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There is a constantly growing demand for monoclonal antibodies for therapeutic and diagnostic applications. Due to this trend the development of new and cost-efficient purification methods is of high importance. This study investigates the separation of a monoclonal antibody from model impurity bovine serum albumin using anionic polyelectrolytes. Different types and molecular weights of anionic polyelectrolytes were screened for their applicability as precipitation agents. Polyanetholesulfonic acid was identified as most promising candidate. Furthermore, the influence and interactions of various parameters on antibody precipitation were investigated by means of Design of Experiments. Design of Experiments provided a fast identification of influencing parameters and parameter interactions. The strongest influence on yield and selectivity of precipitation showed the polyelectrolyte concentration and its interaction with the pH value and the sodium chloride and bovine serum albumin concentration. The interactions identified showed, that parameters should be investigated and optimized together, not stand-alone.

This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP–ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP–ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 μg/l concentration of sulfite in the loaded sample (0.9 μl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99–101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH).

This work deals with zone electrophoresis (ZE) separations of proteins on a poly(methyl methacrylate) chip with integrated conductivity detection. Experiments were performed in the cationic mode of the separation (pH 2.9) with a hydrodynamically closed separation compartment and suppressed electroosmotic flow. The test proteins reached the detector in less than 10 min under these working conditions and their migration times characterized excellent repeatabilities (0.1-0.6% RSD values). The chip-to-chip agreements of the migration times, evaluated from the ZE runs performed on three chips, were within 1.5%. The conductivity detection provided for protein, loaded on the chip at 10-1000 microg/ml concentrations, detection responses were characterized by 1-5% RSD values of their peak areas. Such migration and detection performances made a frame for reproducible baseline separations of a five-constituent mixture (cytochrome c, avidin, conalbumin, human hemoglobin and trypsin inhibitor). On the other hand, a high sample injection channel/separation compartment volume ratio of the chip (500 nl/8500 nl) restricted the resolution of proteins of very close effective mobilities in spite of the fact that in the initial phase of the separation an electric field stacking was applied. A maximum macroconstituent/trace constituent ratio attainable for proteins on the chip was assessed for cytochrome c (quantifiable when its concentration in the loaded sample was 10 microg/ml) and apo-transferrin (containing a trace constituent migrating in the position of cytochrome c detectable when the load of apo-transferrin was 2000 microg/ml). This assessment indicated that a ratio of 1000:1 is attainable with the aid of conductivity detection on the present chip.

Isotachophoretic (ITP) separation and determination of a group of 13 organic and inorganic acids, currently present in wines, on a poly(methyl methacrylate) chip provided with on-column conductivity detection was a subject of a detailed study performed in this work. Experiments with the ITP electrolyte systems proposed to the separation of anionic constituents present in wine revealed that their separation at a low pH (2.9) provides the best results in terms of the resolution. Using a 94 mm long separation channel of the chip, the acids could be resolved within 10-15 min also in instances when their concentrations corresponded to those at which they typically occur in wines. A procedure suitable to the ITP determination of organic acids responsible for some important organoleptic characteristics of wines (tartaric, lactic, malic and citric acids) was developed. Concentrations of 2-10 mg/l of these acids represented their limits of quantitation for a 0.9 microl volume sample loop on the chip. A maximum sample load on the chip, under the preferred separating conditions, was set by the resolution of malate and citrate. A complete resolution of these constituents in wine samples was reached when their molar concentration ratio was 20:1 or less. ITP analyses of a large series of model and wine samples on the chip showed that qualitative indices [RSH (relative step height) values] of the acids, based on the response of the conductivity detector, reproduced with RSD better than 2% while reproducibilities of the determination of the acids of our interest characterized RSD values better than 3.5%.

The use of a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to electrophoretic separations of a group of inorganic anions (chloride, nitrate, sulfate, nitrite, fluoride and phosphate) that need to be monitored in various environmental matrices was studied. The electrophoretic methods employed in this study included isotachophoresis (ITP) and capillary zone electrophoresis (CZE) with on-line coupled ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the separations performed by these methods. ITP separations on the chip provided rapid resolutions of sub-nmol amounts of the complete group of the studied anions and made possible rapid separations and reproducible quantitations of macroconstituents currently present in water samples (chloride, nitrate and sulfate). However, concentration limits of detection attainable under the employed ITP separating conditions (2-3 x 10(-5) mol/l) were not sufficient for the detection of typical anionic microconstituents in water samples (nitrite, fluoride and phosphate). On the other hand, these anions could be detected at 5-7 x 10(-7) mol/l concentrations by the conductivity detector in the CZE stage of the ITP-CZE combination on the CC chip. A sample clean-up performed in the ITP stage of the combination effectively complemented such a detection sensitivity and nitrite, fluoride and phosphate could be reproducibly quantified also in samples containing the macroconstituents at 10(4) higher concentrations. ITP-CZE analyses of tap, mineral and river water samples showed that the CC chip offers means for rapid and reproducible procedures to the determination of these anions in water (4-6 min analysis times under our working conditions). Here, the ITP sample pretreatment concentrated the analytes and removed nanomol amounts of the macroconstituents from the separation compartment of the chip within 3-4 min. Both the ITP and ITP-CZE procedures required no or only minimum manipulations with water samples before their analyses on the chip. For example, tap water samples were analyzed directly while a short degassing of mineral water (to prevent bubble formation during the separation) and filtration of river water samples (to remove particulates and colloids) were the only operations needed in this respect.