Weak Cation Exchange Resin Research Articles

PH effects on non-ideal protein size-exclusion chromatography on Superose 6

The retention behavior of five globular proteins (ribonuclease, lysozyme, β-lactoglobulin, serum albumin, γ-globulin) was determined on Superose 6 in low ionic strength (0.02–0.04 M) mobile phases of high, intermediate and low pH (10.0, 7.0, 4.3). Quantitative assessments of attractive or repulsive solute-packing interactions were made by comparing the chromatographic partition coefficient to the value obtained for non-interacting spherical solutes on the same column. The dependence of this “non-ideal” adsorption or exclusion on net protein charge is complex, but not very sensitive to protein type. The results suggests that on size-exclusion chromatography packings that behave as weak cation-exchange resins the electrostatic solute-packing interaction is not governed by a highly localized set of a few charged sites.

DETERMINATION OF ION EXCHANGE EQUILIBRIUM CONSTANTS FOR WEAKLY DISSOCIATING ION EXCHANGE RESINS

ABSTRACT In this paper the general progressive neutralization technique has been developled to evaluate the ion exchange equilibrium constant for weakly dissociating ion exchange resin. In substance, this technique is the potentiometric titration procedure of the weak acid resin weak base resin ) with the mixture solutions containing various ratios of base and salt, M(OH)n — MpXn( acid and salt, HpX—MpXn), where Mn+is metal cation, Xp−anion. According this technique, the rational equilibrium constants of the ion exchange reactions for the weak acid cation exchange resin D725 and for the weak base anion exchange resin D705 have been determined. This technique has proved useful in the determination of rational equilibrium constant of ion exchange reaction for weakly dissociating ion exchange resin.

DETERMINATION OF ION EXCHANGE EQUILIBRIUM CONSTANTS FOR WEAKLY DISSOCIATING ION EXCHANGE RESINS

ABSTRACT In this paper the general progressive neutralization technique has been developled to evaluate the ion exchange equilibrium constant for weakly dissociating ion exchange resin. In substance, this technique is the potentiometric titration procedure of the weak acid resin (weak base resin ) with the mixture solutions containing various ratios of base and salt, M(OH)n—MpXB(( acid and salt, HpX-MpX^), where M°+is metal cation, X^anion. According this technique, the rational equilibrium constants of the ion exchange reactions for the weak acid cation exchange resin D725 and for the weak base anion exchange resin D705 have been determined. This technique has proved useful in the determination of rational equilibrium constant of ion exchange reaction for weakly dissociating ion exchange resin.

Complexity of sea urchin embryo nuclear proteins that contain basic domains.

We describe a quantitative two-dimensional gel electrophoretic analysis of nuclear extract from 24-hr sea urchin embryos. The extract was fractionated by using a weak cation-exchange resin, and eight known DNA-binding proteins were shown to be entirely included in a salt eluate that releases proteins containing basic domains. This fraction and a lower-salt fraction containing the majority of the protein species were mapped two-dimensionally by using new algorithms that permit reproducible spot identification, storage of intensity and map-position data, and subtractive comparison of one pattern with respect to another. By reference to a previously characterized DNA-binding factor, spot intensity could be interpreted in terms of the number of molecules per embryo nucleus. A map was constructed displaying all nuclear proteins containing basic domains that are present within the concentration range per nucleus of a set of known DNA-binding factors of the sea urchin embryo. The map includes 265 spots that fulfill both of these criteria, probably representing about 100 different protein species.

Optimization of the sensitivity of high-performance liquid chromatography in the detection of heterocyclic aromatic amine mutagens

Solid-phase extraction with the weak cation-exchange resin Fractogel TSK CM650(S) (TSK CM) was used to optimize the sensitivity and chromatographic resolution by HPLC of the mutagenic heterocyclic aromatic amines MeIQx, 4,8-DiMeIQx, IQ, MeIQ, PhIP, Trp-P-1, Trp-P-2, amino-α-carboline and the co-mutagens norharman and harman. The clean-up using cation exchange was applied after purification on Extrelut and propylsulphonic acid—silica gel cartridges (PRS tandem purification). Only the clean-up with TSK CM enabled low detection limits of the mutagenic compounds to be achieved (in the order of ng heterocyclic amines/g experimentally overheated process-flavour sample).

Weak acidic cation exchange resins for treatment of solutions of high salt concentration

Weak acidic cation exchange resins for treatment of solutions of high salt concentration

A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland

A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland.

Liquid chromatographic determination of free serotonin in human plasma.

The complex background of interfering substances makes plasma pretreatment necessary before performing liquid-chromatographic quantitation of circulating serotonin. We compared two isolation procedures (extraction into butanol and isolation on a weak cation-exchange resin) before chromatography for measuring free serotonin from human plasma. Sensitivity, specificity and recovery were all evaluated. The cation-exchange resin yielded chromatograms with no interfering peaks, was cheaper and took less processing time than butanol extraction. We conclude that the clean-up of human plasma by a cation-exchange resin is suitable and accurate for routine liquid-chromatographic determination of circulating serotonin.

A Rapid Preparative Procedure for the Treatment of Free Urinary Catecholamines

Abstract We report a preparative and liquid chromatographic method for the determination of free urinary catecholamines in urine. An extraction column device (1) was used to isolate norepinephrine, epinephrine on a weak cation exchange resin (Bio-Rex 70) using a table centrifuge forcing the sample through the resin material. The preparation time for a 8 sample size was 50 minutes. The extraction recovery was 80.33% and 80.89% for norepinephrine (NE) and epinephrine (E). The curve was linear up to 400 ug/24 H. The within-run CV was 3.84% and 4.22% for NE and E; the between-run CV was 5.58% and 6.27% respectively for NE and E. The mobile phase consisted of a 90% phosphate buffer (0.05M NA2HP04) and 10% methanol adjusted to pH. 3.8. Chromatographically well separated on a C-8 reverse phase column, the eluent was monitored with a coulometric electrochemical detector set up at 0.40 V.

Analysis of urinary catecholamines by high-performance liquid chromatography in the presence of labetalol metabolites

Common sample preparation methods for catecholamines lead to contamination with metabolites of labetalol, an anti-hypertensive drug. When the extracts are analyzed by cation-exchange high-performance liquid chromatography, these metabolites are separated from the catecholamines, but their strong retention lengthens the analysis time. A procedure has been developed for complete removal of these drug metabolites from acidified urine by the use of XAD-4 resin. Loss of catecholamines is monitored by an internal standard. This pretreatment can be combined with extraction by weak cation-exchange resin and borate elution to simplify catecholamine analysis for patients receiving labetalol.

Determination of histamine concentrations in plasma by liquid chromatography/electrochemistry.

Histamine was extracted from deproteinized plasma with Amberlite CG 50 weak cation-exchange resin (analytical recovery of [3H]histamine, 60.5%). The eluate was evaporated and histamine in the redissolved sample was condensed with o-phthalaldehyde and 2-mercaptoethanol at pH 11.5. This adduct was separated by liquid chromatography under isocratic conditions and oxidized on a glassy carbon electrode at +0.4 V for electrochemical detection. 3-Methylhistamine was used as internal standard. As little as 0.45 pmol of standard histamine condensate was detected. The histamine concentration in 88 human plasma samples appeared to be normally distributed; its mean value was 7.20 (SD 2.61) nmol/L. Authentic and extracted histamine produced similar hydrodynamic voltammograms, and exogenous and endogenous histamine gave identical chromatographic characteristics with different mobile phases or different chromatographic columns. Standard and extracted histamine had similar degradation rates when samples were incubated with diamine oxidase (EC 1.4.3.6). Analytical recovery of known amounts of histamine added to pooled plasma was 97.7% (SD 22.3%). The inter- and intra-assay CVs for histamine determinations were 9.0% and 8.6%, respectively.

Normal reference intervals for free catecholamines and their acid metabolites in 24-h urines from children, as determined by liquid chromatography with amperometric detection.

We used two methods of reversed-phase liquid chromatography with amperometric detection, one for assay of unconjugated norepinephrine, epinephrine, and dopamine in urine, the other for determination of unconjugated vanillylmandelic acid and homovanillic acid. The catecholamines were extracted from urine by passage through a weak-cation exchange resin, then through alumina. Vanillylmandelic acid and homovanillic acid were isolated from urine by a two-step solvent-extraction procedure. The between-day CV was 5% for norepinephrine, 3% for epinephrine, 3% for dopamine, 4.5% for vanillylmandelic acid, and 6.4% for homovanillic acid. Reference ranges for these analytes in 76 children, ages 3-16 years, are reported.

Quantification of tryptamine in brain using high-performance liquid chromatography

The concentration of endogenously formed tryptamine in central nervous system tissue was determined after extraction into ethyl acetate, purification on a weak cation-exchange resin and analysis using high-performance liquid chromatography with fluorometric detection. Final chromatographic separation of this indoleamine was achieved using a μBondapak C 18 reversed-phase column under isocratic conditions. Using this method, the concentration of tryptamine in the whole brain of normal rats was found to be 0.60 ± 0.06 ng/g of tissue, while pretreatment with dl-p-chlorophenylalanine, trnylcypromine and l-tryptophan increased the concentration to 96.7 ± 21.9 ng/g.

Bioanalysis and Disposition of α-Fluoromethylhistidine, a New Histidine Decarboxylase Inhibitor

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of α-fluoromethylhistidine (α-FMH) in human biological samples. The plasma assay required isolation of the drug using a weak cation-exchange resin prior to HPLC analysis with UV detection. The urine assay employed postcolumn derivatization with o-phthalaldehyde (without a thiol) and fluorescence detection. The extent of metabolism of α-FMH in humans was studied in four healthy volunteers using tritium-labeled material. No significant differences in the plasma and urine concentrations of radioactivity and unchanged drug were detected. In addition, the radiochromatograms of selected urine samples revealed a single peak with a retention time corresponding to the unchanged drug. The evidence presented suggests negligible biotransformation of α-FMH in humans.

Preparation of urine samples for liquid-chromatographic determination of catecholamines: bonded-phase phenylboronic acid, cation-exchange resin, and alumina adsorbents compared.

We compared results for the liquid-chromatographic determination of free norepinephrine and epinephrine in urine after purifying the catechols by the following methods: (a) acid-washed alumina, (b) weak cation-exchange resin (WCX), (c) a combination of weak cation-exchange resin followed by alumina (WCX-alumina), and (d) commercially available phenylboronic acid adsorbent. We evaluated analytical specificity, sensitivity, recovery, and turnaround time. The WCX-alumina combination produced the most sensitive and specific chromatograms for urinary catecholamines; the other methods took less processing time. Neither WCX nor alumina alone was suitable for routine work because of chromatographic interferences in a significant proportion of urines. The phenylboronic acid method is adequately sensitive and specific for norepinephrine and epinephrine, and samples can be assayed faster. Thus it provides a compromise between the high analytical performance of the WCX-alumina method and the speed of the WCX and alumina methods.

Ammonia Volatilization from Surface‐Applied Urea: Effect of Hydrogen Ion Buffering Capacity

AbstractThe influence of soil pH and soil cation exchange capacity on ammonia volatilization from surface applied ammoniacal nitrogen has been reported in previous studies. Since the hydrolysis of ureacontaining N fertilizers causes an increase in alkalinity, a soil's inherent H+ buffering capacity (defined as the soil's total acidity, comprised of exchangeable acidity plus non‐exchangeable, titratable acidity), should exert a dominant influence on the maximum soil pH at the site of urea fertilizer application. The objective of this study was to demonstrate the importance of a soil's H+ buffering capacity in affecting NH3 volatilization from surface‐applied urea. The H+ buffering capacity of two soils was increased by adding hydroxy‐Al polymers to one soil, and weak and strong acid cation exchange resins to the other soil. Care was taken to keep cation exchange capacity and initial pH close to the same on amended and unamended (control) soils. Urea was surface‐applied to amended and unamended soils and ammonia volatilization and soil surface pH were measured. The increase of H+ buffering capacity of soils was found to reduce soil surface pH and NH3 volatilization after application of urea. It is concluded from this work that H+ buffering capacity of a soil is a better indicator of NH3 loss potential than a soil's initial pH.

Gas chromatographic analysis of endogenous catecholamines, phenolic amines and derived isoquinolines using short glass capillary columns and electron-capture detection

A gas chromatographic method is described for the concomitant separation and analysis of catecholamines, catecholamine or 3,4-dihydroxyphenylethylamine condensation products (tetrahydroisoquinolines), and their isomeric mono-O-methyl (phenolic) metabolites which may be present in neuronal tissues, utilizing short glass capillary columns and electroncapture detection. Isomeric phenolic amines that were not generally separable with conventional-packed gas chromatographic columns were rapidly resolved on the capillary system, and with their catecholamine or catechol isoquinoline precursors, quantitated with high sensitivity (0.25–7.0 pg) and reproducibility. Key steps in the approach with tissues include initial amine isolation with a weak cation-exchange resin (BioRex-70), fluoracyl derivative formation, and brief washing of the derivatives with ammonium phosphate buffer (pH 5.8)just prior to capillary analysis; overall recoveries of amines or alkaloids added to rat brain homogenates ranged from 79% to 89%. Application of the method is demonstrated in an assay of endogenous dopamine in rat corpus striatum and hypothalamus. This new procedure should complement and in some instances may be preferred over liquid chromatographic assays for catecholic and phenolic amines and isoquinolines, and ought to be applicable to mass spectrometric detectors as well.

Resin-Contact-Time Method for Determination of Protein-Bound Calcium in Milk and Model Systems

A theory for exchange of calcium between milk or model milk-protein system and an appropriately buffered weak cation-exchange resin was developed which could be used for determination of protein-bound or colloidal calcium. With suitable preconditioning of the resin, the resin-contact-time method yielded results for ionic and protein-bound calcium in a model β-casein system which not only conformed to the proposed theory but also agreed with those by the murexide method of Sundararajan and Whitney. β-Casein dispersions (3%) in calcium-potassium chloride solutions at pH 7.0, Γ/2 .14, and 2°C bound calcium as if there were 11.20 binding sites per molecule, each with an intrinsic association constant of 76.62 liters per mole. Data from skim milk samples at 23°C and 2°C by the resin-contact-time method also were consistent with the proposed theory and yielded protein-bound or colloidal calcium in reasonable agreement with reported results.

Fluorometric determination of peripheral tissue histamine levels using Bio-Rex 70.

Bio-Rex 70, a weak cation exchange resin, has been incorporated into a column chromatographic extraction procedure for the determination of peripheral tissue levels of histamine. The extracted histamine is quantified fluorometrically after condensation with o-phthaldialdehyde (OPT). The sensitivity of the procedure allows for the chromatographic extraction, using Bio-Rex 70, of 10 ng histamine to give fluorescence twice that of the blank. Because this extraction procedure does not separate histamine from spermidine it cannot be applied in determining levels of brain histamine. However, because spermidine in concentrations of up to 400 times that of histamine does not interfere with OPT-histamine fluorescence, the histamine levels of all peripheral tissues (including nerves) and fluids may be confidently examined. The relative simplicity of the procedure allows for the determination of 30 tissue samples within a working day. The method has been applied to determine peripheral tissue levels of histamine in naïve Wistar rats and the effects of compound 48/80 on these levels in several selected tissues have been investigated. Compound 48/80 decreased the levels of histamine in the heart, kidney and ileum, whereas these levels were initially elevated in the liver and lung without subsequent depletion. Since the reliability of existing histamine extraction procedures is questionable under certain conditions, it is suggested that the use of Bio-Rex 70 is a valuable addition in evaluating the possible physiological role(s) of tissue histamine.

A modified method for the isolation and determination of brain histamine using the Bio-Rex 70.

Bio-Rex 70, a weak cation exchange resin, has been used to specifically isolate histamine from rat brain tissue. This method compares favourably to other extraction procedures with respect to selectivity, reproducibility and time taken to perform the procedure. However, because it combines the optimum of these properties, it appears more adaptable for routine laboratory use. Following isolation, histamine is quantified fluorometrically after condensation with o-phthaldialdehyde (OPT). The sensitivity of the procedure allows for the chromatographic isolation, using Bio-Rex 70, of 12.5 ng histamine to give fluorescence twice that of blank. In addition, the use of Bio-Rex 70 enables the selective separation of histamine from fluorescent contaminants such as spermidine. The stability and the reproducibility of the adsorption and elution characteristics of Bio-Rex 70 enables the determination of 30 brain samples in a working day. This method has been applied to determine whole brain and regional brain levels of histamine in control and L-histidine-treated rats. The whole brain level of histamine, which was 50 ng/g, was increased by L-histidine and the highest concentration of histamine was found in the hypothalamus. Since the reliability of existing histamine extraction procedures is questionable, under certain conditions, it is suggested that the use of Bio-Rex 70 is a valuable addition in evaluating the possible physiological role of brain histamine.