Analysis of the influence of interdetector volume on local calibration in size exclusion chromatography with dual multiangle-light-scattering/concentration detection

A comparison of the s- and w-detections of molecular weight, a necessary condition for precise determination of interdetector volume in size exclusion chromatography of polymers with the dual light scattering--concentration detection, is examined. From the theoretical analysis for a polymer with the log-normal molecular weight distribution it follows that the error in determination of the local calibration, i.e., of a logarithmic dependence of molecular weight on elution volume obtained by the s-detection, from the known dependence of radius of gyration on elution volume and of molecular weight on the radius of gyration, remains linear and is slightly shifted with respect to that obtained by the w-detection (dual detection giving the weight-average molecular weight) towards higher molecular weights, the difference being below the experimental error. It also follows that the error in the slope of local calibration found by the w-detection is given by the ratio of the error in interdetector volume and the variance of the elution curve. This is demonstrated on several polymer samples with symmetric elution curves of polymer samples differing in polydispersity indices. The range of this rule depends on the broadness and symmetry of elution curves. The precision of the interdetector volume determination depends therefore strongly on the Mw/Mn ratio of the sample.     

Size-exclusion chromatographic determination of polymer molar mass averages using a fractal calibration

The characterization of polymers by size-exclusion chromatography basically consists of the determination of the weight-average molar mass (Mw), number-average molar mass (Mn), and polydispersity index (I). An accurate estimation of these magnitudes requires the use of a reliable and trusted calibration curve. Three procedures for building up a calibration curve are analyzed in this work. The first is the classical universal calibration (UC), based on the elution of tetrahydrofuran-polystyrene in a system as reference. The second is based on the proper calibration curve made with standards of the sample under study. However, two main drawbacks arise when using these methodologies: the nonfulfilment of the UC when secondary mechanisms, other than pure size-exclusion, are present in the separation process; and the lack of a broad set of narrow standards of the sample under analysis in the second procedure. In order to circumvent these difficulties, a third, recently-proposed approach based on fractal considerations is applied. The accuracy and reliability of this method is proven through the calculation of the deviations observed in the estimation of the Mw values for polymer samples in different solvent-gel chromatographic systems. Whereas the classical UC shows a mean deviation of approximately 80% relative to the values given by the manufacturer, the fractal calibration yields a mean deviation of approximately 16%, similar to that obtained from the proper calibration. Moreover, the fractal procedure only needs one polymeric sample to generate the calibration curve.     

Relative matrix effects: A step forward using standard line slopes and ANOVA analysis

One of the alternative methods to identify and study the matrix effect is by determination of “relative” matrix effect. In this experiment % coefficient of variance of standard line slopes are calculated. First, six standard lines are prepared from single plasma lot. In another experiment standard line slopes are compared from six different lots of plasma. All these standard curves are prepared by using three different types of IS (internal standard). From all these experiments it is observed that using SIL-IS (stable isotope labeled-internal standard) is one of the best approach in methods having matrix effects. Alternatively, analog IS is a cost effective approach. After comparing a large number of calibration curve slopes, it can be recommended that during every bioanalytical method validation, where the sample size is >50, scientist should perform the “relative” matrix effect experiment by standard line slope method. In selected cases, the precision of standard line slopes in six different lots of a biofluid was compared with precision values determined six times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 5% for the method to be considered reliable and free from the relative matrix effect liability.     

Size Exclusion Chromatography of Poly(vinylpyrrolidone)

Abstract

Poly(vinylpyrrolidone) and poly(ethylene oxide) separate by hydrodynamic volume on Toyo Soda TSK-PW columns in a mixed solvent mobile phase of 50:50 (v/v) MeOH/H₂O containing 0.1M LiNO₃. From this separation a single universal calibration curve based on hydrodynamic volume [η]M can be obtained. Accurate weight average molecular weights of PVP were obtained by both SEC/LALLS and universal calibration showing good agreement between the two methods. SEC/LALLS overestimates the number average molecular weight for broad distribution polymers due largely to the lack of sensitivity of the LALLS detector to the low molecular weight portion of the polymers, while the universal calibration method slightly underestimates the number average molecular weight as compared to osmometric values.     

Kinetic-catalytic determination of vanadium(IV) using methyl orange-bromate redox reaction.

Determination of V(IV) based on its catalytic effect on the reaction between Methyl Orange and bromate in thepresence of citric acid was studied. The calibration curve obtained by fixed-time method was linear in the range of 2.5-300 ng ml(-1). By use of slope method, a calibration curve containing two linear portions were obtained. Using fixed-time and slope methods, we obtained detection limits of 0.8 and 1.5 ng ml(-1), respectively. Fe2+, As(III), V(V) and Hg2+ interfered. The method was successful for analysis of water samples.     

Ein verfahren zur eichung der gelchromatographie (GPC) mit präparaten beliebiger,bekannter molekulargewichtsverteilung

If, in GPC calibration, the mean elution volume (or centre of gravity volume) of the elution curves obtained with monomolecular samples is used, the mean elution volume of a polymolecular mixture is a weight-average. According to the calibration curve, a molecular weight average can be attributed to the mean elution volume. For the special case of a logarithmic-linear calibration curve, this is the geometric (weight) mean of the molecular weight. Thus one can perform broad-standard calibration of GPC without interference by column dispersion. The potential application for non-linear calibration curves is discussed. It is considered how calibration functions for the higher moments of the monomolecular elution curves could be determined and how universal calibration should be performed if the standards are polydisperse relative to hydrodynamic volume. Absolute determination of polydispersity becomes possible with an additional z-detector.     

A new kinetic photometric method for the simultaneous determination of metals based on catalytic and activating effects

Summary The potential use of the activating effect of Pb(II) on the Mn(II)-catalysed oxidation of Tiron by hydrogen peroxide in the presence of 1,10-phenanthroline for the simultaneous determination of the two metals was investigated. The results obtained allowed the development of a new kinetic photometric method for the simultaneous determination of Pb(II) and Mn(II). The catalysed reaction was monitored by the initial rate method, which was applied to absorbance-time curves. Different Mn(II) concentrations were used to construct calibration graphs by plotting the slopes of the photometric curves obtained against the Mn(II) concentration at each Pb(II) concentration assayed. A new calibration graph was obtained in terms of the Pb(II) concentration from the slopes of such graphs. By applying the standard-addition method to the sample to be assayed a third graph was obtained, the slope and intercept of which provided the analytical concentration of Pb(II) and Mn(II), respectively. The optimized values of the different variables involved were used to determine Mn(II) and Pb(II) over the concentration ranges 1–5 and 200–800 ng/ml, respectively.     

The determination of selenium in serum and urine by inductively coupled plasma mass spectrometry: comparison with Zeeman graphite furnace atomic absorption spectrometry

An inductively Coupled Plasma Mass Spectrometric (ICPMS) method for the determination of selenium in both serum and urine is described. ⁷⁸Se is used analytically in spite of ³⁸Ar⁴⁰Ar isobaric interference at mass 78. Initially ⁸²Se was monitored but, limited isotope abundance and therefore limited detection capability for urine selenium precluded continued use. An ethanol–Triton X-100-nitric acid diluent was used to dilute serum and urine and enhance selenium ionization so that both serum and urine can be analyzed with the same calibration curve. Results derived by the ICPMS method were compared with Zeeman Graphite Furnace Atomic Absorption Spectrometry (ZGFAAS) using nickel as the matrix modifier. Detection limits for ZGFAAS and ICPMS using mass 78 are 2.9 and 0.25 μg/l, respectively. ICPMS and ZGFAAS instrument responses were recorded for additions of inorganic selenium, trimethylselenonium iodide, seleno-dl-methionine, and seleno-dl-cystine to urine and serum. ICPMS slopes for all compounds added to urine and serum were found to be nearly identical. ZGFAAS response for each compound was more variable than ICPMS. ZGFAAS response for trimethylselenonium iodide was approximately 3-fold lower than for the other compounds. ZGFAAS regression slopes and correlation coefficients were 0.72 and 0.8139 for reference urine samples. ICPMS regression slope and correlation coefficient vs. the reference target values were 0.95 and 0.9700 for the same urines. Regressions slopes and correlation coefficients for reference sera were 1.01 and 0.9912 for ICPMS and 1.12 and 0.9648 for ZGFAAS. We conclude that ICPMS produced more accurate results than ZGFAAS for selenium in serum and urine.     

Quantification in MALDI-TOF mass spectrometry of modified polymers

MALDI-TOF mass spectrometry quantification is hampered by the poor reproducibility of the signal intensity and by molecular-mass and compositional discrimination. The addition of a suitable compound as an internal standard increases reproducibility and allows a calibration curve to be constructed. The concept was also verified with synthetic polymers but no instructions for practical implementation were given [H. Chen, M. He, J. Pei, H. He, Anal. Chem. 75 (2003) 6531-6535.], even though synthetic polymers are generally non-uniform with respect to molecular mass and composition and access to the polymer of the same molecular mass distribution and composition as that of the quantified one is thus the exception rather than rule. On the other hand, relative quantification of polymers e.g., the content of the precursor polymer in a batch of a modified polymer, is usually sought. In this particular case, the pure precursor is usually available and the modified polymer can serve as an internal standard. However, the calibration curve still cannot be constructed and the use of the internal standard has to be combined with the method of standard addition in which the precursor polymer is added directly to the analyzed sample. The experiments with simulated modified polymers, mixtures of poly(ethylene glycol) (PEG) and poly(ethylene glycol) monomethyl ether (MPEG) of similar molecular-mass distribution, revealed a power dependence of the PEG/MPEG signal-intensity ratio (MS ratio) on the PEG/MPEG concentrations ratio in the mixture (gravimetric ratio). The result was obtained using standard procedures and instrumentation, which means that the basic assumption of the standard-addition method, i.e., the proportionality of the MS and gravimetric ratios, generally cannot be taken for granted. Therefore, the multi-point combined internal-standard standard-addition method was developed and experimentally verified for the quantification of the precursor in modified polymers. In this method, the two parameters of the power-type calibration curve - the proportionality constant and the exponent-are assumed. If the exponent strongly deviates from unity the minority component can be significantly underrepresented in the spectrum. Therefore, the absence of the precursor polymer signals in the MALDI-TOF mass spectrum of a modified polymer sample does not prove the absence of the precursor in the sample. Such a conclusion has to be corroborated by the standard-addition method.     

Electrocatalytic Activity of Bamboo‐Structured Carbon Nanotubes Paste Electrode Toward Hydrogen Peroxide

Abstract

In this report, we describe the finding that bamboo‐structured carbon nanotubes (CNT) showed superior electrocatalytic activity toward hydrogen peroxide. The slope of the calibration curve for hydrogen peroxide obtained with the bamboo‐structured CNT paste electrode was more than 20 times as large as the slopes obtained with hollow‐structured CNT paste and glassy carbon electrodes at an operating potential of ?0.1 V, with no interfering reactions. Incorporation of glucose oxidase within the bamboo‐structured CNT paste electrode allows the selective detection of glucose in the presence of common interferents without using any permselective membranes. This excellent ability of the bamboo‐structured CNT paste electrode toward hydrogen peroxide is applicable to the development of other enzymatic biosensors.     

Calibration of an evaporative light-scattering detector as a mass detector for supercritical fluid chromatography by using uniform Poly(ethylene glycol) oligomers

The quantitativeness of an evaporative light-scattering detector (ELSD) for supercritical fluid chromatography (SFC) was evaluated by using an equimass mixture of uniform poly(ethylene glycol) (PEG) oligomers. Uniform oligomers, in which all molecules have an identical molecular mass, are useful for the accurate calibration of detectors. We calibrated the SFC-ELSD system for various concentrations and molecular masses by using an equimass mixture of PEG oligomers. ELSD not only showed a good linear response to the injected concentration over a wide concentration range, from 10(-4) to 10(-1)g/mL, but also showed a strong dependence on the molecular mass of the solute. By using chromatograms of the equimass mixture of uniform oligomers to calibrate SFC-ELSD, it was possible to determine exact values of not only the average mass but also the molecular-mass distribution for a PEG 1540 sample. The average molecular mass was shifted to a higher value by several percentage points after calibration of the ELSD.     

Investigation of artifacts caused by deuterium background correction in the determination of phosphorus by electrothermal atomization using high-resolution continuum source atomic absorption spectrometry

The artifacts created in the measurement of phosphorus at the 213.6-nm non-resonance line by electrothermal atomic absorption spectrometry using line source atomic absorption spectrometry (LS AAS) and deuterium lamp background correction (D₂ BC) have been investigated using high-resolution continuum source atomic absorption spectrometry (HR-CS AAS). The absorbance signals and the analytical curves obtained by LS AAS without and with D₂ BC, and with HR-CS AAS without and with automatic correction for continuous background absorption, and also with least-squares background correction for molecular absorption with rotational fine structure were compared. The molecular absorption due to the suboxide PO that exhibits pronounced fine structure could not be corrected by the D₂ BC system, causing significant overcorrection. Among the investigated chemical modifiers, NaF, La, Pd and Pd + Ca, the Pd modifier resulted in the best agreement of the results obtained with LS AAS and HR-CS AAS. However, a 15% to 100% higher sensitivity, expressed as slope of the analytical curve, was obtained for LS AAS compared to HR-CS AAS, depending on the modifier. Although no final proof could be found, the most likely explanation is that this artifact is caused by a yet unidentified phosphorus species that causes a spectrally continuous absorption, which is corrected without problems by HR-CS AAS, but which is not recognized and corrected by the D₂ BC system of LS AAS.     

High-precision pH measurement by means of electrochemical cells with transference

pH Measurements were carried out by means of cells containing Pt/hydrogen and glass electrodes, both equipped with the same type of ground glass sleeve junction carefully selected with respect to the leak rate. The cells were calibrated with the same set of standard buffers using multiple point calibration with linear regression. The slopes of the regression lines were compared with the theoretical (Nernst) slope of the E versus pH function and yielded the electromotive efficiency of the two cells. The accuracy of the measurements was obtained from the residual liquid junction potential errors.     

Estimation of the standard uncertainty of a calibration curve: application to sulfur mass concentration determination in fuels

The construction of a calibration curve using least square linear regression is common in many analytical measurements, and it comprises an important uncertainty component of the whole analytical procedure uncertainty. In the present work, various methodologies are applied concerning the estimation of the standard uncertainty of a calibration curve used for the determination of sulfur mass concentration in fuels. The methodologies applied include the GUM uncertainty framework, the Kragten numerical method, the Monte Carlo method (MCM) as well as the approximate equation calculating the standard error of prediction. The standard uncertainty results obtained by all methodologies agree well (0.172?C0.175?ng???L^?1). Aspects of inappropriate use of the approximate equation of the standard error of prediction, which leads to overestimation or underestimation of calculated uncertainty, are discussed. Moreover, the importance of the correlation between calibration curve parameters (slope and intercept) within GUM, MCM and Kragten approaches is examined.     

Multi-element X-ray fluorescence analysis of thin films using a mono-standard calibration procedure

Summary A semi-fundamental approach to calibration for multi-element X-ray fluorescence analysis of thin films is described. The curve, calibration line slope vs. atomic number, is calculated using known values for mass absorption coefficients, fluorescent yields and detector efficiency. This curve is then standardised for a particular matrix by determining the slope of at least one matched standard or via a standard addition procedure for one element. The technique is applied to the analysis of industrial waste water.

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Multielementanalyse von dünnen Schichten durch Röntgenfluorescenz mit Hilfe einer Monostandard-Eichmethode

     

Direct spectrophotometric determination of zirconium with spadns

A method has been developed for the spcctrophotometric determination of microgram amounts of zirconium using SPADNS as the colour-forming agent. The method is sensitive and quantities as small as 0.07 γ zirconium can be estimated in 25 ml of final volume. A standard calibration curve lias been prepared; it conforms to Beer's law. The stoichiometric composition of the coloured complex has been determined spcctrophotomctrically by adopting the 'slope ratio method'.     

Determination of water in ethanol and acetone by direct injection enthalpimetry based on the heat of dilution

Two analytical procedures based on the magnitude of the heat of dilution and direct injection enthalpimetry have been developed for determination of water in ethanol and acetone. The results obtained by means of the heat of dilution were compared with the values given by pycnometry, and gave a relative difference in the range 0.2-3%. The precision depends on the slope of the calibration curve, which varies with the procedure and the concentration of the sample.     

Determination of sodium dodecylbenzene sulfonate in ternary mixtures by curie-point pyrolysis gas chromatography coupled to multivariate analysis

Summary Multivariate analysis was used to develop a viable method for determination of sodium dodecylbenzene sulfonate (DBS) by Curie-point pyrolysis gas chromatography. The pyrograms obtained were normalized against a maximum peak area and peak height. Normalized values were used for Quantification IV, which is one of the multivariate analysis methods, to select useful values initially. Then cluster analysis was carried out using both the selected values and their deviations. This method corresponds to qualitative analysis and indicates which data-base is similar to the sample. On the basis of this data-base, calibration data-bases are chosen. Principal component analysis (PCA) was performed using the calibration data-base and a set of sample data simultaneously. The principal component scores and contribution coefficients obtained were used to construct a calibration curve from which the DBS content of the sample was calculated. The results are in fair agreement with theoretical values.     

Screening of octanol-water partition coefficients for pharmaceuticals by pressure-assisted microemulsion electrokinetic chromatography

A rapid screening assay for the determination of octanol-water partition coefficients (log P(OW)) of pharmaceuticals was developed by using pressure-assisted microemulsion electrokinetic chromatography (MEEKC). The microemulsion system contains 50 mM sodium dodecyl sulfate, 0.87 M l-butanol, 82 mM heptane, and 50 mM borate-phosphate (2:3) at pH 10. Ten standard compounds with known log P(OW) values from -0.26 to 4.88 were used for constructing the calibration curve of log P(OW) against the MEEKC retention factor, log k. The log P(OW) values of the compounds were calculated based on the log k values measured by MEEKC and the slope and intercept of the calibration curve. For 13 literature and 32 Roche compounds, about 90% of the log P(OW) values measured by MEEKC are within 0.5 log units of the values from the literature and potentiometric titration. The throughput is about 2 samples/h using +20 kV voltage plus 5 mbar air pressure for separation. This MEEKC method is applicable for log P(OW) screening of weakly basic, weakly acidic, and neutral pharmaceuticals with log P(OW) = 0-5 and pKa < or = 10.