Eluent Solution Research Articles

Removal of chromium from aqueous solutions by diatomite treated with microemulsion

In order to evaluate the sorption of heavy metals, a crude diatomite was impregnated with a microemulsion which showed remarkable increase in chromium sorption capacity as compared to untreated diatomite. Samples with two different granulometries were investigated, both yielding practically complete adsorption. The adsorption process is pH dependent and the best results for the initial Cr (III) concentration of 1.5 g/L were obtained at pH 2.95. The effect of the concentration of the chromium synthetic solution was also investigated. The adsorption isotherms were obtained (30, 40 and 50°C) and the Freundlich and Langmuir models were used to determine the adsorption capacity of the adsorbent. Following the adsorption step, a desorption process was carried out using several eluant solutions. The best results were obtained using hydrochloric acid (100%) as eluant.

On-line deproteinization by adsorption of proteins on a polyethylene powder pre-column for the determination of Na, K, Mg and Ca in human serum by high-performance liquid chromatography

Deproteinization of human serum was carried out on-line using a polyethylene powder cartridge as pre-column. The serum sample, after dilution, was injected into the chromatograph, and when passing through the cartridge the proteins were adsorbed by the polyethylene. A protein-free eluate was carried to the analytical column while the pre-column was washed with methanol and water to elute the adsorbed proteins by changing the pump channels. After washing, the pre-column was conditioned with the eluent to receive the next sample. Deproteinization was evaluated off- and on-line using pooled serum, and testing the protein residue after passing through a polyethylene cartridge with the Comassie Brilliant Blue reaction. Parameters such as serum volume, eluent and washing solution were investigated. Sodium, potassium, magnesium and calcium determinations were performed by high-performance liquid chromatography with conductimetric detection. The proposed method is suitable for the determination of these cations in serum samples without further treatments. The total analysis time was about 20 min and a linear range from 0.3 to 1.2 mg/l for sodium, potassium, magnesium and calcium was observed. The method was characterized by a precision of about 95% and recoveries from spiked samples were between 96 and 102%. The results for serum samples analysed by the proposed method were compared with photometry and atomic absorption spectrometry.

A single calibration graph for the direct determination of ascorbic and dehydroascorbic acids by electrogenerated luminescence based on Ru(bpy)(3)2+ in aqueous solution

Ascorbic (H2A) and dehydroascorbic (DA) acids were for the first time directly determined in a single chromatographic run by means of the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)2+) based electrogenerated chemiluminescence (ECL) detection. For the first time, it was demonstrated that DA, a nonelectroactive compound, is ECL active and is responsible for the ECL behavior of H2A. This fact, together with the lack of a DA standard, suggested the use of a calibration graph obtained for H2A, for determining both analytes. The proven ECL activity of DA, together with literature data relative to the standard redox potentials of the different species coming from H2A, led to a reconsideration of the proposed ECL reaction mechanism for H2A. The role of the OH- ion in the reaction mechanism of the two analytes appeared to be crucial. H2A and DA could be separated by a suitable C18-reversed-phase HPLC column using an aqueous 30 mM H3PO4 solution as the mobile phase. The optimal ECL response was achieved by polarizing the working electrode at 1.150 Vvs SCE (standard calomel electrode) (oxidation diffusion limiting potential for both H2A and Ru(bpy)(3)2+). The Ru(bpy)(3)2+ solution, at pH 10 for carbonate buffer, was mixed to the eluent solution in a postcolumn system, obtaining, still at pH 10, the final 0.25 mM Ru(bpy)(3)2+ concentration. The detection limit found for the two analytes was 1 x 10(-7) M. The method was successfully applied to the determination of the analytes in a commercially available orange fruit juice.

Use of molecularly imprinted solid-phase extraction for the selective clean-up of clenbuterol from calf urine

A feasibility study was performed in order to study the possibilities in using molecularly imprinted polymers (MIPs) as sorbent material in solid-phase extraction (MISPE) for clean-up of clenbuterol from urine. A binding study of clenbuterol in several solvents was performed on a clenbuterol imprinted polymer as well as on a blank polymer. These binding experiments were used to find suitable loading, washing and elution solvents for the MISPE procedure. Extraction of clenbuterol from calf urine was performed by directly loading a 10-ml urine sample onto the MIP column. Thereafter the column was washed with 10 ml of acetonitrile containing 1% acetic acid, and finally clenbuterol was eluted with 6 ml of methanol containing 10% acetic acid. A recovery of 65% was obtained. This recovery could be increased up to 75% if a sample volume of 1 ml was used or up to 100% if urine was freeze-dried and the residue was dissolved in acetonitrile and spiked with clenbuterol prior to analysis. Chromatograms of the wash and eluate solutions show an efficient clean-up, which supports the potential of MISPE for clean-up of trace amounts of clenbuterol from calf urine.

Liquid chromatographic determination of aniline in table-top sweeteners based on pre-column derivatization with 1,2-naphthoquinone-4-sulfonate

A liquid chromatographic method for the determination of aniline in cyclamate sweeteners based on a pre-column derivatization with 1,2-naphthoquinone-4-sulfonate (NQS) is proposed. Aniline traces were extracted from the cyclamate samples using dichloromethane. After solvent evaporation, the dry residue was derivatized with NQS at pH 9.5 and 85°C for 1 min. The aniline derivative, which was extracted from the reacting mixture, was redissolved in the eluent solution and injected into the chromatographic system. The separation of aniline derivative from other amine impurities was carried out in a C 18 column using a 2% acetic acid–methanol (40:60, v/v) mobile phase. Results from the analysis of aniline in the sweetener samples with the proposed method were compared with those from the standard method. A good concordance between the two methods was observed.

Isocratic separations of closely-related mono- and disaccharides by high-performance anion-exchange chromatography with pulsed amperometric detection using dilute alkaline spiked with barium acetate

Mixtures of closely related mono- and disaccharides may be efficiently separated by high-performance anion-exchange chromatography (HPAEC) only when relatively dilute alkaline eluents are employed (i.e., <20 m M NaOH). The main drawbacks of these eluent solutions are (i) column regeneration between runs, (ii) poor reproducibility of the retention times, and (iii) the need for post-column base addition for enhancing sensitivity. Here, we describe some examples of isocratic separations of carbohydrates by HPAEC coupled with pulsed amperometric detection (PAD) accomplished by carbonate-free alkaline eluents (i.e., 5–20 m M NaOH) obtained upon addition of Ba(OAc) 2 (1–2 m M). These separations include aldohexoses (i.e., galactose, glucose, and mannose), aminohexoses (i.e., glucosamine and galactosamine) and their N-acylated derivatives (i.e., N-acetylglucosamine and N-acetylgalactosamine) along with some isomeric disaccharides (i.e., lactose, lactulose and epilactose). The separation of closely related isomers of trehalose, α,α, α,β, and β,β, is also presented. It is recommended to add Ba(OAc) 2 to NaOH solutions several hours before using the alkaline eluent (i.e., 12–24 h) to ensure complete barium carbonate precipitation in the eluent reservoir. Adopting such a simple strategy can be especially useful for performing carbohydrate separations under isocratic conditions in which no regeneration and or re-equilibration of column between runs is required. Excellent repeatability of retention data throughout a three-day working session was observed, with relative standard deviations ranging from 2.0 to 3.7%, and from 0.5 to 2.0%, as day-to-day and within-day values, respectively. In addition, there was no need for postcolumn addition of strong bases to the eluent, and successful applications of the present approach confirmed its validity and practicability with detection limits of simple carbohydrates in the picomole range.

Leaching of 137Cs and 90Sr Contaminated Soils by FeCl3 and NH4Cl Solutions

The leaching of 137Cs and 90Sr radionuclides contaminated soils by solutions of FeCl3 and NH4Cl in batch (static), column (continuous-flow) and field (Chernobyl region) experiments is investigated. The eluent solution is a 1:1 mixture of 0.01–0.02 mol l−1 FeCl3 and NH4Cl. The equilibrium distribution coefficients of 137Cs and Fe3+ between a Chernobyl sandy loam sample and the eluent are determined to be 25 and 3800, respectively. The first order rate constants of ion exchange reactions for the 0.01M eluent were 10−6 s−1 for 137Cs and 10−4 s−1 for 90Sr. In the Chernobyl region, a 10×10m plot with radioactivity up to 2000 Mbq km−2 (50 Ci km−2) was selected for decontamination by an ‘in situ’ leaching technology developed within the plot. For example, the leaching time to 30–40% residue radioactivity to a depth of at least 0.5 m is 15 days; in addition, the consumption of the eluent is 3–4 m3 m−2 for 137Cs and 1.5–2 m3 m−2 for 90Sr, at a flux of 8–10 l h−1 m−2.

Assessment of the genotoxicity of mine-dump material using the Tradescantia-stamen hair (Trad-SHM) and the Tradescantia-micronucleus (Trad-MCN) bioassays

The Tradescantia-stamen hair (Trad-SHM) and -micronucleus (Trad-MCN) bioassays were used to determine the genotoxicity of two eluates derived from mine tailings. The goal was to test the suitability of the Tradescantia bioassays as screening tools for this kind of waste material. Leachates obtained using the current standard German leaching test methods (S4 eluate) as well as leachates obtained using a new eluation method (pH stat4) were tested and compared. Concentration of heavy metals in the pH stat4 eluate were much higher than in the S4 eluate. The chemical analysis corresponded well with the results of the bioassays. Exposure to solutions containing more than 1% pH stat4 eluate caused a significantly higher number of micronuclei. The Trad-SHM bioassay also showed an increased pink mutation rate when plants were exposed to 8 or 16% eluate solutions. In contrast, the S4 eluate only caused increased mutation rates when solutions containing more than 32% eluate were used. The low pH of the pH stat4 eluate was not responsible for the genotoxicity observed using both bioassays, as indicated by the lack of significant mutation rates in the nitric acid controls. This demonstrates that the Tradescantia bioassays can be used as tools to assess the genotoxic potential of environmental samples with a wide range of pH values, without the need for sample modification.

Automated extraction chromatographic separations of actinides using separation-optimized sequential injection techniques

A sequential injection (SI) separation system has been developed for automated analytical separations of actinides using an actinide specific extraction chromatographic material (TRU-resin, Eichrom Industries, Inc., USA). On-line liquid scintillation counting was used to observe eluting species during method development, and fraction collection and alpha energy analysis were used for quantification. Several procedures for individual and group actinide elution are demonstrated and discussed, including elution of actinides as a single group; elution as groups based on valence state; selective separation of Pu using on-column redox chemistry; selective separation of Th; and various sequential actinide elution schemes. Eluent solution compositions and reagent chemistries were investigated with regard to elution peak shapes, selectivity, recoveries, carryover, and suitability for rapid automated procedures. The SI separation methods described serve as the basis for an automated actinide separation work station. An automated actinide separation procedure has been applied towards the analysis of Am, Cm, and Pu isotopes in three types of aged nuclear waste samples. Results from automated analytical separations followed by quantification by alpha spectroscopy were in good agreement with results obtained using manual separation techniques.

Flow injection determination of selenium by successive retention of Se(IV) and tetrahydroborate(III) on an anion-exchange resin and hydride generation electrothermal atomization atomic absorption spectrometry with in-atomizer trapping: Part 1. Method development and investigation of interferences

A sample solution was passed at 20 ml min −1 through a column (150×4 mm 2) of Amberlite IRA-410Stron anion-exchange resin for 60 s. After washing, a solution of 0.1% sodium borohydride was passed through the column for 60 s at 5.1 ml min −1. Following a second wash, a solution of 8 mol l −1 hydrochloric acid was passed at 5.1 ml min −1 for 45 s. The hydrogen selenide was stripped from the eluent solution by the addition of an argon flow at 150 ml min −1 and the bulk phases were separated by a glass gas–liquid separator containing glass beads. The gas stream was dried by passing through a Nafion® dryer and fed, via a quartz capillary tube, into the dosing hole of a transversely heated graphite cuvette containing an integrated L’vov platform which had been pretreated with 120 μg of iridium as trapping agent. The furnace was held at a temperature of 250°C during this trapping stage and then stepped to 2000°C for atomization. The calibration was performed with aqueous standards solution of selenium (selenite, SeO 3 2−) with quantification by peak area. A number of experimental parameters, including reagent flow rates and composition., nature of the gas–liquid separator, nature of the anion-exchange resin, column dimensions, argon flow rate and sample pH, were optimized. The effects of a number of possible interferents, both anionic and cationic were studies for a solution of 500 ng 1 −1 of selenium. The most severe depressions were caused by iron (III) and mercury (II) for which concentrations of 20 and 10 mg 1 −1 caused a 5% depression on the selenium signal. For the other cations (cadmium, cobalt, copper, lead,. magnesium, and nickel) concentrations of 50–70 mg 1 −1 could be tolerated. Arsenate interfered at a concentration of 3 mg −1, whereas concentrations of chloride, bromide, iodide, perchlorate, and sulfate of 500–900 mg l −1 could be tolerated. A linear response was obtained between the detection limit of 4 ng 1 −1, with a characteristic mass of 130 pg. The RSDs for solutions containing 100 and 200 ng 1 −1 selenium were 2.3% and 1.5%, respectively.

The kinetics of metal uptake by microbial biomass: implications for the design of a biosorption reactor

The kinetics of copper ion biosorption by free cell suspensions of inactivated Penicillium biomass was investigated. During the first 15 seconds of the metal uptake reaction copper biosorption was extremely rapid, becoming progressively slower until equilibrium was achieved. At a biomass concentration of 5.48 g dry weight/l, the copper biosorption reaction reached approximately 90% of the equilibrium position in one minute. This is in contrast with biosorption onto immobilised biomass which is known to take up to 24 hours or longer to reach equilibrium. However, the conventional reactor design for free cell suspensions, the stirred tank, may make inefficient use of the equipment and process due to the extended retention time. A plug-flow biosorption reactor with a limited retention time has been developed and demonstrated in the laboratory. The biomass and metal solution were mixed together using static in-line mixers and pumped along a length of pipe before discharging the mixed liquor into a settling tank to achieve phase separation. Desorption of the bound copper ions was achieved by drawing an eluant solution through metal-loaded biomass immobilised as a filter cake. The combination of metal uptake using free cells, and desorption using immobilised cells, optimises the kinetic and mass transfer effects for both processes and minimises the equipment requirements.

Solvent impregnated hollow fibre for a selective preconcentration of Pb(II) in an on-line determination by flame atomic absorption spectrometry

A solvent impregnated hollow fibre (SIHF) module has been developed for the preconcentration of lead by using bis(2-ethylhexyl) phosphoric acid (DEHPA) dissolved in kerosene as extractant. The module has been designed for an on-line determination of trace amounts of lead(II) at mg l −1 (ppm) level by flame atomic absorption spectrometry (FAAS). The SIHF system is based on the metal liquid–liquid distribution between aqueous solutions of different acidity and the mentioned organic solution. The highest enrichment factor of Pb(II) was determined at pH=4.0 using a formic acid/formiate buffer solution. Preconcentration experiments were carried out at low lead(II) concentration (mg l −1 level) by using the SIHF module. This study includes the influence of hydrodynamic and chemical conditions on the loading and elution of Pb(II) on the SIHF, i.e., flow rate through the fibres, acidity of the eluent (as nitric acid concentration) and the chemical nature of the acid used in the elution. Breakthrough curves were determined for different sampling flow rates, 0.54 ml min −1 was selected to minimise the loading volume of Pb(II) sample. 0.1 M nitric acid was chosen as eluent solution, and perchloric acid also shows appropriate elution characteristics. The degree of concentration obtained for Pb(II) are of 10 fold the original concentration. The quantification limit for Pb(II) achieved with this preconcentration system is 0.17 mg l −1. The results obtained indicate that the SIHF system can be applied for on-line determination of trace amounts of lead(II) by FAAS.

Chromatographic Separation and Sample Preparation in One Step for MALDI Mass Spectrometric Analysis of Subpicomole Amounts of Heterogeneous Protein Samples

A rapid and efficient sample preparation method is described that allows analysis of biopolymer mixtures by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Very low amounts of complex biopolymer mixtures can be analyzed by MALDI MS without any apparent loss of material and with a substantial gain in time by combining chromatographic separation and sample preparation in a single operation. This micropurification method is based on both the formation of a submicroliter reversed-phase chromatographic bed and the use of matrix-containing eluent solutions. In this way, desalting, matrix mixing, and micropurification of a biological sample could be performed in one step. A proteolytic mixture containing Ca2+ ATPase fragments ranging from ∼1000 to ∼30 000 Da, obtained after endoproteinase AspN cleavage of sarcoplasmic reticulum vesicles, was analyzed by MALDI MS. Direct analysis by MALDI MS of this peptidic mixture did not provide any signal for the higher molecular mass species. When o...

Dry deposition modeling of nitrate and sulfate by using particle size distribution data

Dry deposition and air sampling: were undertaken simultaneously in the ambient air of Ping Tung area by using several dry deposition plates, two MOUDIs (Micro‐orifice Uniform Deposit lmpactors) and one NRI (Noll Rotary Impactor) from December 1995 to May 1996 in Southern Taiwan. The NRI and MOUDI were used to collect ambient coarse and fine particulate, respectively. Dry deposition plate was applied to collect particle deposition flux. An ion chromatography (Dionex 2000i/SP) equipped with 4 mm AG4A‐SC and AS4A‐SC column was employed to analyze the anion species (NO, and SO4 2). The eluent solution is 1.8 mM sodium carbonate/ 1.7mM sodium bicarbonate. The calculated dry deposition flux for each particle size range was obtained by using measured mass‐size distribution data between 0.18 and 100 μm diameter and a dry deposition model. The mean modeled/measured (Md/ Ma) ratio of dry deposition flux varied between 1.11 and 1.28, between 1.28 and 1.42 and between 0.88 and 0.97 for nitrate, sulfate and total particle mass, respectively. The results indicated that nitrate and sulfate were slightly overestimated by a dry deposition model, while total particle mass was slightly underestimated by this model. In general, by using the particle size distribution data, this dry deposition model can provide a good predication for the dry deposition flux of nitrate, sulfate and total particle mass. More than 87.5 % of nitrate and more than 92.9 % of sulfate dry deposition flux are contributed by particle diameters larger than 10 μm. This is due to the fact that particle size larger than 10 μm have higher dry deposition velocities (>4.65 cm/sec) and control the majority of the dry deposition flux.

Evaluation of an anion‐exchange membrane for extracting plant available phosphorus in soils

Anion‐exchange resins (AER) have been used to determine plant available phosphorus (P) since the fifties and their results have shown strong relationships with plant growth and P uptake irrespective of soil properties. However, this procedure is still not widely used by laboratories because of difficulties in handling resin beads under routine conditions. New kinds and different shapes of resins are being produced each with specific characteristics that must be evaluated before use in laboratory procedures. Thus the objective of this work was to evaluate an AER manufactured in membranes reinforced with a Modacrylic fabric. These anion‐exchange membrane (AEM) sheets are commercially available, making them suitable for soil testing. The membranes were cut in pieces (1.0×7.5 cm) identified as AEM‐strips. The AEM‐strips were soaked in 0.5M HCl for a few days and transferred, after being rinsed with deionized water (DI), to 0.5M NaHCO3 to convert them to HCO3 ‐ form. The AEM‐strips and resin beads in nylon bags recovered 98.4 and 98.0% of the P content in an aqueous P solution, respectively. Three eluent solutions were evaluated with different shaking times. The 0.1M H2SO4 and 1.0M NaCl in 0.1M HCl were equally suitable for the molybdenum blue color development without any pH adjustment, while the pH of the 0.5M HCl was too low. The elution of P from the AEM‐strips was independent of time with a 15‐min shaking being adequate for removal of all P from the strips. A comparison of soil sample preparation demonstrated that it was not necessary to vigorously grind or sieve the soil to improve the repeatability of the results. The AEM‐strips were compared with other methods (Pi impregnated filter paper, Mehlich I and Bray 1) using 32 soils from Guatemala with widely varying physico‐chemical and mineralogical properties. Phosphorus extracted by the AEM and Pi procedures (similar principle) were highly correlated and gave similar results irrespective of soil type. The acid extraction (Mehlich I and Bray 1 methods) attacked soil components (apatites) resulting in higher and inconsistent amounts of P extracted which may not be available to plants; the correlation between these methods within soils of similar properties was good, but when all soils were considered together the relationship was not significant. This demonstrated that the acid extraction method for P is not suitable for soils containing apatites, while those based on a sink for P (AEM and Pi) can be applied irrespective of the type of soil.

Influence of the concentration of adsorbate and modifier in the mobile phase on retention in high-performance liquid chromatography

A method for the determination of the monomolecular adsorption constants from binary (adsorbate–eluent) solution directly from chromatography data (retention volume and concentration) is suggested. The constants of the adsorption equation are calculated from an anisole chromatogram measured on hydroxylated silica using n-hexane in the low concentration region. The adsorption isotherm of anisole is constructed. Good agreement between isotherms calculated by the suggested and by Glueckauf's methods is shown. The influence of the modifier concentration on retention of benzene derivatives is studied for the chromatographic system: n-hexane–tetrahydrofuran–aminopropyl silica. The deviation from linear dependence of the reciprocal of the retention volume on mole fraction of the polar modifier is explained by molecular modifier–modifier interaction in eluent. Taking into account this interaction and using the quasi-chemical model of retention allows one to describe the dependence of chromatographic data of benzene derivatives on the modifier concentration in the eluent.

The kinetics of metal uptake by microbial biomass: Implications for the design of a biosorption reactor

The kinetics of copper ion biosorption by free cell suspensions of inactivated Penicillium biomass was investigated. During the first 15 seconds of the metal uptake reaction copper biosorption was extremely rapid, becoming progressively slower until equilibrium was achieved. At a biomass concentration of 5.48 g dry weight/l, the copper biosorption reaction reached approximately 90% of the equilibrium position in one minute. This is in contrast with biosorption onto immobilised biomass which is known to take up to 24 hours or longer to reach equilibrium. However, the conventional reactor design for free cell suspensions, the stirred tank, may make inefficient use of the equipment and process due to the extended retention time. A plug-flow biosorptiOn reactor with a limited retention time has been developed and demonstrated in the laboratory. The biomass and metal solution were mixed together using static in-line mixers and pumped along a length of pipe before discharging the mixed liquor into a settling tank to achieve phase separation. Desorption of the bound copper ions was achieved by drawing an eluant solution through metal-loaded biomass immobilised as a filter cake. The combination of metal UPtake using free cells, and dcsorption using immobilised cells, optimises the kinetic and mass transfer effects for both processes and minimises the equipment requirements.

Ion chromatographic determination of borate in aqueous samples together with other common anions

The conversion of boric acid (pKa ≈ 9) in a more acidic complex, using mannitol or sorbitol as ligands, has been exploited to obtain a sensitive and accurate method for the determination of boric acid by ion chromatography. Both separation and detection conditions have been selected on the basis of available thermodynamic data on the relevant equilibria, and then experimentally optimised on a conventional ion cromatograph equipped with a suppressed conductivity detector. The performances of the method are practically identical for the two eluent solutions adopted, containing mannitol or sorbitol: the detection limit is about 1 µmol dm–3, the linear response range extends up to 200 µmol dm–3, the repeatability is better than 5% and the analysis time is less than 6 min. Moreover, other common anions (i.e., acetate, fluoride, and chloride) can be determined together with borate. The comparison of results with those obtained by conventional analytical techniques applied to real samples at known concentration of borate allows the estimation of a negligible bias for the ion chromatographic method.

Affinity enzymometric assay for detection of organophosphorus compounds

A new method for the detection of organophosphorus compounds has been developed. Organophosphorus compounds are recognized by binding of an excess of bienzyme conjugate (BEC) built up by butyrylcholinesterase as a receptor and horseradish peroxidase as the signal generating and amplifier element. The separation of the analyte-free bienzyme conjugate from the analyte-loaded bienzyme conjugate is performed on an affinity support formed by a Langmuir film containing an amphiphilic paraoxon derivative (B 1). The analyte-free bienzyme conjugate binds irreversibly to the affinity support. Bienzyme conjugate with bound analyte is prevented from binding to the B 1 film modified support and can thus be detected in the eluate solution by the horseradish peroxidase activity. This activity is directly proportional to the concentration of organophosphorous compounds. The assay procedure was developed for both the steady-state and the flow-injection mode. In both cases the lower limit of detection was about 1 pM for diisopropylflurophosphate.

Postcolumn Immunodetection Following Conditioning of the HPLC Mobile Phase by On-Line Ion-Exchange Extraction

Postcolumn immunodetection can provide enhancement of selectivity and sensitivity when used in combination with HPLC. Typical reversed-phase HPLC employs a variety of organic modifiers and buffers, and immunoassays generally perform best in a low ionic strength aqueous matrix with a pH between 7 and 8. There is therefore a need for removal of an analyte from a typical mobile phase into an environment compatible with an immunoreactor. An on-line system utilizing an ion-exchange column along with a switching valve to sequester an analyte from a HPLC mobile phase and apply a counterion eluent solution was designed to provide the proper matrix for immunoassay. An on-line HPLC−ion-exchange system was developed for the model analyte procainamide hydrochloride. The eluents from the on-line HPLC and ion-exchange system were collected and analyzed off-line using fluorescence polarization immunoassay. The fluorescence polarization immunoassay was calibrated using standards in a buffer matrix, and the system was validated using controls at 2.0, 7.0, and 15.0 μg/mL in mobile phase. Procainamide and its metabolite were extracted from spiked plasma using liquid−liquid extraction, and validation of the system with plasma samples demonstrated 15.60, 15.63 and 15.70% (n = 6) RSD for the three controls and accuracies of 6.42, 10.30, and 17.70% (n = 6) difference from spiked, respectively.