Hplc Method for Kinetic Study of Mutarotation of Sugars

Abstract

A high-performance liquid chromatographic (HPLC) method was established for kinetic studies of the interconversion (mutarotation) between pyranose sugar anomers in solution. At very low temperature, HPLC chromatograms of each sugar indicated the concentrations of each anomer in the state prior to the HPLC analysis. Thus, when one of the pyranose anomers obtained by recrystallization was dissolved into water and was analyzed by HPLC repeatedly, gradual changes in the chromatogrphic patterns were observed. The equilibrium concentrations of each sugar anomer in different solvents were found to vary, which was also investigated by HPLC.     

Selectivity issues in targeted metabolomics: Separation of phosphorylated carbohydrate isomers by mixed-mode hydrophilic interaction/weak anion exchange chromatography

Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2‐ and 3‐phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3‐phosphate, as well as glucosamine 1‐ and 6‐phosphate utilizing mixed‐mode chromatography with reversed‐phase/weak anion‐exchangers and a charged aerosol detector. The best results were obtained when the reversed‐phase/weak anion‐exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion‐exchange processes. Employed acidic conditions have led to the complete separation of α‐ and β‐anomers of glucose 6‐phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on‐column interconversion with a plateau in‐between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10^?4 s^?1. Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely.     

Kinetics of the Polymerization of Aldohexoses

Abstract

Thermogravimetric procedures are used to obtain the kinetic parameters for the intermolecular condensation of the anomers of D-glucose, D-mannose, and D-galactose. The condensation rates in the temperature range 176–220°C are found sufficiently rapid compared to the rates of mutarotation that significant differences between anomers can be observed.     

Separation of Ethyl Anthranilate Azopigments from Germ-Free Rat Feces by Reversedphase High-perform Ance Liquid Chromatography

Abstract

A simple method was developed for the separation of ethyl anthranilate azopigments derived from conjugated bilirubins prepared from germfree (GF) rat feces. The Δ and α₀ azopigments were separated and these two azopigments were also separated into their endo-vinyl- and exo-vinyl isomers, respectively. The reverse-phase, high-performance liquid chromatographic (HPLC) separation was achieved by using a μBondapak C₁₈ column and a mixture of acetonitrile, distilled water and sodium acetate as the mobile phase.     

Parallel factor analysis of HPLC-DAD data for binary mixtures of lidocaine and prilocaine with different levels of chromatographic separation

A set of 17 samples containing a constant amount of lidocaine (667 μM) and a decreasing amount of prilocaine (667-0.3 μM) was analysed by LC-DAD at three different levels of separation, followed by parallel factor analysis (PARAFAC) of the data obtained. In Case 1 no column was connected, the chromatographic resolution (R_s) therefore being zero, while Cases 2 and 3 had partly separated peaks (R_s=0.7 and 1.0). The results showed that in Case 1, analysed without any separation, the PARAFAC decomposition with a model consisting of two components gave a good estimate of the spectral and concentration profiles of the two compounds. In Cases 2 and 3, the use of PARAFAC models with two components resolved the underlying chromatographic, spectral and concentration profiles. The loadings related to the concentration profile of prilocaine were used for regression and prediction of the prilocaine content. The results showed that prediction of prilocaine content was possible with satisfactory prediction (RMSEP<0.01). This study shows that PARAFAC is a powerful technique for resolving partly separated peaks into their pure chromatographic, spectral and concentration profiles, even with completely overlapping spectra and the absence or very low levels of separation.     

Liquid Chromatographic Analysis of Some N-Alkyl-3,4-methylenedioxyamphetamines

Abstract

The liquid chromatographic separation of a series of low molecular weight N-alkyl derivatives of 3,4-methylenedioxyamphetamine (MDA) is reported. The N-methyl-, N-ethyl- and N,N-dimethyl-derivatives of MDA have appeared as street drugs in recent years. These compounds are becoming significant forensic problems due to their unique pharmacological properties and relative ease of chemical synthesis. These amines were synthesized via reductive amination of the corresponding ketone with alkylamines. The UV absorption spectra for these compounds produced almost equally intense absorbance at 234 and 285 nm. The compounds were separated by reversed-phase (C₁₈) HPLC procedures using a mobile phase of aqueous methanol at low pH.     

Rapid and Complete Separation of Inorganic Polyphosphates from Monomer to 35-Mer by Hplc

Abstract

The on-line coupling system of high-performance liquid chromatography (HPLC) and flow injection analysis (FIA) was developed for the rapid separation and sensitive detection of inorganic polyphosphates. Effects of gradient elution conditions on the chromatographic behavior were studied. The column temperature as well as a chloride concentration gradient were found to be very effective for the improvement of resolution(1). To minimize analysis time and to maximize resolution, the gradient elution conditions were optimized by use of a computer-assisted retention prediction system(2). More than 35 kinds of inorganic polyphosphates could be separated completely within 200 min under the optimum conditions as shown in Fig. 1.     

An Improved Method Fòr Extraction,Partial Purification,Separation and Isolation of Chlorophyll from Spinach Leaves

Abstract

Powdered sugar column chromatographic separation and isolation of chlorophyll were studied. A wet column was employed using a Pharmacia R25 chromatographic tube connected, through a UV monitor, to a fraction collector. Chlorophyll extracted from spinach leaves with acetone was selectively precipitated with dioxane and water, and then was washed with 80% (v/v) aqueous methanol. Use of the further purified chlorophyll as a starting material for chromatographic separation and isolation of chlorophyll, made column chromatographic procedures become extremely effective and easier. Thin-layer chromatographic and high-performance liquid chromatographic tests revealed that chlorophyll preparations in this study did not contain any other photosynthetic pigments and their degradation products.     

Separation Studies of Metal Ions in Sodium Thioglycolate Medium by Thin Layer Partition Chromatography

Abstract

A thin layer partition chromatographic method has been developed for separation of Fe(III), Ni(II), Zn(II), Cu(II), Pb(II) and Mn(II) on thin layers of silica gel-G as an adsorbent. The R_f values were determined using 0.01–0.2 aqueous solution of sodium thioglycolate as a mobile phase. The dependence of R_f values on the migration time, pH and concentration of mobile phase has been studied. The optimum conditions for possible 3-component separation have been determined. Metal ions have been separated, detected, eluted and quantitatively determined by atomic absorption spectroscopy. The present method was applied to the separation and determination of zinc in forensic samples.     

Ion Exchange High Performance Liquid Chromatography of Leukotrienes

Abstract

A novel anion exchange liquid chromatographic system has been developed for isocratic separation of leukotrienes. Hydrophobic as well as ionic forces were found to influence the separation. By optimization of solvent strenght, ionic strenght and pH, amphoteric peptidoleukotrienes could be separated simultaneously with hydroxy fatty acids such as leukotriene B₄ and its ω-oxidized metabolites. To obtain a good buffering capacity of the mobile phase at optimum pH, a multicomponent buffer was developed.     

Isocratic Separation of Fatty Acid Derivatives by Reversed Phase Liquid Chromatography. Influence of the Solvent on Selectivity and Rules for Elution Order

Abstract

The p-bromophenacyl esters of 16 fatty acids (C₁₂-C₂₂) have been separated by isocratic chromatography on a Radial Pak A cartridge (Reverse phase C₁₈ material). The separation factors α were measured using two solvent mixtures of comparable strength and the superiority of methanol-water to acetonitrile-water becomes evident.

Five precise rules are established, which indicates the retention of every fatty acid. They explain the chromatographic process i.e. elution order, resolution and selectivity.     

Influence of Bonded-Phase Column Type,Mobile Phase Composition,Temperature and Flow-Rate in the Analysis of Triglycerides by Reverse-Phase High Performance Liquid Chromatography

Abstract

Two chromatographic systems for RP-HPLC analysis of triglycerides, operating under isocratic conditions using octadecylsilane and octylisilane bonded phases, are described.

The influence of such chromatographic factors as bonded phase column type, mobile phase composition, temperature and flow rate on retention, analysis selectivity and efficiency, and separation of mixtures of homogeneous triglycerides was assessed. Linear relationships were established for the logarithm of the capacity factor and selectivity for each triglyceride in relation to temperature, the proportion of certain mobile phase components and flow rate.

The octadecylsilane bonded phase was more selective when analyzing triglycerides with a partition number below 48, while octylsilane was appropiate for separating mixtures of long chain saturated triglycerides to the detriment of the resolution of triglycerides with low partition numbers. ACN/ACE/THF (58/38/4) was a suitable mobile phase for use with the octadecylsilane bonded phase, and ACN/THF /H₂O (60/40/1) for the octylsilane bonded phase. A column temperature of 30°C and a flow rate of 1.5 mL/min resulted in acceptable resolution and analysis time in both systems.     

A Two-Factor Simplex Optimization of a Programmed Temperature Gas Chromatographic Separation

Abstract

Simplex optimization was used to optimize the temperature programmed gas chromatographic separation of octane, 2-methyltetrahydrofuran, butylcyclohexane, and ethylbenzene. The initial temperature and the programming rate were the factors studied and a 5 minute time constraint was placed on the system. The Chromatographic Response Function (CRF) used by Deming and Morgan¹ was used as an operational measure of performance. Further experiments in the region of the optimum and regression analysis of the data obtained were carried out to further understand the factor effects.     

Rapid HPLC Methods for the Separation and Quantitation of a Mono-, Di-, and Tri-Saccharides Mixture and Applications

Abstract

A simple, rapid method has been developed for the separation and quantitation of mono-, di-, and tri-saccharides. The method utilizes a 30cmx 3.9mm i.d. Microbondapak NH₂ column, refractive index detection and water-acetonitrile elution. Two chromatographic systems are described. The isocratic mode was necessary to develop a procedure. 20 Carbohydrate's retention times were evaluated. To optimize the separation of nine water-soluble sugars, a gradient mode flow-programming was used. Separation was achieved within 28 minutes. The low detection limit (4 micrograms) of the above chromatographic procedure and its different possibilities could be of great interest to the analyst. The method has been successfully applied to quantify the major carbohydrates found in two types of commercial honey.     

Template Imprinted Polymers for HPLC Separation of Racemates

Abstract

A novel approach to the separation of enantiomers using polymeric sorbents imprinted with optically active template molecules has been examined for use in HPLC. The racemate of the template molecule can be separated whereby covalent interactions are responsible for retardation in the chromatographic process. This covalent interaction governs the overall kinetics of the separation. Increasing the reaction rate of this binding reaction by optimizing the experimental conditions improves the separation considerably. The template form of the enantioraeric mixture is thus strongly retarded and separated from the non-template form. A further substantial improvement of the chromatographic pattern is obtained by and it is remarkable that the racemic resolution of mannose n this polymer is possible although the template is different. This finding offers the possibility of the racemic resolution of underivated sugar which s not possible by other methods at the present time. It gives also an insight into the separatión mechanism of template imprinted polymers. It encourages us to postulate that more than the precise shape of the cavities, the spatial arrangement of the interactive groups within the cavities plays a predominant role in the enantioselective separation mechanism of this sorbent type (13).     

Single derivatives for GC-MS assay of reducing sugars and selective detection by the nitrogen-phosphorus detector (NPD)

Reduction of sugar methoximes with sodium cyanoborohydride followed by permethylation with CH₃SOCH₂^?/ICH₃ affords the corresponding deoxy(methylmethoxylamino)alditol O-methyl ethers, which are amenable to capillary gas chromatography. Derivatization proceeds under mild conditions with high yields. Only a single derivative is formed for each reducing sugar. All aldose derivatives can be separated in a single chromatographic run on glass capillaries coated with Carbowax 20 M or Superox 0.1. Good linearity was found. The introduction of the nitrogen atom in the sugar molecule makes these derivatives suitable for selective detection by the NPD. The El-mass spectra contain intense characteristics ions at m/z 74 for aldoses and at m/z 280 for reducing disaccharides, together with a (M - 15)⁺ion. These fragments are very suitable for highly sensitive GC-MS analysis by selective single ion monitoring.     

Optimizing UPLC isocyanate determination through a Taguchi experimental design approach

The development of a chromatographic procedure for an ultra‐performance liquid chromatography can be a very time‐consuming task, as the general approach for finding the appropriate operating conditions has been a trial‐and‐error process. The present study reports a novel approach in the field of ultra‐performance liquid chromatography by using statistical experimental design based on Taguchi's method, which allows a complete separation of nine isocyanates present in a complex matrix. The resolution between two adjacent peaks was considered as a quality characteristic and transformed to a Taguchi signal‐to‐noise ratio. An orthogonal array L₉ (3⁴) was selected to analyze the effect of four chromatographic factors, that is, proportion of solvent, percent triethylamine (v/v), temperature (°C), and flow (mL min⁻¹), with three levels each. The joint analysis performed to the significant factors achieved in the eight analyses of variance allowed to identify two methods to conduct a complete separation of all peaks. Six isocyanates were separated with the first method, with all factors at the lowest level. The remaining three isocyanates were separated with the second method, with the proportion of solvent at the highest level and the other factors at the lowest level. The overall Taguchi experimental design identified the proportion of solvent and the flow rate as major chromatographic factors. Finally, confirmatory experiments were performed with samples prepared with six and three isocyanates, confirming the complete separation of all isocyanates in the study. The Taguchi methods provided a systematic and efficient methodology for this optimization, with considerably less effort than would be required for other optimizations techniques. Copyright © 2013 John Wiley & Sons, Ltd.     

High Performance Liquid Chromatographic Separation of Bryostatins 1–12

Abstract

A reversed-phase high performance liquid chromatographic (RP-8; acetonitrile-water gradient) separation procedure was developed for detecting bryostatins 1–12, using a photodiode array detector system. While bryostatins 6 and 9 were found to co-elute they were easily separated using a silica gel column with 9:1 n-hexane-n-propanol as eluent.     

Simultaneous analysis of six cardiovascular drugs by capillary electrophoresis coupled with electrochemical and electrochemiluminescence detection,using a chemometrical optimization approach

CE coupled with dual electrochemical (EC) and electrochemiluminescence (ECL) detection was optimized for simultaneous analysis of six cardiovascular drugs (alprenolol, propafenone, acebutolol, verapamil, atenolol and metoprolol) via central composite design. Following this study, three critical electrophoretic factors governing the CE separation were investigated: Tris‐H₃PO₄ buffer concentration, buffer pH value and separation voltage. A modified chromatographic response was adopted for evaluating CE separation quality. Optimum conditions were achieved using Tris‐H₃PO₄ buffer 35.6 mM (pH 2.3) separated at 13.9 kV, which was employed experimentally and led to the successful simultaneous separation of the above six drugs. The good agreement of the chromatographic response was observed between predicted data and actual experimental results using these optimized conditions (RSD=3.75%). The proposed method was validated for linearity, repeatability and sensitivity, and subsequently successfully applied to determine six basic drugs in urine samples.     

HPLC Analysis of Oligomeric Ethylene Glyccl Mixtures Via bis(2,4-Dinitrophenylation)

Abstract

Bis(2,4-dinitrophenylation) of oligomeric ethylene glycols of the formula HO-(CH₂CH₂O)_n-H (n=4–16)to the corresponding DNP-C(CH₂CH₂-O) -DNP (where DNP stands for 2,4-dinitrophenyl) provides chromophoric derivatives, which are separated chromatographically on HPLC column.

The bis(2,4-dinitrophenyl) glycols are stable in presence of triethylamine, but undergo ethanolysis in presence of hydroxide ions. The quantitative removal of the DNP groups allows an integrated scheme to pure glycols from commercially available polyethylene glycol mixtures, by bis(2,4-dinitrophenylation), chromatographic separation, end-group removal, using HPLC of the bis-(2,4-dinitrophenyl) glycols for purity monitoring.