Effects of Counter Ions in Ion-Pair Liquid Chromatography of Hydrophobic Amines on Non-Polar Bonded Phases

Abstract

The retention behaviour of alprenolol and related hydrophobic amines in ion-pair adsorption systems has been examined with particular emphasis on the influence of different mono-and divalent counter ions (dihydrogenphosphate, bromide, perchlorate, dimethylcyclohexyl sulphate, sulphate and ethylenediaminetetraacetate). N,N-dimethyloctylamine (DMOA) and 1-pentanol were used as modifiers in the aqueous eluent and LiChrosorb RP-8 as stationary phase.

The retention is evaluated according to a two-site adsorption model and equilibrium constants are given for ion pair adsorption of DMOA. The retention of alprenolol has been evaluated in terms of ion exchange with DMOA and the ion-exchange constants are shown to be of the same magnitude and independent of the nature of the counter ion used. The ion-pair adsorption and the ion-exchange approaches are analogous expressions for the distribution process governing the retention.     

Retention in Reversed-Phase Ion-Pair Chromatography of Amines On Alkyl-Bonded Phases

Abstract

Reversed phase ion-pair chromatography of phenethylamine derivatives (noradrenaline, adrenaline, dopamine, synephrine, tyramine and pholedrine) and lower alkylamines has been performed with octyl sulfate as counter ion in an aqueous eluent with a low content of 1-pentanol. LiChrosorb RP-18 was used as the solid phase. The retention of lithium and potassium in the system has also been studied.

On the basis of adsorption and retention studies a model for the chromatographic behaviour of the amines is proposed that includes interaction with two sites with different binding ability in the stationary phase. The adsorption capacity of the sites has been calculated as well as adsorption constants for the octyl sulfate ion pairs.     

The TcO4-binding to human serum albumin --the stoichiometry and characteristics of the binding equilibrium.

The stoichiometry and the characteristics of the TcO4-binding equilibrium to human serum albumin were investigated by the use of 99TcO4-. Based on the Scatchard plots, the number of binding sites and the association constants were obtained at pH 7.38,6.04, 5.10, 4.18, 2.80 and 0.65, respectively. From the parameters at pH 7.38, it was estimated that 64% of TcO4- added was bounded to human serum albumin under physiological condition. Variance in the values at pH 7.38, 6.04 and 5.10 shows that these bindings are stabilized by electrostatic forces. Below pH 4.18, the number of binding sites increased and the association constants constants diminished. These phenomena may be attributed to the conformation change of human serum albumin.     

Adsorption of Bilirubin-Albumin Complex on the Surface of Highly Dispersed Silica

The interaction of bilirubin with albumin in aqueous buffer solutions is studied and the stability constants of the formed complex are determined. The adsorption of bilirubin and albumin from their individual and mixed solutions on the surface of highly dispersed silica is studied as a function of pH and albumin concentration. It is shown that the character of the adsorption of bilirubin from solutions containing albumin changes significantly compared to the adsorption from an individual solution.     

Study of the complexation of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene with pyridine carboxylic acids by RP HPLC and molecular modelling

Host–guest complexation process of 5,17-bis-(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene with pyridine carboxylic acids by RP HPLC method (mobile phase – MeCN/H₂O, 86/14 by volume, LiChrosorb RP 18, UV detector, λ = 254 nm) had been studied. The binding constants and Gibbs free energies of the complexes 5,17-bis(N-tolyliminomethyl)-25,27-dipropoxycalix[4]arene with the pyridine carboxylic acids are within 584 to 1914 M^? 1 and ? 15.76 to ? 18.69 kJ/mol, respectively. It was shown by the molecular modelling that the complexes are stabilised by hydrogen bonds between carboxylic groups of the acids and nitrogen atoms of imino groups at the upper rim or oxygen atoms of the hydroxyl groups at the lower rim of the macrocycle. Linear dependence of the binding constants from the acid lipophilicity log P indicates the role of solvophobic interactions during the complexation process.     

Studies on Interactions of Antibiotics with Serum Albumin by Surface Plasmon Resonance Biosensor

Introduction Humanserumalbumin(HSA)isawell known transportproteinforavarietyofmoleculesandions[1].Thebindingofadrugtoserumalbuminhasimportant pharmacokineticconsequencesbecauseitinfluences distribution,excretionandpharmacologicaleffectsof thedruginthebody…     

The interaction of cationic and anionic porphyrins with the bovine serum albumin in borate buffer

The interaction of water-soluble porphyrins with the bovine serum albumin in borate buffer at pH 8.6 has been studied. The localization of porphyrins in the protein globule has been determined. It was established that the native conformation of albumin upon binding with the porphyrins is preserved, however, the anionic porphyrins are exhibit wedging effect on the albumin domains. The binding constants were obtained from fluorescence spectroscopy data.     

Binding of benzodiazepine drugs to bovine serum albumin: a second derivative spectrophotometric study

The binding constants (K values) of three benzodiazepine drugs to bovine serum albumin were determined by a second derivative spectrophotometric method. Despite the sample and reference samples were prepared in the same way to maintain the same albumin content in each sample and reference pair, the absorption spectra show that the baseline compensation was incomplete because of the strong background signals caused by bovine serum albumin. Accordingly, further quantitative spectral information could not be obtained from these absorption spectra. On the other hand, the calculated second derivative spectra clearly show isosbestic points indicating the complete removal of the residual background signal effects. Using the derivative intensity differences (ΔD values) of the studied benzodiazepine drugs before and after the addition of albumin, the binding constants were calculated and obtained with R.S.D. of less than 8%. The interactions of drugs with bovine serum albumin were investigated using Scatchard's plot. In addition, the consistency between the fractions of bound benzodiazepine calculated from the obtained K values and the experimental values were established. The results indicate that the second derivative method can be advantageously applicable to the determination of binding constants of drugs to serum albumin without prior separation. Moreover, the validity of the proposed method was confirmed.     

Association Constants in the Bovine Serum Albumin/Human Serum Albumin–Tween 20 System in Aqueous Solutions

The association constants in the bovine serum albumin/human serum albumin–Tween 20 system are determined in aqueous solutions at pH 3.5–8.0 by means of nonpolarized fluorescence and protein fluorescence quenching. It is found that the most efficient association of serum albumin molecules with Tween 20 micelles occurs at pH 5.0 near the isoelectric points of proteins.     

Binding constant determination of drugs toward subdomain IIIA of human serum albumin by near-infrared dye-displacement capillary electrophoresis

Drug binding to serum albumin influences several important pharmacological properties such as toxicity, solubility, activity, distribution, and excretion. It is therefore of interest to have methodologies that allow for the determination of drug-albumin affinity constants while simultaneously providing information on the location of the drug binding site. In the present work we describe a method for the determination of binding constants of drugs known to bind to subdomain IIIA of serum albumin. Drugs used in the study were ketoprofen, ibuprofen, quinidine, naproxen, imipramine, and clofibrate. Binding constants of the drugs were determined by near-infrared dye-displacement capillary electrophoresis. The dye-displacement technique uses a competitive-type interaction between the drug of interest and a dye probe to arrive at a binding constant. A heptamethine cyanine dye was used as a probe for drug binding at subdomain IIIA of serum albumin. The utility of the dye as a noncovalent label for serum albumin was investigated. Additionally, the ability of the method to illustrate enantioselective binding is shown. The dye displacement technique has advantages over current electrophoresis-based techniques in that it is faster and uses less reagent.     

Dependence of the constants of binding for nanomarkers of the fluorescein family with human serum albumin on Ph

The effective constants of binding for probes of the fluorescein family (fluorescein and its halogen derivatives, eosin and erythrosin) with human serum albumin (HSA) at different pH were determined. It was found that the introduction of halogen groups into the structural formula of fluorescein changes the character of the dependence on pH of the effective constant of binding of a nanomarker with HSA: the nonlinear dependence of the effective constants of binding with its protein typical for fluorescein, eosin, and erythrosine is characterized by an almost linear reduction with increasing pH dependence of the effective constants of their binding with human serum albumin. It was shown that the presence of more electronegative atoms in the structural formula of nanomarker leads to decrease of values of effective constants of nanomarker binding with HSA.     

Systematic approach to the determination of cephalosporins in biological fluids by reversed-phase liquid chromatography

The chromatographic behaviour of some cephalosporins as a function of pH and ionic strength of the mobile phase was studied on 10-microns LiChrosorb RP-18. Acidic cephalosporins were retained longest in their neutral form with an acidic eluent. Amphoteric cephalosporins were retained longest in their protonated form with an acidic eluent of low ionic strength. Cefotiam was retained longer with an alkaline mobile phase. LiChrosorb RP-18, Nucleosil C18 and muBondapak C18 gave rise to different selectivities when an acidic eluent, methanol-water (25:75) containing 0.2% of 1.8 M H2SO4 was used. This may be related to interactions with residual silanol groups. The studied cephalosporins (with the exception of cefotiam and cefsulodin) were separated from compounds present in biological fluids on 5-microns LiChrosorb RP-18 using the mobile phase 0.2% of 1.8 M H2SO4 in a mixture of methanol and water with various methanol contents. The determination of cefotiam in biological fluids was performed with an alkaline mobile phase. The preparation of the sample was simple and rapid: precipitation of plasma proteins or dilution of urine. The method was applied to the determination of ceftizoxime in human plasma and urine. Concentrations down to 0.2 micrograms/ml of plasma and 25 micrograms/ml of urine could be determined with good reproducibility and accuracy.     

黄芩类药物与人血清白蛋白相互作用的研究

本文利用荧光猝灭技术和紫外可见分光光度法研究了黄芩苷和汉黄芩苷与人血清白蛋白的结合性质,由药物对血清白蛋白的荧光猝灭作用求出了其结合常数. 根据热力学参数,确定了结合力的性质. 并进一步研究了Zn2+, Mg2+, Al2+,和 Cu2+存在时对结合性质的影响. 不仅对于揭示体内药物动力学问题,指导临床合理用药具有一定意义,而且对于进行药物分子设计、开发新药等也具有重要指导意义.     

芦荟大黄素与血清白蛋白的相互作用

利用荧光光度法研究了中药有效成分芦荟大黄素与血清白蛋白的相互作用机制 ,求得两者的形成常数 ,讨论了某些金属离子对其形成常数的影响 ,并根据热力学常数确定了它们之间的作用力类型 ,利用F rster非辐射能量转移技术求出了两者的结合位置。     

Separation and determination of inorganic anions by means of ion-pair chromatography

Bromide, iodide, bromate, iodate, thiocyanate, nitrite and nitrate can be separated by means of ion-pair chromatography on a reversed-phase column (LiChrosorb RP2), using the tricaprylylmethylammonium ion (Aliquat-336) as counter ion in an acetonitrile-water mobile phase. The elution is monitored with an ultraviolet detector at 205 and 226 nm. The detection limits are in the low nanogram range.     

Binding constant measurement by hyper-Rayleigh scattering: bilirubin-human serum albumin binding as a case study

In this paper, a new application of the hyper-Rayleigh scattering technique in determining multiple binding constants of a small molecule like bilirubin to a macromolecule like the protein human serum albumin has been demonstrated. Human serum albumin has two binding sites for bilirubin, and the binding constants have been measured by carrying out a second harmonic titration of the protein against bilirubin and vice versa. The measured binding constants K(1) = 1.5 +/- 0.43 x 10(7) M(-1) and K(2) = 1.01 +/- 0.16 x 10(6) M(-1) agree well with the reported values obtained by other methods.     

Liquid chromatographic retention of homologous alkanes in the two states of C18-bonded reversed phase silicas

Summary LiChrosorb Si100 densely grafted with octadecylmonofunctional reagents and the similar commerical LiChrosorb RP 18 have been studied in RP-HPLC, with water-methanol mobile phases at different temperatures. They exhibit a phase transition revealing two different states of bonded film as we have previously shown on densely grafted C18 or C22 macroporous silicas.The measurement of the capacity factors of the alkane homologous series indicated a discontinuity in the plot (logK, N) at a critical number whose value is dependent on temperature. Two different forms of these curves can be observed above and below the transition, revealing the influence of bonded film state on the retention mechanism.     

Analytical affinity chromatography. II. Rate theory and the measurement of biological binding kinetics

Affinity chromatography can be used to measure equilibrium constants and kinetics of biological interactions. The local-equilibrium theory presented in the preceding paper is extended to include mass transfer and kinetic effects. Solutions for both zonal and frontal elution are presented. For highly nonlinear isotherms, the frontal elution method is preferred. Experiments with bovine serum albumin binding to immobilized Reactive Blue show that the binding kinetics inside the porous gel are several orders of magnitude slower than typical biological binding reactions in solution. The temperature dependence of the kinetic constants indicate that the binding may still be diffusion-controlled.     

Ion-interaction chromatography of dithiocarbamates on βBondapak CN and LiChrosorb RP-8 stationary phases in the presence of tetraalkylammonium salts

Dithiocarbamates are examined by high-performance liquid chromatography on βBondapak CN and LiChrosorb RP-8 bonded phases using ion-interaction with tetraalkylammonium salts. The effects of the tetraalkylammonium cation and its inorganic co-anion on solute retention are considered. The response for sodium diethyldithiocarbamate is linear over the range 1–500 μg ml^?1, with a detection limit of 0.1 μ ml^?.     

Characterization of the interaction between a new merocyanine dye and bovine serum albumin

A new merocyanine dye was synthesized, and its acidity constant was determined by spectrophotometric and chemometrics methods. The interactions of the new cyanine dye with bovine serum albumin (BSA) have been studied by fluorescence and UV absorption spectroscopy at pH 7.40. A visual color change from red to blue was observed by addition of BSA to aqueous solution of the dye. The quenching constants and binding parameters (binding constants and number of binding sites) were determined at different temperatures. The calculated thermodynamic parameters confirmed that the binding reaction is mainly entropy-driven, whereas electrostatic interaction plays major role in the reaction. The displacement experiment confirmed binding of the dye to the subdomain IIA (site 1) of albumin. Moreover, synchronous fluorescence spectroscopy studies revealed the dye induces some local conformational change in BSA. The binding distance, r, between donor (serum albumin) and acceptor (dye) was obtained according to Förster’s theory.