only a very limited number of stationary phases is available for aqueous HPLC of polymers, the interactions of polyelectrolytes in such chromatographic systems are not well understood. The present paper addresses the problems related to the application of SEC and HPLC in aqueous mobile phases. For graft copolymers with a polyethylene oxide backbone, e.g. PEG-g-polymethacrylic acid and PEG-g-polyvinyl alcohol, it will be shown that methods can be developed that give accurate molar mass and chemical composition information. Two-dimensional chromatography where aqueous HPLC and SEC are coupled on-line will be shown to be the most powerful analysis tool for the analysis of such copolymers. The hyphenation of the chromatographic separation techniques with spectroscopic detection techniques provides further insight into the molecular complexity of these copolymers.     

Chromatographic behaviour of low molar-mass polyesters in normal-phase high-performance liquid chromatography

Summary The normal-phase chromatographic retention behaviour of polyesters on bare silica and on a polymer-based polyamine (PA) column has been studied with a variely of binary mobile phases under isocratic conditions. The dependence of experimental retention data on the degree of polymerization (p) and on mobile phase composition (φ) was characterized by to an approach developed by Jandera et al. The bulky repeating unit and the relatively highly polar end groups of the polyesters both had a large influence on retention behaviour. The two effects in combination explain the molar-mass-independent retention observed experimentally at a particular mobile phase composition for all the mobile phase—stationary phase combinations investigated. These conditions were found to be independent of the type of end group. End group separation on a silica column improves when the polarity of the less polar solvent is increased. End group separation is better on the PA column because of a greater difference between the adsorption energy of the alcohol and acid end groups. Better prediction of retention data on the PA column was achieved by use of an approach which assumes two different types of adsorption site. Results enabled further understanding of retention behaviour in normalphase gradient polymer-elution chromatography (NPGPEC) and explained both the dependence of the order of elution onp and differences between the end-group selectivity of different systems.     

高效阴离子交换色谱-铜/铂修饰电极安培法测定大观霉素

采用阴离子交换色谱分离,在CuSO4溶液中加入少量镧系化合物,经电沉积制备La3+-Cu/pt/CME工作电极,建立了直流安培电化学法(DC)直接检测硫酸大观霉素的方法。考察了流动相浓度、测定电位等参数对色谱分离和测定的影响。在固定相为CarboPac PA10阴离子交换柱、流动相为26mmol/L NaOH,流速为0.6mL/min的色谱条件下,检测电位为0.68V时,硫酸大观霉素峰面积与其浓度在0.12～12mg/L(r=0.9991)和12～280mg/L(r=0.9995)两个范围内呈线性。本方法不需要柱前和柱后衍生化,能同时测定硫酸大观霉素中的主要组分和杂质。修饰电极制作方法简单,催化稳定性好,可作为电化学传感器测定硫酸大观霉素中的各组分。     

High-performance size-exclusion chromatography of humic substances on the hydroxyethyl methacrylate column

HEMA, the copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, has proven to be a suitable stationary phase for the size-exclusion chromatographic (SEC) characterization of humic substances in a wide pH range. The SEC measurements were performed typically with alkaline borate-based mobile phase: 0.025 mol/l Na2B4O7, 0.025 mol/l NaCl, 0.001 mol/l Na4P2O7, pH 9.1. After calibrating with globular protein standards, apparent molecular weights of typical peat- and coal-derived humic acids were estimated. The average molecular weights determined with the aid of the HEMA column were quite comparable with the values obtained previously by the method of low-pressure gel-permeation chromatography on the Sephadex column.     

An Improved Microanalytical Procedure for the Quantitation of Procainamide and N-Acetyl Procainamide in Serum by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for analysis of procainamide (PA), and N-acetyl procainamide (NAPA) is presented. Sample preparation employs a simple base-acid double extraction procedure and analysis is carried out on a reverse phase chromatographic system using a μBondapak C₁₈ column and buffered aqueous acetonitrile as the mobile phase. The extraction procedure gives quantitative recovery of both PA and NAPA, and chromatographic results show that drug levels of as low as 0.3 mg per liter of serum can be conveniently analyzed without significant background interferences. The small volume (0.2 ml) of serum needed to perform an analysis makes this method suitable for pharmacokinetic studies in humans and animals as well as for clinical therapeutic drug monitoring studies.     

Two-dimensional chromatography of complex polymers 6. Method development for (meth)acrylate-based copolymers

The free-radical copolymerisation of various acrylates and methacrylates resulting in complex copolymers for cosmetic applications were investigated using different chromatographic techniques including HPLC and on-line coupled two-dimensional (2D) liquid chromatography. The complete separation of all polymerisation products was achieved by gradient HPLC. A computated optimisation procedure, using the Polymer Chromatographic Model allowed us to design a step mobile phase gradient to improve resolution of homopolymer chromatographic separation. By combining gradient HPLC and SEC (Size Exclusion Chromatography) in a fully automated two-dimensional chromatography setup, the complex distributions of chemical composition and molar mass could be simultaneously described and fingerprinted.     

Void volume markers in reversed-phase and biomimetic liquid chromatography

A comparative investigation of 15 void volume marker candidates was carried out on two reversed-phase (RP), two immobilized artificial membrane (IAM) and two immobilized protein columns, namely Human Serum Albumin (HSA) and α₁-acid-glycoprotein (AGP), using different mobile phases in respect to pH and buffer composition. Pycnometric analysis of the 6 chromatographic columns was also employed for reasons of comparison. The results revealed the possible overestimation of stationary phase void volume if a general void volume marker is used under different chromatographic conditions. In particular, overestimation of the investigated columns’ void volumes was a common phenomenon when using acidic eluents or High Purity Water (HPW) as mobile phase. For this purpose, a classification of the recommended chemicals according to the column type or the pH of the mobile phase is suggested for the accurate determination of retention factors.     

Micellar bile salt mobile phases for the liquid chromatographic separation of routine compounds and optical, geometrical, and structural isomers

Aqueous solutions of bile salts, i.e. sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium taurocholate (NaTC), are characterized and evaluated as reversed-phase liquid chromatographic (RPLC) mobile phases. The separation of the ASTM-recommended RPLC test mix in addition to more than 50 other compounds on a C18 column demonstrates the viability of these bile salts as HPLC mobile phases. The Armstrong-Nome theory was applied and found to adequately describe the partitioning behavior of solutes eluted with these bile salts at low surfactant concentrations. The effect of alcohol additives on chromatographic retention and efficiency was also assessed. Not only are the bile salt molecules rigid and chiral, but they form helical micellar aggregates as well. Consequently, many isomeric compounds can be easily resolved with this mobile phase additive. The base-line resolution of some binaphthyl-type enantiomers with a standard C18 column and the bile salt micellar mobile phases is also demonstrated. In addition, these bile salt mobile phases may be preferable to conventional hydroorganic mobile phase systems for the separation of many classes of routine compounds. A brief prospectus on the future utilization of bile salts in liquid chromatography is presented.