Single-pump heart-cutting two-dimensional liquid chromatography applied to the determination of fatty alcohol ethoxylates.

A setup for heart-cutting bi-dimensional liquid chromatography (LC-LC), constructed with a chromatograph provided with a single pump, an auxiliary 6-port 2-position valve (V6/2) and a column selector valve (VCS), is described. The possible ways of connecting the two valves for LC-LC, namely with V6/2 first followed by VCS and vice versa, are compared. The possibility of using the setups for preconcentration followed by the backwards transfer of the preconcentrated solutes to the detector or to a second column is also shown. The V6/2-first configuration for LC-LC was applied to the characterization of industrial fatty alcohol ethoxylates (FAEs) using UV-vis detection. For this purpose, the phthalates of the FAE oligomers were first obtained. The hydrocarbon series were separated along the 1st dimension by MeOH/water gradient elution on a C8 column at 60°C. Selected segments of the eluate were transferred to the 2nd dimension, where the EO oligomers of the isolated series were resolved by gradient elution with a complementary ACN/water mobile phase on a C8 column at 25°C. In addition, an average response factor of the hydrocarbon series of FAEs was proposed. To apply the factors, the average EO number of the series is first established by chromatographing one of the series along the 2nd dimension. Then, the factors are used to correct the peak areas of the isolated series which are obtained along the 1st dimension chromatogram, thus allowing the fast and accurate determination of the series in industrial FAEs. The method is particularly useful to characterize FAEs having large average EO numbers or constituted by mixtures of even and odd series.

Determination of non-ionic and anionic surfactants in industrial products by separation on a weak ion-exchanger, derivatization and liquid chromatography.

A method for the determination of priority surfactants, including fatty alcohol ethoxylates (FAE), alkylether sulfates (AES) and linear alkylbenzene sulfonates (LAS) is described. The samples were diluted with 50% methanol at pH 4 prior to solid-phase extraction on a weak anionic exchanger (WAX). The AES and LAS surfactant classes were retained, whereas the non-ionic components, including most FAE oligomers were eluted. After washing the WAX cartridge to remove cations, the remaining hydrophobic FAE oligomers were eluted using hot 80% methanol at pH 4 (at ca. 50°C). These two eluates were combined to constitute the non-ionic fraction. Then, AES and LAS were eluted using 80% MeOH with 3M NH3 followed by 95% methanol with 0.75M NH3. The two eluates obtained in basic media were combined to constitute the anionic fraction. The solvents were evaporated, the residues were dissolved in 1,4-dioxane, and esterification of the alcohols and transesterification of AES with phthalic anhydride was performed. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid and 0.1M NaClO4. The chromatogram of the non-ionic fraction showed the peaks of the resolved FAE oligomers. The chromatogram of the anionic fraction showed the peaks of the LAS homologues well resolved from those of the AES oligomers. The method was applied to laundry and industrial cleaners, shampoos and a shower gel.

Determination of fatty alcohol ethoxylates and alkylether sulfates by anionic exchange separation, derivatization with a cyclic anhydride and liquid chromatography.

A method for the separation, characterization and determination of fatty alcohol ethoxylates (FAE) and alkylether sulfates (AES) in industrial and environmental samples is described. Separation of the two surfactant classes was achieved in a 50:50 methanol-water medium by retaining AES on a strong anionic exchanger (SAX) whereas most FAE were eluted. After washing the SAX cartridges to remove cations, the residual hydrophobic FAE were eluted by increasing methanol to 80%. Finally, AES were eluted using 80:20 and 95:5 methanol-concentrated aqueous HCl mixtures. Methanol and water were removed from the FAE and AES fractions, and the residues were dissolved in 1,4-dioxane. In this medium, esterification of FAE and transesterification of AES with a cyclic anhydride was performed. Phthalic and diphenic anhydrides were used to derivatizate the surfactants in industrial samples and seawater extracts, respectively. Separation of the derivatized oligomers was achieved by gradient elution on a C8 column with acetonitrile/water in the presence of 0.1% acetic acid. Good resolution between both the hydrocarbon series and the successive oligomers within the series was achieved. Cross-contamination of FAE with AES and vice versa was not observed. Using dodecyl alcohol as calibration standard, and correction of the peak areas of the derivatized oligomers by their respective UV-vis response factors, both FAE and AES were evaluated. After solid-phase extraction on C18, the proposed method was successfully applied to the characterization and determination of the two surfactant classes in industrial samples and in seawater.

Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products.

Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 µm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 µm or 3 µm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners.

Comparison on photo-initiators for the preparation of methacrylate monolithic columns for capillary electrochromatography.

The synthesis of lauryl methacrylate monoliths for capillary electrochromatography by UV polymerization using several free-radical initiators (alpha,alpha'-azobisisobutyronitrile, 2,2-dimethoxy-2-phenylacetophenone, dibenzoyl peroxide (BPO) and lauroyl peroxide (LPO)) has been investigated. Using a 1,4-butanediol/1-propanol mixture as porogenic solvent, the influence of each initiator and its content on the morphological and electrochromatographical properties of beds was evaluated. Under their respective optimum content, satisfactory separations of a test mixture of PAHs with similar efficiencies (minimum plate heights of 8.0-12.7 microm obtained from Van Deemter plots) were achieved for the four investigated photo-initiators. The columns photo-polymerized with LPO provided the best compromise between chromatographic performance and analysis time. Moreover, this initiator showed a fine control in the column retention properties. The resulting monolithic columns exhibited a good run-to-run repeatability in the tested chromatographic parameters (RSD<2.4%) for all initiators investigated; and satisfactory column-to-column repeatability (RSD<6.0%), except for beds photo-polymerized with BPO (RSD<10.8%).

Characterization and determination of poly(vinylpyrrolidone) by complexation with an anionic azo-dye and nonequilibrium capillary electrophoresis.

Using capillary zone electrophoresis in nonequilibrium conditions, the complexes of poly(vinylpyrrolidone) (PVP) with anionic azo-dyes dissociate following a first-order kinetics. Two peaks due to the remaining PVP-dye complexes and the equilibrium concentration of the free dye, plus an exponential region due to the dye liberated by the complexes during the electrophoretic run, are obtained. This behaviour was closely similar to that described in the literature for protein-probe and DNA-protein mixtures, upon application of the technique known as nonequilibrium capillary electrophoresis of equilibrium mixtures or NECEEM. Using Congo Red and Acid Blue 113, information about the maximal stoichiometry and average stability of the PVP-dye complexes was obtained. The procedure was also useful to predict the average molecular mass of PVP and to determine PVP in cleaning products and pharmaceutical preparations. By using an appropriate probe, the procedure should be also useful to characterize and determine many other synthetic or natural nonionic polymers, and to study polymer-probe interactions.

Enzyme class identification in cleaning products by hydrolysis followed by derivatization with o-phthaldialdehyde, HPLC and linear discriminant analysis.

The enzymes present in raw materials of the cleaning industry (enzyme industrial concentrates) and in household cleaners were isolated by precipitation with acetone and hydrolyzed with HCl. The resulting amino acids were derivatized with o-phthaldialdehyde, and the derivatives were separated by HPLC. The peaks of 14 amino acids were observed using a C18 column and a multi-segmented gradient of acetonitrile-water in the presence of a 5 mM citric/citrate buffer of pH 6.5. Using either normalized peak areas (divided by the sum of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables, linear discriminant analysis models, capable of predicting the enzyme class, including proteases, lipases, amylases and cellulases, were constructed. For this purpose, both enzyme industrial concentrates and detergent bases spiked with them were included in the training set. In all cases, the enzymes of the evaluation set, including industrial concentrates, spiked detergent bases and commercial cleaners were correctly classified with assignment probabilities higher than 99%.