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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]

Among calibration approaches for organic compounds determination in complex matrices, external calibration, based in solutions of the analytes in solvent or in blank matrix extracts, is the most applied approach. Although matrix matched calibration (MMC) can compensates the matrix effects, it does not compensate low recovery results. In this way, standard addition (SA) and procedural standard calibration (PSC) are usual alternatives, despite they generate more sample and/or matrix blanks consumption need, extra sample preparations and higher time and costs. Thus, the goal of this work was to establish a fast and efficient calibration approach, the diluted standard addition calibration (DSAC), based on successive dilutions of a spiked blank sample. In order to evaluate the proposed approach, solvent calibration (SC), MMC, PSC and DSAC were applied to evaluate recovery results of grape blank samples spiked with 66 pesticides. Samples were extracted with the acetate QuEChERS method and the compounds determined by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS). Results indicated that low recovery results for some pesticides were compensated by both PSC and DSAC approaches. Considering recoveries from 70 to 120% with RSD <20% as adequate, DSAC presented 83%, 98% and 100% of compounds meeting this criteria for the spiking levels 10, 50 and 100 μg kg −1 , respectively. PSC presented same results (83%, 98% and 100%), better than those obtained by MMC (79%, 95% and 97%) and by SC (62%, 70% and 79%). The DSAC strategy showed to be suitable for calibration of multiresidue determination methods, producing adequate results in terms of trueness and is easier and faster to perform than other approaches. [ABSTRACT FROM AUTHOR]

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]