High Analyte Concentrations Research Articles

Dilution Protocols for Detection of Hook Effects/Prozone Phenomenon

The prozone or (high-dose) hook effect, documented to cause false-negative assay results >50 years ago (1), still remains a problem in one-step immunometric assays (2)(3)(4)(5)(6)(7)(8)(9), immunoturbidimetric assays (10), and immunonephelometric assays (11) for immunoglobulins. To detect the prozone effect, samples are often tested undiluted and after dilution (9). If the result on dilution is higher than for the undiluted sample, then the undiluted sample most likely exhibited the prozone effect. Unfortunately, this approach increases labor and reagent costs for assays that may only rarely encounter extremely high analyte concentrations. An alternative approach involves pooling patient samples and measuring the pool and a 10-fold dilution of the pool (12). If one or more of the samples in the pool is falsely low because of the prozone effect, then the results from the undiluted and diluted pools (after correcting for the 10-fold dilution) will differ significantly (12). Other approaches to eliminate the prozone effect include using two-step immunoassays that have a wash step between the addition of …

Fate of free selenium atoms in externally heated quartz tube atomizers for hydride generation atomic absorption spectrometry and their reatomization at tube ends studied by means of the determination of longitudinal free atom distribution

The longitudinal distribution of Se free atoms in externally heated quartz tube hydride atomizers was studied under analytical conditions, i.e. selenium concentrations of 10–100 μg l −1 and argon flow rates of 25–400 ml min −1. The selenium signal was measured through the walls of the optical tube with the tube perpendicular to the radiation beam at various positions along the tube length. The highest concentration of free atoms was found near the middle of the optical tube, where atomization proceeds. Under typical analytical conditions, all free atoms disappear before they reach the ends of the atomizer. The free atom decay rate is greatly enhanced at higher analyte concentrations. This is a major source of calibration graph curvature. The atomization efficiency was influenced much less. The presence of zones where once reacted free atoms are reatomized at the optical tube ends was proven by the spatially resolved observation. Using the measures originally meant to prevent flame ignition on the tube ends, such as quartz windows or prolonged unheated parts of the tube, no reatomization takes place. The effect of reatomization on the analytical performance of the atomizer was shown: the sensitivity is improved and the non-linearity of the calibration graph is substantially reduced, while the baseline noise remains at the same level. Recommendations concerning the quartz tube atomizer design and dimensions are given.

Lifetime of ion-selective electrodes based on charged ionophores

The lifetime of solvent polymeric ion-selective electrodes (ISEs) is limited by leaching of the membrane components into the sample solutions. In this article, leaching of charged ionophores is discussed. Because of the electroneutrality principle, the loss of the charged ionophore into the sample must be accompanied by parallel transport of an ion of the opposite charge sign into the sample or by ion exchange with a sample ion of the same charge sign. Because ionic sites of high lipophilicity are available, the loss of ionic sites is, in general, not a concern. Therefore, it is assumed here that the cotransported or ion-exchanging ions are primary or interfering ions forming complexes with the ionophore. A general theory that allows quantification of ionophore lipophilicities and a discussion of changes in the membrane composition and selectivity with time is presented. A high complex stability and high analyte concentrations diminish the rate of ionophore loss into the sample if a charged ionophore is coextracted from the membrane into the sample together with an analyte ion of opposite charge. On the other hand, if the charged ionophore has the same charge sign as the ion that it binds, a large binding constant and high analyte concentrations enhance ionophore leaching into the sample. The model is applied to interpret results for an electrically charged ionophore, for which selectivity changes as a function of the leaching time were observed and the lipophilicity was determined with potentiometric measurements. Using the lipophilicities of neutral ionophores, as described previously, and the lipophilicities of charged ionophores, as described here, a direct comparison of the expected leaching rates of charged and neutral ionophores has become possible.

Experimental Design for Analysis of Complex Kinetics Using Surface Plasmon Resonance

Using BIAcore surface plasmon resonance technology, we found that the real-time association kinetics of Fabs specific for hen egg-white lysozyme did not conform to a 1:1 Langmuir association model. Heterogeneity of the components is not the source of the complex kinetics. Informed by independent structural data suggesting conformational flexibility differences among these antibodies, we chose global mathematical analysis based on a two-phase model, consistent with the encounter-docking view of protein–protein associations. Experimental association times (Ta) from 2 to 250 min revealed that initial dissociation rates decreased with increasing Ta, confirming a multiphasic association. The relationship between observed dissociation rate and Ta is characteristic of each antibody–antigen complex. We define a new parameter, T50, the time at which the encounter and final complexes are of equimolar concentration. The observed T50 is a function of analyte concentration and the encounter and docking rate constants. Simulations showed that when the ligand is saturated at high analyte concentrations, T50 reaches a minimum value, T50(MIN), which can be used to compare antigen–antibody complexes. For high-affinity complexes with rapid rearrangement to a stable complex, T50(MIN) approaches T1/2 of the rearrangement forward rate constant. We conclude that experiments with a range of Ta are essential to assess the nature of the kinetics, regardless of whether a two-state or 1:1 model is applicable. We suggest this strategy because each Ta potentially reveals a different distribution of molecular states; for two-step analysis, a range of Ta that brackets T50 is optimal.

Effects of mobile-phase additives, solution pH, ionization constant, and analyte concentration on the sensitivities and electrospray ionization mass spectra of nucleoside antiviral agents.

The effects of various mobile-phase additives, solution pH, pKa, and analyte concentration on electrospray ionization mass spectra of a series of purine and pyrimidine nucleoside antiviral agents were studied in both positive and negative ion models. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H]+ ions. In the negative ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H]- ions. The sensitivities as [M + H]+ ions were significantly larger than the sensitivities as [M - H]- ions for purine antiviral agents. Vidarabine monophosphate and pyrimidine antiviral agents, however, showed comparable or greater sensitivities as [M - H]- ions. The sensitivity as [M + H]+ showed no systematic variation with pH; however, the sensitivity as [M - H]- did increase with increasing pH. At constant pH, the ion intensity of the protonated species increased with increasing pKa. At higher analyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na]+ adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4]+ adduct ions, but [M + NH4]+ ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was close to that of NH3. Abundant [M + OAc]- ions were observed for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic separations.

Characterization of alpha-pinene-degrading microorganisms and application to a bench-scale biofiltration system for VOC degradation

A study was conducted to isolate and characterize monoterpene-degrading microorganisms and apply them to a biofiltration unit for use in degrading high levels of alpha-pinene. Soil from a monoterpene-contaminated site was used with enrichment culture techniques to recover a consortium of bacteria able to utilize alpha-pinene as the sole source of carbon and energy. The Biolog system was utilized to identify the bacteria as Pseudomonas fluorescens and Alcaligenes xylosoxidans. Aerobic growth and biodegradation studies confirmed that rapid growth and biodegradation were being achieved with alpha-pinene. Complete degradation of alpha-pinene was achieved in 36 h with a maximum rate of degradation of 3.9 mg/L/h. The microorganisms were placed in a biofiltration column and demonstrated good removal of alpha-pinene from an air stream at concentrations averaging 295 ppmv. A nitrogen test was performed and confirmed that the removal of alpha-pinene was due to biological activity. Given the ability of these microorganisms to utilize high levels of alpha-pinene, they will be used in a coupled treatment system using a physical/chemical adsorption/desorption unit coupled to a biofiltration column. Often, biofiltration studies are performed using much lower levels of analyte in the influent air stream. However, the ability of these microorganisms to utilize higher levels of compounds expands the capabilities for future coupled biofiltration systems. During future studies, high flow rates with low levels of analyte will be concentrated so that a higher analyte concentration and lower flow rate can be utilized with the biofilter.http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p151.html

Fundamental baseline variations in aqueous near-infrared analysis

Distinctive, relatively small, analyte extinction coefficients in the near-infrared (NIR) spectral region, have led to NIR methodologies to probe the composition of concentrated solutions. However, as exemplified with alkaline hydroxide solutions, it is shown that the analyte strongly affects the conventional aqueous baseline of these measurements, and this baseline behavior must be addressed for effective NIR analysis. In the domain of molal and higher analyte concentration, the water baseline changes drastically as solute interacts and confines bulk solvent. At 967 nm an (hydroxide) absorption peak increases smoothly with increasing hydroxide activity in LiOH and NaOH electrolytes. Yet at this same wavelength, there is an unexpected absorption decrease in concentrated cesium (>5 m CsOH) and potassium (>13 m KOH) electrolytes, evidently due to solvent bridged anion–cation association. A peak for bulk water occurs nearby at 976 nm, with a unit pathlength absorbance of 0.24. This absorbance diminishes by an order of magnitude at high analyte concentration, as water activity decreases. A broad strong (0.53 cm −1) series of absorbances centered near 1200 nm diminish smoothly with decreasing bulk water activity. However, a broad strong absorption (14 cm −1) centered near 1450 nm, is resolved into two peaks, one disappearing, the other increasing at high hydroxide concentrations. Recognition of these variations permits effective NIR determination of analyte (in this case hydroxide). Second-order differentiation, ∂ 2 A/ ∂λ 2, at either 967 or 1421 nm, minimizes the effects of the near-lying bulk water absorption, and of baseline shift, and provides a linear variation with hydroxide concentration.

Spatially resolved absorption measurements of antimony atom formation and dissipation in quartz tube atomizers following hydride generation

The cross-sectional distribution of free antimony atoms generated from admission of stibine into quartz tube atomizers was measured by atomic absorption spectrometry. A CCD camera was used for the spatially resolved detection. In the unheated flame-in-tube atomizer, the highest free atom concentration was found near the tube axis, decreasing towards the walls. The free atom distribution was not influenced by atomization conditions such as purge gas flow rate and oxygen delivery. Significant changes in the free atom distribution were obtained by changing the position of the oxygen delivery capillary tip. Analyte reactions within the tube were revealed from an analysis of the curvature of the calibration curve. In the externally heated atomizer (900°C), the free atom distribution was much more homogeneous compared to the unheated atomizer under analytical conditions. However, pronounced inhomogeneity (higher concentration of free atoms near the tube axis and in the regions close to the walls) was obtained at high Sb concentrations in a roll-over part of the calibration curve (over 300 ng ml −1). This is explained on the basis of free atom decay on the surface of polyatomic particles formed at high analyte concentrations. From a practical point of view, no effects caused by the inhomogeneous free atom distribution are to be expected in the heated `flameless' tubes, the most widely used in routine analysis, since cross-sectional inhomogeneity observed under typical working conditions was negligible.

Electrospray ionization mass spectrometry of cyclodextrin complexes with amino acids in incubated solutions and in eluates of gel permeation chromatography

1:1 complexes of beta-cyclodextrin (CD) with three amino acids (Gly, Phe and Trp) have been detected as ions in the gas phase using infusion positive and negative ion electrospray ionization mass spectrometry (ESI-MS). In contrast with the positive ion ESI mass spectra of simple aqueous solutions, the aggregates and adducts usually formed in the ESI process did not appear in the positive ion ESI spectra of solutions buffered with ammonium acetate (NH4Ac), even at higher analyte concentrations, These studies suggest that addition of buffer and/or use of a low analyte concentration should be used to overcome formation of aggregates and metal ion adducts in such mass spectrometry studies. Also, the deprotonated complexes are dissociated by collision induced dissociation (CID) to form an abundant product ion, the deprotonated CD, requiring transfer of a proton to the amino acid carboxyl group, To understand formation of complexes in the gas phase, gel permeation chromatography (GPC) was used to separate free amino acids (AAs) from complexes in an incubated solution. The ESI mass spectra of the GPC fractions show the presence of 1:1 complexes of both CD-aromatic amino acids and CD-aliphatic amino acids. Compared with CD-aliphatic amino acid complexes, CD-aromatic amino acid complexes appear to be destabilized in the gas phase, possibly because the hydrophobic interaction which binds the aromatic group of amino acids in the CD cavity in solution may become repulsive when solvent evaporates from the droplets during the electrospray process, whereas those complex ions formed as proton bound dimers are stabilized by electrostatic forces, the major binding force for such complexes in the gas phase. In addition, the GPC technique coupled with off-line ESI-MS can rapidly separate CD complexes by size, and provides some information on the character of the complexes in solution. (C) 1998 John Wiley & Sons, Ltd.

Evaluation of the analytical performance of the Boehringer Mannheim Elecsys 2010 immunoanalyzer.

We evaluated the analytical performance of the Elecsys 2010 immunoanalyzer (Boehringer Mannheim Canada), which is based on a new detection technology, electrochemical luminescence. We used six representative assays from the initial launch menu of the instrument: thyroid stimulating hormone (TSH), free thyroxine (FT4), troponin T, human chorionic gonadotropin (hCG), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA). Within-run and between-day imprecision were evaluated using pools of human specimens at low, mid and high concentrations. Linearity was evaluated by diluting specimens with high analyte concentrations with specimens that had a low level of this analyte. Carry over and hook effect were investigated using specimens with high concentrations of hCG. Functional sensitivity was studied by running low TSH specimens over 21 daily runs, and by comparing the scatterplot of FT4 as a function of TSH. Over 100 specimens distributed across the analytical range were analyzed with two comparison methods: ES 300 (Boehringer Mannheim Canada) and AxSYM (Abbott Laboratories). Within-run and between-day imprecisions were less than 4% and 10%, respectively, for most assays. All assays were linear over the whole analytical range. Carry over was minimal (< 0.0002%). A hook effect was present for hCG levels greater than 560,000 U/L. The functional sensitivity of the TSH assay was lower than 0.02 mlU/L. Correlation coefficients were all > 0.94. Small proportional errors were observed in comparison studies for the CEA and PSA assays. The Elecsys 2010 system was shown to have an acceptable analytical performance for the rapid analysis of a wide variety of analytes. The hook effect observed with hCG assay would imply that the laboratory informs the clinicians of the possibility of falsely low values in trophoblastic diseases or that all specimens with values greater than 3000 U/L are reassayed after dilution.

A predictive model for matrix and analyte effects in electrospray ionization of singly-charged ionic analytes.

In electrospray ionization (ESI), droplets with a surface excess charge are created. The rate of production of surface excess charge is a constant and is equal to the rate of ion production. The ions appearing in the mass spectrum are postulated to be those that formed the surface excess charge at the time of droplet formation (or their collision products). An equilibrium model based on competition among the ions in the solution for the limited number of excess charge sites has been developed. This model accurately predicts the response curves of singly-charged ionic analytes as a function of the concentration of electrolyte and other analytes and provides an explanation for the selective effectiveness of ESI. At low concentrations of total analyte (micromolar and less), the response curves are linear, indifferent to the presence of other low concentration analytes, and suppressed by electrolyte concentrations in excess of the minimum required. At higher analyte concentrations, the response becomes independent of analyte concentration but highly affected by the presence of other analytes.

On cluster ions, ion transmission, and linear dynamic range limitations in electrospray (ionspray) mass spectrometry

The ion transmission in Electrospray (Ionspray) Mass Spectrometry (ESMS) was studied in order to examine the instrumental factors potentially contributing to observed ESMS linear dynamic range (LDR) limitations. A variety of means used for the investigation of ion transmission demonstrated that a suspected loss in tetraalkylammonium ion signal in favour of formation of analyte ion-analyte ion-pair clusters is negligible. The ion/ion-pair cluster abundance continues to rise after the core analyte ion reaches the 10 −5M concentration LDR plateau. The relative cluster ion abundance changes observed with increasing concentration appear to reflect solution phase ion/molecule clustering reactions at the surface of charged droplets produced in the electrospray. The simultaneous measurement of the ES capillary spray current and ion currents at the first three stages of ion sampling revealed that the nozzle orifice which separates the atmospheric pressure ion source from the first vacuum stage receives a current that rises continuously with sample concentration. The skimmer current rises to a plateau then falls off at higher analyte concentrations, closely matching the final mass spectrometric response. Complete coverage of the charged droplet surface can explain the plateau, but a simple model that explains tetraalkylammonium ion signal suppression cannot be given. Dynamic range limitation was less pronounced with higher atmospheric pressure chemical ionization total ion currents, which showed linearity over greater total MS ion current ranges. MS signal suppression observed at high (> 0.2 mM) analyte concentrations may thus be attributed to unique ES ion formation/instrumental/space charge effects.

An analysis of transport resistances in the operation of BIAcore™; implications for kinetic studies of biospecific interactions

The mass-transfer processes that affect kinetic measurements of biospecific interactions between one species in a flowing solution and another species immobilized in a thin hydrogel instrument were analysed by means of convection-diffusion-reaction models. The specific purpose was to identify experimental design considerations for kinetic measurements using the BIAcore™ instrument. Numerical solutions identified three different regimes of operation. A kinetic regime exists at low values of Damköhler number (Da), when the intrinsic kinetics are slow and the diffusion is relatively fast. This allows for the accurate determination of kinetic constants. A limiting value of Da, above which mass-transfer limitations appear, is presented as a function of Peclet number, Pe. At higher Da values, the reaction occurs in the mass-transfer-controlled regime where the reaction-rate is independent of the intrinsic kinetics. It was observed that, frequently, the reaction occured in an intermediate regime where, although the mass-transfer rate was not strictly limiting, substantial concentration gradients were present. Analysing the data in this regime by direct application of kinetic equations underestimates the association rate constant. Even when the reaction was not limited by mass-transfer in the flow channel, it may have been affected by steric hindrance to transport in the hydrogel, if a large concentration of capturing antibody or ligand was immobilized. The primary effect of the hindrance was to lower the soluble-species (analyte) concentration in the hydrogel when compared to the bulk solution. Non-uniformity of conditions within the hydrogel in the presence of steric hindrance had a significant effect on the observed reaction. The effect was most prominent at higher analyte concentration, when the rate constant showed an apparent reduction as the reaction progressed.

Analyte and label binding assay read by flow cytometry

A new immunometric two-site sandwich assay is introduced, in which a label-scavenging binding partner is added to the sample in addition to the analyte-binding partner. The scavenger binding partner binds excess label antibody, giving a signal proportional to the amount of excess label antibody in the sample solution. A set of two calibration curves is obtained from the two binding partners simultaneously, and a combination of the two signals gives an unambiguous determination of the analyte concentration, even for high analyte concentrations where the hook effect may occur. Two-particle immunofluorometric assays developed for placental alkaline phosphatase and human chorionic gonadotropin on the basis of this principle and yielding signals measured by flow cytometry gave rapid results (2 h) and had working ranges in excess of 5 and 6 orders of magnitude for the respective analytes.

Comparison of two dry chemistry analyzers and a wet chemistry analyzer using canine serum.

Canine serum was used to compare seven chemistry analytes on two tabletop clinical dry chemistry analyzers, Boehringer's Reflotron and Kodak's Ektachem. Results were compared to those obtained on a wet chemistry reference analyzer, Roche Diagnostic's Cobas Mira. Analytes measured were urea nitrogen (BUN), creatinine, glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholesterol and bilirubin. Nine to 12 canine sera with values in the low, normal, and high range were evaluated. The correlations were acceptable for all comparisons with correlation coefficients greater than 0.98 for all analytes. Regression analysis resulted in significant differences for both tabletop analyzers when compared to the reference analyzer for cholesterol and bilirubin, and for glucose and AST on the Kodak Ektachem. Differences appeared to result from proportional systematic error occurring at high analyte concentrations.

Time-integrated spectra from a flame photometric detector

A rotating, variable-wavelength interference filter has been used to acquire spectra from peak or baseline of a dual-channel, high-sensitivity flame photometric detector. On its spectral channel, the device monitors 100 data points from 400 to 700 nm ten times a second, and ensemble-averages them over seconds to (if needed) hours of acquisition time. The spectra can be smoothed, adjusted for background, corrected for filter transmission and photocathode profiles, and plotted with energy or photon ordinates. The extent of spectral fluctuation suggests the emergence of flicker noise at high analyte concentrations.

On-line coupling of isolation/in situ concentration integrated with derivative synchronous spectrofluorimetry for the simultaneous determination of epinephrine and norepinephrine in urine

An unsegmented flow system for the automated determination of epinephrine and norepinephrine in urine is proposed. The simultaneous determination involves the following on-line steps: 1. (i) matrix isolation/analyte preconcentration in an exchange minicolumn located in the loop of a injection valve; 2. (ii) elution-injection into the dynamic system and derivatization during the transport to the detector; 3. (iii) in situ concentration in the flow cell integrated with the synchronous spectrofluorimetric scan; 4. (iv) data acquisition and derivation by computer. The method thus developed enables a linear determination range between 2 and 60 ng ml −1 for both catecholamines with R.S.D.s of 1.0 and 2.0% for norepinephrine and epinephrine, respectively, and a sample throughput of 12 samples h −1. The utility of the method has been proved by applying it to urine samples with low and high concentrations of the target analytes.

Ion source for electrohydrodynamic mass spectrometry

AbstractEarly in the development of electrohydrodynamic mass spectrometry (EH‐MS), it was realized that ion source performance improved when the sample liquid was supplied discontinuously to the capillary emitter. Based on this realization, an ion source was designed that operates without an external sample supply system. Its essential part is a metal capillary that serves both as the liquid reservoir and the field anode and is mounted exchangeably on a pushrod. As the liquid flow is field induced, the flow rates are extremely low, in the range of 0.2 nl min−1. Thus, in spite of high analyte concentrations needed, sample consumption is only a few picomoles per mass scan. Moreover, the emission of larger droplets is avoided, which contributes to the stability of the ion signal. As the capillary emitter is mounted on a standard emitter carrier known from field desorption (FD) MS, the new ion source is compatible with conventional FD ion sources. EH mass spectra of analytes could be obtained from substances whose solubility in glycerol was&gt;0.5 mol l−1. The substances investigated with the new ion source include sugars, amino acids, peptides and an antibiotic. The general characteristics of the mass spectra are essentially the same as those known from other EH ion sources. Besides abundant solvent cluster ions, only cationated or protonated molecules of the analytes could be detected, frequently with one or more solvent molecules attached to the ions. Fragment ions were not observed.

Characterization and analysis of soils using mid-infrared partial least-squares .2. Correlations with some laboratory data

Infrared partial least squares (PLS) analysis is shown to provide a simple, rapid chemometric technique for the simultaneous analysis of soil properties. The method is capable of extracting both qualitative and quantitative information from soil spectra. A number of the mineral and organic components which are responsible for certain soil properties have been identified and the prediction of these properties assessed. Diffuse reflectance infrared Fourier-transform (DRIFT) spectra of whole soils were recorded to form a large training data set. The spectral information from this set was compressed into a small number of subspectra (called weight loadings) which contained positive and negative peaks reflecting correlations between the soil mineral and organic components and corresponding analytical data. Positive peaks in the weight loadings corresponding to organic components including alkyl, carboxylic and amide species were highly correlated with OC and N. Likewise, smectite, kaolinite and gibbsite clay minerals, together with organic alkyl and carboxylic species, contributed either positively or negatively to pH, sum of cations and clay content. Positive peaks due to calcite were well resolved in the first carbonate weight loading. Quartz was identified as an 'interference' for all analyses, with a series of negative peaks in the weight loadings. Implications were that quartz exerted a strong spectral signal for the majority of soil spectra, although it was not directly related to particular analyses. The usual PLS method, in which there is assumed to be a linear relationship between the loading intensities and soil property values, was found to give nonlinear prediction regression. The nonlinearity was assumed to be due to the effects of nonlinear response of the DRIFT signal and to significant compositional variability between calibration samples with high and low analyte concentrations. An alternative strategy of using a locally linear PLS model was tested, where small subsets of the total span of analytical values were independently used for PLS analysis. This approach improved prediction linearity and precision improved significantly for most analyses. The PLS method was thus shown to provide a useful surrogate technique for the study of soils, with which the PLS analysis of a single spectrum could provide simultaneous qualitative and quantitative information on a number of widely different soil analyses.

Evaluation of Chloroparaffin/PVC Phases for ATR/FT-IR Analyses

A significant limitation to the use of ATR/FT-IR analysis for aqueous solutions is the relatively high bulk concentration of analyte that is required. One approach to improving the detection of an analyte is to incorporate a thin polymeric phase at the surface of the ATR element. The purpose of the polymeric phase is to extract the analyte of interest and concentrate it within the depth of penetration of the evanescent wave. With the use of a very high molecular weight polyvinyl chloride phase, the time necessary to reach equilibrium for a 0.05% (v/v) nitrobenzene in a 1.5% (w/v) methanol/water solution was over 60 min. A study was undertaken to determine whether incorporating a chloroparaffin plasticizer into the polymeric phase would reduce the time required to reach the maximum level of absorbance achieved for the analyte. Specifically, the ability of phases which are mixtures of PVC and chloroparaffin plasticizers to concentrate the analyte from aqueous solutions has been investigated. The results indicate that incorporating chloroparaffin into a PVC phase reduces the time required to reach the maximum absorbance for the analyte in an aqueous solution containing 1.5% methanol by approximately 45%. The addition of chloroparaffin also results in an increase in the magnitude of absorbance observed for the analytes investigated in this study.