Determination of equilibrium constants from chromatographic and electrophoretic measurements

Chemical interactions, such as acid-base, complex-forming, ion association and other equilibria, are widely exploited to improve the separation efficiency in liquid chromatography as well as in electrophoresis. On the other hand, these techniques can be advantageously used to study the chemical equilibria affecting the separations. If the equilibium is sufficiently fast in comparison with the separation process, then the retention characteristics in chromatography (retention factors) or the migration characteristics in electrophoresis (effective mobilities) may be expressed as functions of the composition of mobile phase or background electrolyte (BGE), respectively. Using a proper experimental arrangement, the dependencies of retention (migration) characteristics on the mobile phase (background electrolyte) composition can be measured and utilized to calculate the equilibrium constants for equlibria taking place in the mobile phase (background electrolyte). Although principles of these measurements have been known for a long time, only more recent studies utilizing HPLC and capillary electrophoretic techniques are reviewed in this paper.     

Development of an HPLC method for the monitoring of tricyclic antidepressants in biofluids

A simple, rapid and sensitive HPLC method was developed and validated for the determination of four tricyclic antidepressants (TCAs): amitriptyline, doxepin, clomipramine (CLO) and imipramine, in pharmaceutical formulations and biological fluids. A Kromasil C(8 )analytical column (250 x 4 mm, 5 microm) was used for the separation, with a mobile phase consisting of 0.05 M CH(3)COONH(4) and CH(3)CN (45:55 v/v) delivered at 1.5 mL/min isocratically. Quantification was performed at 238 nm, with bromazepam (1.5 ng/microL) as the internal standard. The determination of TCAs in blood plasma was performed after protein precipitation. Urine analysis was performed by means of SPE using Lichrolut RP-18 Merck cartridges providing high absolute recoveries (> 94%). Direct analysis of urine was also performed after two-fold dilution. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between-day precision (n = 5) revealed RSD <13%. Recoveries from biological samples ranged from 91.0 to 114.0%. The absolute detection limit of the method was calculated as 0.1-0.6 ng in blood plasma and 0.2-0.5 ng in extracted urine or 0.4-0.7 in diluted urine. The method was applied to real samples of plasma from a patient under CLO treatment.     

Cyclic electrophoretic and chromatographic separation methods

A review is given of the application of cyclic analytical methods in capillary electroseparation (CE) and liquid chromatography (LC) systems. Cyclic methods have been used since the early sixties in chromatographic systems to overcome pressure limitations to resolution. From the early nineties on they have also been applied in capillary electroseparation systems to overcome voltage limitations. Some basic theory is given, outlining the temporal development of resolution in cyclic CE and LC systems and calculating the maximal resolution that can be obtained as a function of the operational parameters of pressure and electrical field. Simple equations are given for the temporal change in the peak capacity and the loss of peaks from the systems as it occurs in some cyclic systems. Finally, a circular open tubular chromatographic system is proposed using integrated pumping and continuous detection. The performance of such a system is discussed using magnetohydrodynamic and alternating current electroosmotic pumping as examples of integrated pumps and Shah Convolution Fourier transform detection as an example of a continuous detection method.     

Interplay of chromatographic and electrophoretic processes in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography and capillary zone electrophoresis, i.e., chromatographic retention and electrophoretic migration. The adsorption of the different sample components on the stationary phase can be modified by the presence of the electric field across the column. Here, we use our previously published approach to decouple chromatographic retention from electrophoretic migration that allows us to investigate the "modification" of the retention process in CEC. This paper presents a methodology for characterization of changes in the retention of neutral and charged sample components, under identical conditions of stationary and mobile phase.     

Liquid chromatographic/mass spectrometric determination of desmedipham and phenmedipham and their metabolites in soil

A simple method is described for the simultaneous determination of residues of 2 carbamate herbicides (phenmedipham and desmedipham) and related metabolites (m-aminophenol, aniline, and m-toluidine) in soil. The analytes are extracted from spiked soils with methanol. The solvent/soil suspension is centrifuged, and the supernatant is directly injected, without any further cleanup, into a reversed-phase liquid chromatography/mass spectrometry apparatus equipped with a TurbolonSpray interface. The method was tested on 5 soils having different physicochemical properties. Recoveries from the soil types, spiked over the range of 50-200 ppb, were essentially quantitative for each analyte. The detection limits of the method are < or = 25 ng/g.     

Liquid chromatographic analysis of prednisolone, prednisone and their 20-reduced metabolites in perfusion media

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20 alpha-dihydro and 20 beta-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6 alpha-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0-1 microgram/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20 alpha-dihydroprednisone) to 8.8% (20 beta-dihydroprednisolone) and 4.1% (20 beta-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20 alpha-dihydroprednisone) to 9.1% (20 beta-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C18 columns which were linked together and maintained at 40 degrees C.     

Reversed-phase high-performance liquid chromatographic, size exclusion chromatographic and polyacrylamide gel electrophoretic studies of glycinin: evidence for molecular species and their association-dissociation

Isolation and purification of glycinin and its molecular species from an Indian soybean variety (JS-335) was achieved using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). Glycinin was found to have two molecular species (glycinin I and II), and only glycinin I underwent reversible dissociation-association system into alpha and beta species. Glycinin I and II were not found to constitute a dissociation-association system. Glycinin II also did not dissociate under varying conditions of time, pH and ionic strength of buffer. Various species so dissociated were isolated, purified and characterized.     

Trace analysis of surfactants using chromatographic and electrophoretic techniques

Because of the widespread use, increased application of new formulations and immense impact on organisms and ecology surfactants are still in the focus of analytical chemistry. The development of methods with higher selectivity and lower detection limits is important to meet the requirements of greater responsibility for health of people and environment. Efficient separation methods, like HPLC, GC and CE, in combination with sensitive detection, like MS, are to be preferred over collective techniques which can suffer from interfering components. A review on trace analysis of ionic and neutral surfactants including sample preparation steps is presented, considering especially those methods which provide information about homologous and isomeric distribution of surfactant mixtures. Examples for the determination of linear alkylbenzene sulfonates in river water by HPLC and CE are discussed to show the capability of these methods for environmental analyses. As future trends increased applications of LC/MS (very high sensitivity) and also of CE (robustness and possibility for rapid method development) can be predicted.     

Trace analysis of surfactants using chromatographic and electrophoretic techniques

Because of the widespread use, increased application of new formulations and immense impact on organisms and ecology surfactants are still in the focus of analytical chemistry. The development of methods with higher selectivity and lower detection limits is important to meet the requirements of greater responsibility for health of people and environment. Efficient separation methods, like HPLC, GC and CE, in combination with sensitive detection, like MS, are to be preferred over collective techniques which can suffer from interfering components. A review on trace analysis of ionic and neutral surfactants including sample preparation steps is presented, considering especially those methods which provide information about homologous and isomeric distribution of surfactant mixtures. Examples for the determination of linear alkylbenzene sulfonates in river water by HPLC and CE are discussed to show the capability of these methods for environmental analyses. As future trends increased applications of LC/MS (very high sensitivity) and also of CE (robustness and possibility for rapid method development) can be predicted. Received: 30 July 1998 / Revised: 28 October 1998 / Accetped: 1 November 1998     

Precolumn derivatization for chromatographic and electrophoretic analyses of carbohydrates

Sensitive detection and high chromatographic resolution are required for analysing the structures of oligosaccharides available in only limited amounts. Precolumn derivatization is one of the most suitable methods for this purpose as it can compensate for the shortcomings of the intrinsic nature of oligosaccharides. Recently published methods of precolumn derivatization are reviewed and advantages and problems are discussed.     

High-performance liquid chromatographic determination of azaarenes and their metabolites in groundwater affected by creosote wood preservatives

Polynuclear azaheterocyclic compounds (azaarenes) are nitrogen-containing analogs of polycyclic aromatic hydrocarbons (PAHs). The nitrogen atom in the ring system causes these compounds to be slightly polar and considerably more water soluble than related PAHs. A method using a solid-surface sorption technique to extract and concentrate azaarenes and their principle metabolites present in groundwater that contains creosote waste is described. Analyte isolation and concentration is accomplished by solid-phase extraction on n-octadecyl cartridges followed by instrumental determination involving high-performance liquid chromatography. Separations and detection are achieved using flexible-walled, wide-bore columns with ultraviolet and fluorescence photometric detectors connected in series. Fluorescence detection alone is insufficient because the fluorescence response produced by two-ring azaarenes is limited. Short wavelength (229 nm) absorbance detection provides improved sensitivity for these compounds and peak rationing for more definitive identification. In this study, oxygen-containing metabolites of quinoline, isoquinoline, and acridine are detected in groundwater from hazardous waste sites in Pensacola, Florida and St. Louis Park, Minnesota. Concentrations ranging from mg/L to ng/L are measured. The dependence of measured octanol-water partition coefficients on pH is discussed in the context of the isolation chemistry. As a direct bacterial degradation product of acridine with a relatively long environmental persistence, 9-acridinone may serve as a biogenic marker signaling creosote contamination of groundwater.     

水溶性电泳漆溶剂中有机成分的气相色谱分析

异丙醇,正丁醇,乙二醇单丁醚均为水溶性电泳漆溶剂中添加的有效成分,其含量的大小影响金属件着漆效果及经济成本,其中乙二醇单丁醚是电泳漆中高沸点溶剂,用作分散剂、湿润剂[1,2]。本文在文献[3,4]的基础上,考察了三种成分的气相色谱分离,确定了测定条件。1　实验部分1 1　仪器与测试条件日本产岛津GC-9A型气相色谱仪带C-R3A型色谱数据处理机。FID为检测器,1 6m×3 2mm玻璃柱,固定相GDX-103(60 80目),气化温度200℃,检测温度250℃,柱温144℃。载气:氮气50ml/min,氢气40ml/min,空气450ml/min。以保留时间定性,采用内标乙二醇单乙醚…     

Liquid-liquid and solid-phase extractions of phenols from virgin olive oil and their separation by chromatographic and electrophoretic methods

The high oxidative stability of virgin olive oil is related to its high monounsaturated/polyunsaturated ratio and to the presence of antioxidant compounds, such as tocopherols and phenols. In this paper, the isolation of phenolic compounds from virgin olive oil, by different methods, was tested and discussed. Particularly liquid-liquid and solid-phase extraction methods were compared, assaying, for the latter, three stationary phases (C8, C18 and Diol) and several elution mixtures. Quantification of phenolic and o-diphenolic substances in the extracts was performed by the traditional Folin-Ciocalteau method and the sodium molybdate reaction, respectively. Furthermore, the quantification of phenolic compounds in the extracts and in a standard mixture was carried out both with diode array and mass spectrometric detection and capillary zone electrophoresis.     

Effect of some parameters on the liquid chromatographic separation of biogenic amines and their metabolites

The liquid chromatographic separation of some biogenic amines and their metabolites with a reversed-phase ion-pair system has been optimized. The effects of the nature of the stationary phase, the column temperature, the pH, and the ion-pair reagent concentration of the mobile phase have been investigated. The most striking result is the influence of the column temperature on selectivity.     

Collaborative studies of zeta-potential measurements and electrophoretic measurements using reference sample

Simultaneous measurements of zeta-potential for two standard latex suspensions were carried out so as to assess the reliability of each of these measurement techniques and find means for their improvement. Furthermore, syntheses of a reference particle dispersion stabilized sterically in an aqueous medium without any electrostatic effects and measurements of zeta-potential using the reference dispersion as a standard were performed under various experimental conditions. It became apparent that the dense adsorption layer of hydroxypropylcellulose (HPC), with a lower critical solution temperature (LCST), formed on latex particles with a low surface charge density at temperatures higher than the LCST, plays a role in completely shielding the electrostatic effect arising from the surface charge on the bare particles. Such reference particles with zero zeta-potential allow us to determine the electrophoretic mobility of unknown samples at the one-half depth in the electrophoretic cell by subtracting the mobility of the reference sample at the same level. Furthermore, the zeta-potential of the cell wall can be easily determined from the mobility of the reference sample, because the apparent velocity profile of the reference sample indicates the liquid flow velocity in the cell.     

Liquid chromatographic determination of sulindac and metabolites in serum

An improved liquid chromatographic procedure is described for the quantitative determination of sulindac, sulindac sulfone, and sulindac sulfide from serum. The procedure makes use of acetonitrile extraction of the compounds of interest from acidified serum samples. Under these conditions extraction efficiencies in the 85 percent range are obtained for each of the compounds. The liquid chromatographic separation of the compounds of interest and the internal standard (indomethacin) is accomplished in an isocratic elution procedure using a nitrile (CN) stationary phase. The HPLC separation procedure is completed in less than 10 minutes, giving excellent resolution and peak shape.     

High-pressure liquid chromatographic determination of some 1,4-benzodiazepines and their metabolites in biological fluids: a review

In recent years the need for rapid, sensitive and specific assays for benzodiazepines has resulted in the publication of a number of high-pressure liquid chromatographic (HPLC) methods for their determination. This paper reviews the methods available to date for the determination of chlordiazepoxide, clonazepam, diazepam, flurazepam, lorazepam, nitrazepam, oxazepam and their metabolites in biological fluids.