Liquid Chromatographic Characteristics of Ethylenethiourea with HPLC Carbon,Chiral, Polymer and Reverse-Phase Bonded Silica Columns

Abstract

Of the columns investigated, the graphitised carbon column provided the best chromatographic characteristics for the highly water-soluble compound ethylenethiourea (ETU). The stability of the carbon column in strongly acidic media permitted the incorporation of the phosphoric acid electrolyte into the 5% acetonitrile-in-water mobile phase. ETU eluted from the column in 200 s as a sharp symmetrical peak at a mobile phase flow rate of 1 mL/min and a column temperature of 35°C. The k' value was 1.72. ETU peak retention times and responses showed excellent repeatability with coefficients of variation of 0.28 and 1.40%, respectively, for 6 replicates with the high performance liquid chromatographic-electrochemical system using the graphitised carbon column. Although ETU eluted as a sharp symmetrical peak with the cyclodextrin chiral columns, their instability at low pH required post-column addition of the phosphoric acid electrolyte solution. ETU chromatographed poorly or degraded on the polymer columns. The chromatographic separation of ETU on the C-8 reverse-phase bonded silica column appeared to be due mainly to residual silanol groups. With the NH₂ bonded silica column ETU eluted immediately after the injection solvent.     

反相离子对色谱/蒸发光散射检测器分离唑来膦酸及其有关物质

采用反相离子对高效液相色谱／蒸发光散射检测法研究了唑来膦酸及其有关物质的色谱分析与分离方法。优化了色谱条件，固定相为Hypersil C8柱，以含10 mmol／L正戊胺的5mmol／L乙酸铵缓冲液（用乙酸调节pH至7．0）-甲醇（体积比为97：3）为流动相，流速为1．0mL/min，蒸发光散射检测器检测。在该色谱条件下，唑来膦酸与其有关化合物（包括合成过程中残余的原料咪唑乙酸及分解产物亚磷酸、磷酸盐）的分离良好，唑来膦酸色谱峰与最近杂质峰的分离度大于1．5。本法简便快速，为唑来膦酸的常规分析提供了有效可靠的方法。     

Determination of Suramin in Plasma and Urine by Ion-Paired Reverse-Phase High-Performance Liquid Chromatography

Abstract

A high-pressure liquid chromatographic (HPLC) assay has been developed for the quantification of the anti-viral drug suramin (SUR) in human plasma and urine. This assay is simple and accurate, using an isocratic mobile phase in a reverse-phase ion-pairing mode.

This assay was developed for the determination of high levels of suramin in the urine and plasma of patients being treated for the acquired immunodefficiency Syndrome (AIDS). The limit of detection of this assay was 0.25 μg/mL. Congo red (CR) was used as the internal standard with a retention time of 5.9 min, while suramin had a retention time of 13 min.     

Effect of increasing concentration of ammonium acetate as an additive in supercritical fluid chromatography using CO2–methanol mobile phase

The effects of increasing concentrations of ammonium acetate additive in supercritical fluid chromatography were studied on silica, 2-ethyl-pyridine and endcapped 2-ethyl-pyridine stationary phases. The study involved the addition of increasing concentrations of the ammonium acetate either in the mobile phase modifier (methanol) or in the sample solvent. The effects of ammonium acetate on retention and peak shape of the analytes were evaluated. Compounds that exhibited satisfactory chromatographic behaviour in the absence of the additive were virtually unaffected by its presence in the mobile phase or sample solvent. Nevertheless, compounds that exhibited late elution and strongly tailing peak shapes when pure methanol was used showed dramatically improved chromatographic behaviour in the presence of the additive. Shorter retention was observed not only when the modifier was introduced in the mobile phase but also when it was in the sample solvent.     

A Reverse Phase HPLC Method to Determine Six Food Dyes Using Buffered Mobile Phase.

Abstract

A reverse phase high performance liquid chromatographic (HPLC) method to determine six food dyes (Sunset Yellow (E-110), Carminic acid (E-120) Carmoisine (E-122), Amaranth (E-123), Ponceau 4R (E-124) and Erythrosine (E-127) is developed in this paper. The separation was made on a Nova-Pack C18 column using methanol -NaH₂PO₄/Na₂HPO₄ pH=7 buffer solution 0.1M as mobile phase with an elution gradient system. The detection was made with a variable UV-Vis. detector fixed at 520 nm.

The effect of mobile phase composition such as the percentage of methanol or acetonitrile, pH value and ionic strength on retention times of the dyes was investigated. In the chromatographic conditions selected, the dyes were eluted in four minutes. Two calibration graphs for each dye were established by measuring the peak area and the peak height in the chromatograms. Determination limits ranging from 0.8 to 9.2 ng were obtained when the peak area was measured.

Several commercial products containing some of these dyes were analyzed.     

Development of a novel,fast, simple,nonderivative HPLC method with direct UV measurement for quantification of memantine hydrochloride in tablets

Fast, simple, accurate, and reproducible reverse phase‐high‐performance liquid chromatography method with direct ultraviolet measurement of memantine hydrochloride in tablets was developed, without any chemical derivatization pretreatment. Three main problems appear during chromatographic analysis of memantine: detection, achieving appropriate column retention, and limited choice of mobile phase components, as a result of memantine molecular structure. Among more than 35 tested columns, the best retention and peak symmetry yielded two C8 and three C18 columns with different characteristics, at a temperature of 30°C, mobile phase composed of 1%, v/v, acetonitrile and 99%, v/v, of 0.05–0.1% phosphoric acid or 2.5–5 mmol phosphate buffer, at flow rate of 1 mL/min and injection volume of 5 µL. The retention time of memantine was between 2.6 and 4 min. Both mobile phase concepts showed perfect linearity, precision, and accuracy. This is the first successful and reproducible direct reverse phase‐high‐performance liquid chromatography–ultraviolet quantification method for memantine.     

Multivariate Methods to Evaluate the Role of Mixed Supports in Reversed-Phase Thin-Layer Chromatography

Abstract

Hydrophobic properties of 17 aniline and phenol derivatives were characterized by reversed-phase thin-layer chromatographic and high performance liquid chromatographic retention data.

In order to elucidate the role of thin-layer chromatographic supports in the hydrophobicity determination paraffin coated silica, aluminium oxide, cellulose, diatomaceous earth and their mixtures were used. Water, water-methanol 7:3 and 1 M NaCl served as mobile phases. The retention data were analyzed by spectral mapping technique.

The potency values differed from support to support proving that the composition of support has a deciding role in the hydrophobicity determination of aniline and phenol derivatives. The eluents did not influence considerably the potency order of supports.     

Adsorption of the anionic surfactant sodium dodecyl sulfate on a C18 column under micellar and high submicellar conditions in reversed‐phase liquid chromatography

Micellar liquid chromatography makes use of aqueous solutions or aqueous‐organic solutions containing a surfactant, at a concentration above its critical micelle concentration. In the mobile phase, the surfactant monomers aggregate to form micelles, whereas on the surface of the nonpolar alkyl‐bonded stationary phases they are significantly adsorbed. If the mobile phase contains a high concentration of organic solvent, micelles break down, and the amount of surfactant adsorbed on the stationary phase is reduced, giving rise to another chromatographic mode named high submicellar liquid chromatography. The presence of a thinner coating of surfactant enhances the selectivity and peak shape, especially for basic compounds. However, the risk of full desorption of surfactant is the main limitation in the high submicellar mode. This study examines the adsorption of the anionic surfactant sodium dodecyl sulfate under micellar and high submicellar conditions on a C₁₈ column, applying two methods. One of them uses a refractive index detector to obtain direct measurements of the adsorbed amount of sodium dodecyl sulfate, whereas the second method is based on the retention and peak shape for a set of cationic basic compounds that indirectly reveal the presence of adsorbed monomers of surfactant on the stationary phase.     

“PRISMA” Model for Computer-Aided HPLC Mobile Phase Optimization Based on an Automatic Peak Identification Approach

Abstract

The tripartite “PRISMA” optimization model, as part of the “PRISMA” system, includes all possible solvent combinations between 1–4 solvents, with a possible fifth one as modifier. The solvent composition is characterized by the solvent strength (S_T) and the selectivity points (P_S).

At a constant S_T the correlation between the P_S and the retention data (horizontal function) can be described by a quadratic function. For constant P_S the solvent strengths and retention data correlate (vertical function) with a logarithmic function. These correlations are used to formulate a mathematical model for the dependence of retention times (capacity factor) on the mobile phase composition. Unknown compounds are estimated in the mathematical model from a sequence of standard chromatograms after having identified individual peaks by an automatic procedure. Only retention times, relative peak areas, and information about the mobile phase compositions are required as input for the identification approach. The approach involves a combination of statistical methods which exploit both the basic properties of retention data and the mathematical relation between retention data, selectivity points, and solvent strength as derived from the “PRISMA” model. Diagnostic information for checking the identification is generated as a by-product. The mathematical model completed by the estimated constants predicts the expected retention times for each possible mobile phase combination. Peak start and peak end times are predicted in a way similar to the retention times, once the identification has been performed. The most important aspects of a chromatogram can thus be predicted for arbitrary mobile phases.

The separation quality of predicted chromatograms is assessed by the chromatographic response function (CRF). The optimal mobile phase combination is that which theoretically generates the chromatogram with the maximal CRF value. This optimal composition is found by a simple mathematical procedure, which maximises the CRF in dependence upon the mobile phase combination. The optimum found is a local one if the starting set of chromatograms contains no variation of the solvent strength, and a global one if, in the set of starting chromatograms, the solvent strength is varied in a suitable way. Recommendations for the starting position are given.

Twelve measurements are necessary for a local optimum, and 15 for the global one. To increase the accuracy, six measurements at three different solvent strength levels are proposed. Generally the highest and the lowest solvent strength level differ by ±(5)% from the middle level.

This strategy is also relevant when modifiers are used in constant amounts. The chromatographic behavior of substances to be separated can be predicted with 1% accuracy from correlations of k' values and selectivity points. Based on these relationships, an automatical mobile phase optimization strategy for isocratic separations is suggested with the “PRISMA” model.     

Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions

The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5-9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides.     

Chromatographic behavior of new deazapurine ribonucleosides in hydrophilic interaction liquid chromatography

The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide‐ and cyclofructan‐based stationary phases. The effects of buffer concentration, pH and acetonitrile‐to‐aqueous‐part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan‐based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan‐based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides.     

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.     

Development and validation of stability indicating RP-HPLC and HPTLC for determination of Niclosamide in bulk and in synthetic mixture

Two simple, specific, sensitive, accurate and precise stability indicating methods were described for quantitative determination of the anthelmintics drug Niclosamide. The first method was high performance liquid chromatographic with the use of a reversed phase hibar^R C-18 column (250 mm × 4.66 mm, 5 μm) and mobile phase of methanol: 1 mM ammonium phosphate buffer (85:15 v/v) at a flow rate of 1.2 mL/min. The retention time of drug was found to be 6.45 ± 0.02 min. Quantification of drug was achieved with diode array detection (DAD) at 332 nm. Linear calibration curve was obtained in concentration range 0.01–100 μg/mL with r² value of 0.999. The limit of detection and limit of quantification were found to be 0.048 μg/mL and 0.01 μg/ml respectively. The second method involved a high performance thin layer liquid chromatographic. Chromatographic separation was carried out with precoated silica gel G60 F254 aluminum sheets using toluene:ethyl acetate (7:3% v/v) as a mobile phase. Linearity of proposed method was found to be 200–700 ng/band at 332 nm with retention factor of 0.59 and r² value of 0.998. The limit of detection and limit of quantification were found to be 36.21 ng/band and 109.7 ng/band respectively. Both the developed methods were successfully validated as per International Conference on Harmonization guideline (ICH). Niclosamide was subjected to different stress conditions. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time. Stress samples were successfully assayed by developed high performance liquid chromatographic and high performance thin layer liquid chromatographic method. Statistically analysis proves that there were no statistical significant differences between two developed methods.     

Chromatography of sterols, alkaloids and other drugs using steam as the mobile phase.

The chromatographic separation of steroids, alkaloids and other drugs with water vapour as the mobile phase has been studied. It is shown that this technique facilitates the analysis and reduces the retention times. The determination of the substances in low concentrations in aqueous solutions or dispersions also appears to be possible.     

对氨基苯胂酸及其氧化物的色谱研究

用紫外分光光度计分析了对氨基苯胂酸（PABAA）及其氧化物的光谱特征后,在十八烷基键合相硅胶柱上,以甲醇-缓冲液作流动相,研究了二者的容量因子随流动相离子强度、柱温、甲醇含量变化的规律。用季铵盐作离子对试剂,反相离子对色谱法分离PABAA时,分离机理符合高子对机理,在适当条件下,所试验的化合物都可有所保留。对保留值作出贡献的有固定相排阻作用、分配作用以及居次要地位的PABAA与固定相表面剩余硅醇基的相互作用。排阻作用及分配作用的相对重要性与流动相中甲醇和离子对试剂的浓度有关。     

Hydrophilic interaction liquid chromatographic tandem mass spectrometric determination of atenolol in human plasma

A hydrophilic interaction liquid chromatographic method with tandem mass spectrometry for the determination of atenolol, a beta-blocking agent, in human plasma has been developed and validated over the curve range of 10--2000 ng/mL. The assay was based on protein precipitation followed by evaporation of the extraction solvent, reconstitution with acetonitrile, and chromatography on an Hypersil silica column (50 x 4.6 mm) using a low aqueous--high organic mobile phase. The mobile phase consists of 85% acetonitrile, 15% water, 0.5% acetic acid and 0.04% trifluoroacetic acid and runs isocratically at a flow rate of 2.0 mL/min. The column ef fluent was split so that 50% of it was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 2.0 min per injection. Atenolol and the internal standard, atenolol-d(7), showed a retention time of 1.0 min. The inter-day and intra-day precision and accuracy of the quality control samples were <5.3% relative standard deviation and <8.0% relative error, respectively. To explore the application of the current method for the analysis of other beta-blocking agents, propranolol and metoprolol were tested under the same chromatographic conditions with retention times of 0.68 and 0.75 min, respectively. The present method could be used for therapeutic drug monitoring, pharmacokinetic and drug--drug interaction studies of beta-blocking agents.     

Occurrence and behavior of system peaks in RP HPLC with solely aqueous mobile phases

System peaks are important but often also disturbing phenomena occurring in separation systems. Behavior of system peaks was studied in reversed phase high performance liquid chromatography (RP HPLC) systems consisting of an RP Amide C16 column and aqueous solutions of organic acids with alkaline metal hydroxides as mobile phases. Binary mobile phases, composed of benzoic acid and lithium hydroxide (LiOH) or cesium hydroxide (CsOH), yielded two system peaks. The first peak was stationary and the second one moved with dilution of the mobile phase or with changes of the alkaline metal hydroxide concentration. The latter changes affected dissociation of the benzoic acid present in the mobile phase and thereby its retention. The presumption that the first system peak is not influenced by the type of alkaline metal cation and that it is related to the non‐adsorbed component of the mobile phase was confirmed by a cyclic procedure. Three‐component mobile phases composed of benzoic acid, tropic acid, and a hydroxide gave rise to three system peaks as expected. The first peak was again stationary and the two others shifted depending on the concentration variation of both acids. Resonance causing a zigzag peak, well described in capillary zone electrophoresis (CZE), was observed if 1‐pentanol was injected into a chromatographic system with one‐component mobile phase.     

对氨基苯胂酸及其氧化物的色谱研究

 用紫外分光光度计分析了对氨基苯胂酸（PABAA）及其氧化物的光谱特征后,在十八烷基键合相硅胶柱上,以甲醇-缓冲液作流动相,研究了二者的容量因子随流动相离子强度、柱温、甲醇含量变化的规律。用季铵盐作离子对试剂,反相离子对色谱法分离PABAA时,分离机理符合高子对机理,在适当条件下,所试验的化合物都可有所保留。对保留值作出贡献的有固定相排阻作用、分配作用以及居次要地位的PABAA与固定相表面剩余硅醇基的相互作用。排阻作用及分配作用的相对重要性与流动相中甲醇和离子对试剂的浓度有关。     

Specific retention behaviour of metal (III) tetraphenylporphyrins in non-aqueous,reversed high-performance liquid chromatography

Summary The Co(II), Ni(II), Fe(III) and V(IV) complexes of tetraphenylporphine (TPP) can be eluted at short retention times from a LiChrosorb RP-18 column with pure ethanol. However, both Mn(III) and Co(III) complexes of metal TPP chloride type are so strongly retained on the column that they cannot be eluted. While the retention of other metal teraphenylporphine complexes was not affected, that of the metal(III) complexes of the TPP chloride type especially MnTPPCl and CoTPPCl, decreases dramatically with an increase in the concentration of NH₄Cl added into the mobile phase; a linear relationship between logk' and log[NH₄Cl], with the slope of about–1, has been observed for these two metal(III) complexes in the NH₄Cl concentration range from 2.5×10^–4 to 1.3×10^–2 mol/l. Thus, the specific control of the retention of the metal(III) complexes is enabled by conditioning the NH₄Cl content of the mobile phase, and the chromatographic separation is demonstrated.     

Optimization of micro‐HPLC peak focusing for the detection and quantification of low hepcidin concentrations

Micro‐high‐performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 μL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile‐phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.