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• A novel SEC technique is proposed for the MWD characterization of ethyleneamines. • Narrow standard SEC calibration is applied using ethyleneamines. • The positively-charged packing material prevents adsorption of the ethyleneamines. • Good repeatibility of MW averages and MW distribution was achieved. Ethyleneamines have been produced and commercialized for decades in the chemical industry for a diverse range of applications. The presence of amine functional groups provides them opportunity to adsorb onto surfaces which can make them a very challenging sample matrix to analyze using separation techniques. In the present report, a new aqueous SEC-RI method, which enables MWD characterization of higher ethyleneamines, is described. The sample preparation was based on the dilute-and-shoot methodology. A surface-modified SEC column with positively charged groups attached to the stationary phase was used. The mobile phase composition (salt concentration, pH) was optimized to suppress interaction between the ethyleneamines and the packing material. Very symmetrical peak shapes were achieved for low MW monodisperse ethyleneamines despite their high primary amine content. MWD calculations were conducted using conventional narrow standard calibration with partial linear extrapolation of the calibration curve. The narrow standards were of the same chemistry as the samples of interest. Consequently, the standard components display a consistent behaviour towards the column packing as the sample components which makes the present method more robust and the interpretation of the quantitative results more convenient. Effect on the measured MW averages and MW distribution due to various experimental parameters (e.g., system variability, mobile phase preparation, sample concentration) were investigated showing good repeatability (RSD < 2%) for M n , M w , and M z. [ABSTRACT FROM AUTHOR]

Highlights • Multi-way chromatographic calibration protocols are reviewed. • The relation between chemometric models and data is discussed. • Application examples in various fields are detailed. Abstract This review discusses recent advances in both theory and experiment regarding multi-way chromatographic calibration. The focus is directed towards the chemometric processing of multi-way data arrays in which one or more instrumental modes describe the elution time behavior across liquid or gas chromatographic columns, and the remaining ones are of spectroscopic nature, e.g. UV–vis absorption, fluorescence emission (either as vectors or matrices) or mass spectrometry. Successful multi-way data decomposition into profiles for the contributing components allows one to perform quantitative analysis of complex samples. Quantitative applications usually make appropriate use of the second-order advantage which is inherent to multi-way data processing, and reduce the complexity of the data to a virtual univariate calibration which is typical of classical analytical chemistry. A number of recent experimental works will be discussed to illustrate the above concepts. [ABSTRACT FROM AUTHOR]

Highlights • A Bayesian approach can be used for model calibration and quality assessment. • The approach enables a prediction-based model uncertainty assessment. • Mechanistic models can perform reliable extrapolations despite substantial parameter uncertainty. • A sole parameter uncertainty assessment is insufficient for evaluating model quality. Abstract Mechanistic modeling of chromatography has been around in academia for decades and has gained increased support in pharmaceutical companies in recent years. Despite the large number of published successful applications, process development in the pharmaceutical industry today still does not fully benefit from a systematic mechanistic model-based approach. The hesitation on the part of industry to systematically apply mechanistic models can often be attributed to the absence of a general approach for determining if a model is qualified to support decision making in process development. In this work a Bayesian framework for the calibration and quality assessment of mechanistic chromatography models is introduced. Bayesian Markov Chain Monte Carlo is used to assess parameter uncertainty by generating samples from the parameter posterior distribution. Once the parameter posterior distribution has been estimated, it can be used to propagate the parameter uncertainty to model predictions, allowing a prediction-based uncertainty assessment of the model. The benefit of this uncertainty assessment is demonstrated using the example of a mechanistic model describing the separation of an antibody from its impurities on a strong cation exchanger. The mechanistic model was calibrated at moderate column load density and used to make extrapolations at high load conditions. Using the Bayesian framework, it could be shown that despite significant parameter uncertainty, the model can extrapolate beyond observed process conditions with high accuracy and is qualified to support process development. [ABSTRACT FROM AUTHOR]

The rational strategy in the evaluation of analytical measurement uncertainty is to combine the “whole method” performance data, such as precision and recovery, with the uncertainty contributions from sources not adequately covered by those data. This paper highlights some common mistakes in evaluating the uncertainty when pursuing that strategy, as revealed in current chromatographic literature. The list of the uncertainty components usually taken into account is discussed first and fallacies with the LOD- and recovery uncertainties are noted. Close attention is paid to the uncertainty arising from a linear calibration normally used. It is demonstrated that following a well-known formula for the standard deviation of an analytical result obtained from a straight line calibration leads to double counting the precision contribution to the uncertainty budget. Furthermore, the precision component itself is often estimated improperly, based on the number of replicates taken from the precision assessment experiment. As a result, the relative uncertainty from linear calibration is overestimated in the budget and may become the largest contribution to the combined uncertainty, which is clearly shown with an example calculation based on the literature data. [ABSTRACT FROM AUTHOR]

Biomolecular and pharmaceutical downstream processing is dominated by chromatographic separation, which is associated with high product quality, low capacity and high costs. The separation can be optimized to minimize the costs while achieving a high purity. This paper presents an experimental validation of a discretized multi-level elution (DiME) trajectory, implemented on commercially available chromatography equipment. The tertiary protein separation of ribonuclease A, cytochrome C and lysozyme was used as a case study. A mechanistic model was calibrated using step and linear gradient experiments. The model was simulated together with the state sensitivities with respect to model parameters, which was used in the Levenberg–Marquardt algorithm to fit the model response to the experimental data. The model was used to solve the dynamic optimization problem of maximizing the yield of cytochrome C given a 95% purity requirement, 1000 s processing time and 50 salt concentration levels in the elution trajectory. The model was spatially discretized using finite volumes and temporally discretized using direct collocation. The corresponding non-linear programming problem was solved with IPOPT. Once the optimal salt trajectory was found it was experimentally implemented on an ÄKTA Pure using an OPC interface. The optimal trajectory was analyzed in-line by UV absorbance measurements and off-line by analysis of collected fractions. The results presented in this study show the successful experimental realization of DiME trajectories and how to use model calibration, optimization and control to realize DiME trajectories for any chromatography separation problem. [ABSTRACT FROM AUTHOR]

Models to predict the total antioxidant capacity (TAC) of rooibos tea infusions from their chromatographic fingerprints and peak table data (content of individual phenolic compounds), obtained using HPLC with diode array detection, were developed in order to identify potential antioxidant markers. Peak table data included the content of 12 compounds, namely phenylpyruvic acid-2- O -glucoside, aspalathin, nothofagin, isoorientin, orientin, ferulic acid, quercetin-3- O -robinobioside, vitexin, hyperoside, rutin, isovitexin and isoquercitrin. The TAC values, measured using the oxygen radical absorbance capacity (ORAC) and DPPH radical scavenging assays, could be predicted from the peak table data or the chromatographic fingerprints (prediction errors 9–12%) using partial least squares (PLS) regression. Prediction models created from samples of only two production years could additionally be used to predict the TAC of samples from another production year (prediction errors < 13%) indicating the robustness of the models in a quality control environment. Furthermore, the uninformative variable elimination (UVE)-PLS method was used to identify potential antioxidant markers for rooibos infusions. All individual phenolic compounds that were quantified were selected as informative variables, except vitexin, while UVE-PLS models developed from chromatographic fingerprints indicated additional antioxidant markers, namely ( S )-eriodictyol-6- C -glucoside, ( R )-eriodictyol-6- C -glucoside, aspalalinin and two unidentified compounds. The potential antioxidant markers should be validated prior to use in quality control of rooibos tea. [ABSTRACT FROM AUTHOR]

Highlights: [•] The precision of the final or reportable result can be optimised. [•] An equation is developed to optimise also the format of calibration. [•] Optimisation is possible by varying the number of determinations. [•] Reliable variances are the basis for optimisation. [•] Obtaining reliable variance contributions is the aim of precision studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] A new method was proposed for maintaining the long term stability of LC–MS/MS calibration models. [•] The method can obtain accurate quantitative results even if variation occurs in sensitivity of LC–MS. [•] It offers significant advantages over other methods in terms of performance and implementation simplicity. [Copyright &y& Elsevier]

Abstract: Size exclusion chromatography (SEC) is often applied for characterization of protein–polyethylene glycol (PEG) conjugates regarding the number of attached PEG chains (PEGamers). SEC analysis is advantageous as it is precise, robust, and straightforward to establish. However, most SEC based assays have a maximal throughput of a few samples per hour. We present a strategy to increase analytical throughput based on combining a short column with a fast flow rate, and finally multivariate calibration in order to compensate for the resolution lost in the trade off for speed. Different multivariate approaches were compared and multilinear regression was shown to result in the most precise calibrations. Further, a dynamic calibration approach was developed in order to account for changes in column performance over time. In this way, it was possible to establish a highly precise assay for protein PEGamer quantification with a throughput of 30 samples per hour. [Copyright &y& Elsevier]

Abstract: Polycyclic aromatic sulfur heterocyclics (PASHs) consist mainly of thiophene class compounds, and are the most important organosulfur compounds in crude oils and sediment extracts. Dibenzothiophene (DBT) and its methylated homologues were identified on mass chromatograms by comparison with retention indices published in the literature. Some isomers of dimethyldibenzothiophene and trimethyldibenzothiophene that were tentatively identified in previous reports have been determined here by comparison with calculated retention indices and taking the substitution pattern of the methyl groups into account. The response factors relating that of dibenzothiophene to internal standards were obtained by GC–MS analyses of mixture solutions with different concentration ratios. We concluded that DBT-d8 (octadeutero-dibenzothiophene) is the optimal internal standard for quantitative analyses of the thiophene compound class in oils, coal, and sediment extracts. Calibration experiments for each class of compounds are absolutely necessary when quantifying polycyclic aromatic hydrocarbons and other heterocyclics with a stable isotope labeled internal standard. [Copyright &y& Elsevier]

Abstract: This contribution discusses the difference in chromatographic performance when switching from the customary employed constant flow rate gradient elution mode to the recently re-introduced constant pressure gradient elution mode. In this mode, the inlet pressure is maintained at a set value even when the mobile phase viscosity becomes lower than the maximum mobile phase viscosity encountered during the gradient program. This leads to a higher average flow rate compared to the constant flow rate mode and results in a shorter analysis time. When both modes carry out the same mobile phase gradient program in volumetric units, normally identical selectivities are obtained. However, small deviations in selectivity are found due to the differences in pressure and viscous heating effects. These selectivity differences are of the same type as those observed when switching from HPLC to UHPLC and are inevitable when speeding up the analysis by applying a higher pressure. It was also found that, when using concentration-sensitive detectors, the constant pressure elution mode leads to identical peak areas as the constant flow rate mode. Also the linearity is maintained. In addition, the repeatability of the peak area and retention time remains the same when switching between both elution modes. [Copyright &y& Elsevier]

Abstract: A model commonly used to describe the separation of biomolecules was used to simulate the harsh environment when eluting neodymium, samarium, europium and gadolinium with a hot acid. After calibration, the model was used to optimise the preparative separation of europium, as this is the most valuable of the four elements. A kinetic dispersive model with a Langmuir mobile phase modulator isotherm was used to describe the process. The equilibration constant, the stoichiometric coefficient and the column capacity for the components were calibrated. The model fitted the experimental observations well. Optimisation was achieved using a differential evolution method. As the two objective functions used in optimising the process, productivity and yield, are competing objectives, the result was not a single set point but a Pareto front. [Copyright &y& Elsevier]

Abstract: The selectivity window of size-based separations of macromolecules was tailored by tuning the macropore size of polymer monolithic columns. Monolithic materials with pore sizes ranging between 75nm and 1.2μm were prepared in situ in large I.D. columns. The dominant separation mechanism was hydrodynamic chromatography in the flow-through pores. The calibration curves for synthetic polymers matched with the elution behavior by HDC separations in packed columns with ‘analyte-to-pore’ aspect ratios (λ) up to 0.2. For large-macropore monoliths, a deviation in retention behavior was observed for small polystyrene polymers (M r <20kDa), which may be explained by a combined HDC–SEC mechanism for λ <0.02. The availability of monoliths with very narrow pore sizes allowed investigation of separations at high λ values. For high-molecular weight polymers (M r >300,000Da) confined in narrow channels, the separation strongly depended on flow rate. Flow-rate dependent elution behavior was evaluated by calculation of Deborah numbers and confirmed to be outside the scope of classic shear deformation or slalom chromatography. Shear-induced forces acting on the periphery of coiled polymers in solution may be responsible for flow-rate dependent elution. [Copyright &y& Elsevier]

Abstract: We have developed a method for quantification of a specific monoclonal IgM directed toward embryonic stem cells based on a peptide affinity monolith. A peptide affinity ligand with the sequence C–C–H–Q–R–L–S–Q–R–K was obtained by epitope mapping using peptide SPOT synthesis. The peptide ligand was covalently immobilized by coupling the N-terminal cysteine to a monolithic disk that was previously modified with iodated spacer molecules. The monolithic disc was used for quantification of purified IgM and for IgM present in mammalian cell culture supernatant. We observed 17% unspecific binding of IgM to the monolithic disk and additionally a product loss in the flow through of 20%. Nevertheless, calibration curves had high correlation coefficients and inter/intra-assay variability experiments proved sufficient precision of the method. A limit of quantification of 51.69μg/mL for purified IgM and 48.40μg/mL for IgM in cell culture supernatant could be calculated. The binding capacity was consistent within the period of the study which included more than 200 cycles. The analysis time of less than 2min is an advantage over existing chromatographic methods that rely on pore diffusion. [Copyright &y& Elsevier]

A robust method for reduction of instrument differences, the vector of calibration ratios, was developed to eliminate differences in unit resolution mass spectra of fatty acid methyl esters (FAME) caused by experimental conditions. Mass spectra of FAMEs were analyzed by two different mass spectrometers and after application of different tune procedures. The proposed method could remove 51–95% of the systematic difference in spectra caused by instrumental conditions. Highly similar spectra, which were incorrectly identified because of the contribution from experimental conditions, could be correctly identified after application of the calibration vector. The proposed method is simple, easy to implement and shows robustness when applied on spectra that is outside the range spanned by the calibration sample. [ABSTRACT FROM AUTHOR]

Abstract: High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5min in a concentration range of 23–250μg/ml. The calibration range of the method could be extended from 4 to 1000μg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity. [Copyright &y& Elsevier]

Abstract: Zirconia (ZrO2) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of organo-phosphates. To investigate whether this affinity can be used for selective preconcentration, the retention of methyl, ethyl, and propylphosphonic acid (MPA, EPA and PPA) and inorganic anion matrix components on ZrO2 was investigated. Only organo-phosphates and sulfate exhibited retention on zirconia. After preconcentration, the retained species were eluted from ZrO2 by 0.75mM Na2CO3, and separated by a Dionex Ionpac AS11 anion-exchange column (250mm×2mm I.D.) and a Hypercarb RPLC column (50mm×4.6mm I.D., 3μm) in series followed by suppressed conductivity detection. Calibration curve showed a linear response for MPA, EPA and PPA in the range of 0.01μM to 1μM (R 2 >0.9989). Detection limits after preconcentration of a 10mL sample were 0.16, 0.19 and 0.16μg/L for MPA, EPA, and PPA, respectively. [Copyright &y& Elsevier]

Abstract: A simple, rapid, and low-cost coulometric method for direct detection of glyphosate and aminomethylphosphonic acid (AMPA) in water samples using anion-exchange chromatography and coulometric detection with copper electrode is presented. Under optimized conditions, the limits of detection (LODs) (S/N =3) were 0.038μgml−1 for glyphosate and 0.24μgml−1 for AMPA, without any preconcentration method. The calibration curves were linear and presented an excellent correlation coefficient. The method was successfully applied to the determination of glyphosate and AMPA in water samples without any kind of extraction, clean-up, or preconcentration step. No interferent was found in the water, like this, the recovery was, practically, 100%. [Copyright &y& Elsevier]

Abstract: A new calibration method for high-performance liquid chromatography was validated. The method was called single-injection calibration approach (SICA) because it allowed to obtain a complete calibration curve by means of a single injection of a standard solution containing several non-volatile and semi-volatile organic compounds at different concentration levels. The compounds studied included carboxylic acids, polyalcohols, carbohydrates and water-soluble vitamins. This method allowed a 1–7-fold reduction in the analysis time with regard to conventional calibration methods. The method was applied to three different chromatographic detection methods: refractive index (RI) detection, diode array detection (DAD) and inductively coupled plasma atomic emission detection (ICP-AED). Good linearity was achieved (r 2 >0.999) for the three detection methods but signal correction was required for RI detection and DAD. This fact demonstrated that ICP-AES was the most universal because the signal obtained for non-volatile and semi-volatile organic compounds was not a function of the chemical nature of the compound and only depended on the mass content of carbon. The method was validated by analyzing a reference non-fat milk powder sample as well as several real food samples (three fruit juices, four wines, three candies and a multivitamin complex). [Copyright &y& Elsevier]

Abstract: In common with all gas chromatography (GC) methods, comprehensive two-dimensional gas chromatography (GC×GC) has the potential to provide both qualitative and quantitative analysis. There are fundamental differences in the way one-dimensional (1D-GC) and GC×GC results are interpreted for these parameters. Since 1D-GC produces a single measured peak in the chromatogram, there is a single retention time, and associated with this a single peak response (either area or height). Peak area and height are related by peak width. GC×GC produces a series of modulated peaks at the detector. Thus, the peak metrics of retention, area and height for one component are now not simple single values for one peak, but rather are derived from the multiple peak distribution generated by the modulation process. The peak retention is interpreted in terms of two-dimensional coordinates in a retention plane. In this study, a brief background review to quantification in GC×GC is provided. Previous reviews cover aspects of quantitative GC×GC studies up to the year 2005, including different approaches to quantification, and reports of quantitative analysis with different detectors, for different compounds classes, and in different matrices. Other studies have developed chemometric approaches based on multivariate analysis to provide quantitative reporting of individual compounds. The coverage of the earlier reviews has been updated to include material that has been presented since 2005 and includes considerations of valve-based modulation. Recently the modulation ratio (M R) concept was proposed and intended to clarify the meaning of modulation number (n M) in GC×GC, which was shown to be a rather poorly defined parameter. Based on the prior studies that introduced this concept, the role of quantitative analysis is investigated here through calculation of the peak areas and peak area ratios of selected series of modulated peaks in GC×GC. The application of isotopically labelled reference compounds for polycyclic aromatic hydrocarbon (PAH) analysis is used here to develop the quantitative metric approach. It is shown that by selecting the two or three major modulated peaks for solutes and internal standards, comparing the response ratio with the sum of all modulated peaks and also with the reference non-modulated result, quantification is statistically equivalent. Thus, adequate quantitative analysis and calibration can be accomplished by using selected major modulated peaks for each compound. This may simplify quantitative interpretation of GC×GC data. [Copyright &y& Elsevier]

Abstract: The sample matrix can cause an enhancement in the observed chromatographic response for pesticide residues in a matrix extract compared with the same concentration in a matrix-free solution. The matrix increases the transfer of pesticides from hot vaporizing injectors by reducing the thermal stress for labile compounds and by masking active sites in the injector responsible for the adsorption or decomposition of polar pesticides. The use of different injector types and matrix simplification procedures can reduce matrix-induced enhancement but do not eliminate it. The most effective strategy is to use matrix-matched calibration standards or analyte protectants which equalize the response enhancement for calibration standards and sample extracts. From a practical point of view it is important that the method used to correct for matrix-induced enhancement is compatible with low system maintenance. The different approaches for correcting matrix-induced enhancement for calibration in pesticide residue analysis are discussed and compared in this review. [Copyright &y& Elsevier]

Abstract: Chemometric techniques for calibration with three-way signals are sufficiently developed for their use in routine analysis. The advantage of the second order property (the possibility of quantifying an analyte in the presence of interferents) together with the guarantee of the uniqueness of the decomposition, what means to extract the signal corresponding only to the analyte of interest, make these calibration techniques especially useful for the quantification and identification of analytes in complex samples. This has a particular interest in the identification and quantification of banned substances or substances with a specified maximum limit. The paper describes the theory of the calibration methodology in relation to the signal order and then focuses the analysis on the three-way techniques commonly used in calibration: n-way partial least squares, multivariate curve resolution and parallel factor analysis. The figures of merit needed for the accreditation of analytical methods are analyzed from the viewpoint of n-way calibrations in chromatography. [Copyright &y& Elsevier]

Abstract: This paper reviews calibration-, uncertainty-, and recovery-related documents from 10 consensus-based organizations. The main points from each treatise are summarized. Also included is a critique of the various approaches, as well as recommendations for a statistically sound protocol that is more compatible with chromatographic data. [Copyright &y& Elsevier]

Abstract: The effect of the amount of matrix compounds in comprehensive two-dimensional gas chromatographic (GC×GC) separation of polycyclic aromatic hydrocarbons (PAHs) was investigated. The second dimension column was overloaded by progressively increasing the matrix amount and the separation was evaluated by inspecting peak widths, asymmetries, resolution, retention times, peak areas and volumes of the target compounds. In quantitative analysis, two different calibration methods were tested for both raw sediment extracts and cleaned extracts, i.e. for extracts in which the matrix was removed by a liquid chromatographic clean-up step. The quality of the separation was not significantly disturbed in terms of peak width, asymmetry and resolution when the amount of matrix was increased. However, the depth of the valley between PAH and preceding matrix peak increased with an increase in matrix amount and the repeatabilities of retention times, peak areas and volumes decreased. In quantitative analysis, calibration by using areas had a tendency to underestimate the trace amounts of PAH, especially for the non-cleaned samples. In case of volume calibration, the most accurate results were obtained by external calibration for both cleaned and non-cleaned samples. [Copyright &y& Elsevier]

Abstract: A bi-dimensional (non-orthogonal) chromatographic system made of two columns of different length (longer along the first direction and shorter on the second) but packed with identical (or similar) materials has been employed to perform automated on-line fraction analysis and peak deconvolution of multicomponent overloaded band profiles. Multicomponent overloaded peaks, eluted from the first column, are sampled through a remotely-controlled switching valve and fractions are injected, under analytical conditions, directly into the second direction. This set-up serves a twofold purpose. First of all, it has been possible to use linear calibration curves for detector calibration (DC). Secondly, since single component peaks were resolved on the second direction, individual concentration profiles have been obtained beneath the envelop of overlapping bands. All the information necessary to operate the bi-dimensional system can be achieved by analyzing the chromatographic behavior of the mixture on the shorter column under linear conditions. Therefore, solutions of unknown concentration can be prepared and their composition reconstructed with a simple chromatographic run. Two- and three-component overloaded mixtures have been employed to validate the system. [Copyright &y& Elsevier]

Abstract: Calibration of SPME by the injection of liquid standard solution is a common method for SPME quantitative analysis by gas chromatography. The feasibility of this calibration method relies on the assumption that sample transfer efficiencies are the same for both the liquid injection and the SPME fiber injection. Sample transfer efficiencies for liquid injection and SPME fiber injection were studied in this paper. The results showed that the sample transfer efficiency for liquid injection was affected by several factors, such as the dimensions of the liner, the presence of the wool, and the temperature of the injector. The sample transfer efficiency for SPME fiber injection was affected by the cross-sectional area of the space between the column and the liner, the carrier gas flow rate, and the length of the column inside the liner. An equation was proposed to estimate the sample loss rate for SPME injection. It was found that the use of a direct injection (DI) liner and program temperature vaporizing provides high sample transfer efficiencies, for both liquid injection and SPME fiber injection. When a common SPME straight liner is used, large outer diameter (o.d.) pre-column will help to achieve high sample transfer efficiency. [Copyright &y& Elsevier]

Abstract: Dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) were determined in plant and water samples by capillary gas chromatography using microwave induced-plasma atomic emission spectrometry for detection. The analytes were leached from the solid samples into methanol by using an ultrasonic probe, and a portion of the extract was preconcentrated by means of a purge-and-trap system before being chromatographed. The analytes were directly purged from the water samples in the presence of 6% (v/v) methanol. Element-specific detection and quantification was carried out by monitoring the selenium (196nm) emission line. Calibration curves were obtained by plotting peak area versus concentration and the correlation coefficients for linear calibration were 0.9999 for both analytes. Detection limits of 0.8 and 1.1ngl−1 were obtained for DMSe and DMDSe, respectively, for water samples. For plant materials, the detection limits calculated for 0.5g samples were 0.3 and 0.4ngg−1 for DMSe and DMDSe, respectively. Concentration levels of DMSe ranging from 1.2 to 4.2ngg−1 were found in some of the plant materials analyzed. No DMDSe was found in any of the samples. The accuracy of the method was checked by analyzing different spiked water and plant samples. [Copyright &y& Elsevier]

Abstract: Restricted access material (RAM) has been used in the packing of a solid-phase extraction (SPE) column for on-line extractions under turbulent flow conditions. The bio-compatible RAM material works by the principle of size exclusion in addition to conventional reversed-phase chromatography, thereby allowing the extraction and preconcentration of small analyte molecules from biological samples such as plasma. Using small column dimensions (0.76mm×50mm) and a consequently high linear velocity, turbulent flow was achieved during online sample extractions. The improved mass-transfer rate characteristic of turbulent flow allows fast sample cleanup without decreased extraction efficiency. The novel use of the RAM column, connected upstream to a C18 monolithic column, allowed the direct injection, extraction, separation, and MS/MS detection of plasma samples spiked with rofecoxib in a span of 5min. Calibration curves obtained using this RAM turbulent flow coupled column method showed good linearity (R 2 >0.99) and reproducibility (%RSD≤7%). The lower limit of quantitation of rofecoxib in plasma samples was found to be 40ng/ml. The extraction method showed good recovery of rofecoxib from a plasma matrix with minimal signal loss and robustness after more than 200 plasma injections. [Copyright &y& Elsevier]

Abstract: In many studies of nonlinear or preparative chromatography, chromatographic signals must be recorded for relatively concentrated solutions and the detectors, that are designed for analytical applications and are highly sensitive, must be used under such experimental conditions that their responses are often nonlinear. Then, a calibration curve is needed to derive the actual concentration profiles of the eluates from the measured detector response. It becomes necessary to derive a relationship between the concentration of the eluent and the detector signal at any given time. The simplest approach consists in preparing a series of solutions of known concentrations and in flushing them successively through the detector cell, recording the height of the plateau response obtained. However, this method requires relatively large amounts of the pure solutes being studied and these are not always available or they may be most costly, although these solutions can be recovered. We describe and validate an alternative procedure providing this calibration from a series of peaks recorded upon the injection of increasingly large pulses of the studied compound. [Copyright &y& Elsevier]

Abstract: Applications of solid-phase microextraction (SPME) in the measurement of very hydrophobic organic compounds (VHOCs) are limited, partly due to the difficulty of calibrating SPME fibers for VHOCs. This study used a static SPME strategy with a large sample volume (1.6L) and a five-point calibration procedure to determine the distribution coefficients for a large suite of polychlorinated biphenyls (PCBs) and chlorinated pesticides between a polydimethylsiloxane (PDMS) phase (100μm thickness) coated on a glass fiber and seawater. An extraction time of 12 days was deemed adequate for equilibrium calibration from kinetic experiments. Two groups of randomly selected fibers divided into three batches (up to nine fibers in each batch) were processed separately with two gas chromatography–mass spectrometry (GC–MS) systems. Matrix effects arising from losses of the analytes to glass container walls and stirring bars were corrected. Relative standard deviations within the same batch were generally smaller than those for the entire group. Furthermore, K f V f (K f and V f are the distribution coefficient of an analyte between the polymer-coated fiber and aqueous phase and the fiber volume, respectively) values determined with two GC–MS systems were statistically different. These results indicate the calibrated K f V f values were less affected by the random selection of SPME fibers than by other experimental conditions, and therefore average K f V f values may be used for the same type of commercially available SPME fibers. The relative accuracy of our calibration method was similar to that of a previous study [P. Mayer, W.H.J. Vaes, J.L.M. Hermens, Anal. Chem. 72 (2000) 459] employing different coating thickness and calibration procedure. The present study also obtained a bell-shaped relationship between log K f and log K ow (octanol–water partition coefficient) for PCB congeners with the maximum log K f corresponding to log K ow ∼6.5. This bell-shaped relationship was attributed mainly to steric effects arising from the interplay between the PDMS thickness and molecular sizes of the target analytes. [Copyright &y& Elsevier]

A HPTLC–densitometric method, based on an external standard approach, was developed in order to obtain a novel procedure for routine analysis of secoisolariciresinol diglucoside (SDG) in flaxseed with a minimum of sample pre-treatment. Optimization of TLC conditions for the densitometric scanning was reached by eluting HPTLC silica gel plates in a horizontal developing chamber. Quantitation of SDG was performed in single beam reflectance mode by using a computer-controlled densitometric scanner and applying a five-point calibration in the 1.00–10.00μg/spot range. As no sample preparation was required, the proposed HPTLC–densitometric procedure demonstrated to be reliable, yet using an external standard approach. The proposed method is precise, reproducible and accurate and can be employed profitably in place of HPLC for the determination of SDG in complex matrices. [Copyright &y& Elsevier]

The determination of the interdetector volume (IDV) in size-exclusion chromatography (SEC) of polymers with dual multiangle light-scattering/concentration detection based on the s-detection is demonstrated. By s-detection the determination is meant of local molecular mass from a known relation between molecular mass and radius of gyration, obtained from the angle dependence of the intensity of scattered light. IDV is found basing on the stipulation of equal slope of local calibrations found by the dual light-scattering/concentration detection, referred to as the w- and s-detection. As the s-detection uses just the light-scattering detector and, therefore, is independent of the value of IDV, this quantity can be found by searching for its value that gives the local calibration dependence obtained by the dual detection closest to that determined independently by the s-detection. [Copyright &y& Elsevier]

Gradient polymer elution chromatography (GPEC) is rapidly becoming the analytical method of choice for determining the chemical composition distribution (CCD) of synthetic polymers. GPEC can be performed in traditional (strict precipitation–redissolution mechanism) or interactive (normal- and reversed-phase) modes, and results may be qualitative, semi-quantitative, or fully quantitative. Quantitative approaches have thus far relied on colligative or end group techniques for determining the values of standards used in constructing the GPEC calibration curve. While the values obtained from said methods are number-averages, they are assigned to the peak apexes of the standards (i.e. assigned as peak averages). This creates a determinate error in the quantitation, referred to herein as “moment bias”. In this paper we determine moment bias for a series of styrene–acrylonitrile (SAN) copolymers, where the distribution and averages of the AN% have been measured using normal-phase (NP) GPEC. We also correct for the effect via statistical treatment of the chromatographic data. [Copyright &y& Elsevier]

The performance of two multivariate calibration measurements, multivariate selectivity (SELs) and scalar net analyte signal (scalar NAS), as chromatographic objective functions (COFs), was investigated. Since both assessments are straightforwardly related to the quantification of analytes in the presence of interferents, they were expected to confer new features in the optimisation of compound resolution, not present in conventional assessments. These capabilities are especially interesting in situations of low resolution, where peak deconvolution becomes an attractive alternative. For comparison purposes, chromatographic resolution (Rs) and peak purity (ps) were used as reference COFs. In order to correlate COFs with the probability of deconvolution error, an artificial peak crossing was used to generate 73 different peak arrangements, which were deconvolved using three different methods. SELs exhibited the best correlation, which allowed predicting properly the risk of obtaining inaccurate deconvolutions. The optimisation of a poorly resolved mixture of 16 aromatic compounds by reversed-phase liquid chromatography with methanol–water and acetonitrile–water mobile phases was examined to investigate the differences in performance among the resolution criteria. In situations like these, SELs tends to consider acceptable mobile phase compositions with partial coelution, which permits however the deconvolution with low errors. In contrast, ps selects compositions where the resolution of some compounds is sacrificed to enhance the separation of others. Scalar NAS was not so favourable as expected, since it depends on sampling frequency and peak widening. SELs was not affected by these factors. [Copyright &y& Elsevier]

We used the Generalized Rank Annihilation Method (GRAM), a second-order calibration method, to quantify aromatic sulfonates in water with high-performance liquid chromatography (HPLC) when interferences coeluted with the analytes of interest. With GRAM, we can quantify in only two chromatographic analyses, one for a calibration sample and one for the unknown sample. The calculated concentrations were not statistically different to those obtained when the chromatographic separation of the unknown sample was modified in order to completely separate the analyte from the interferences before univariate calibration. With GRAM, the concentrations are determined much more quickly because a complete resolution is not required. [Copyright &y& Elsevier]