Construction of polymer monolithic columns in polypropylene ink-pen tubes for separation of proteins by cation-exchange chromatography.

We describe the synthesis of polymer monoliths inside polypropylene tubes from ink pens. These tubes are cheap, chemically stable, and resistant to pressure. UV-initiated grafting with 5 wt% benzophenone in methanol for 20 min activated the internal surface, thus enabling the covalent binding of ethylene glycol dimethacrylate, also via photografting. The pendant vinyl groups attached a poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith prepared via photopolymerization. These tubes measured 100-110 mm long, with 2 mm of internal diameter. The parent monoliths were functionalized with Na2 SO3 or iminodiacetate to produce strong and weak cation exchangers, respectively. The columns exhibited permeabilities varying from 2.7 to 3.3 × 10-13  m2 , which enabled the separation of proteins at 500 µL/min and back pressures <2.8 MPa. Neither structure collapse nor monolith detachment occurred at flow rates as high as 2.0 mL/min, which produced back pressures between 6.9 and 9.0 MPa. The retention times of ovalbumin, ribonuclease A, cytochrome C, and lysozyme in salt gradient at pH 7.0 followed the order of increasing isoelectric points, confirming the cation exchange mechanism. Separation and determination of lysozyme in egg white proved the applicability of the columns to the analysis of complex samples.

Complexing porous polymer monoliths for online solid-phase extraction of metals in sequential injection analysis with electrochemical detection.

A monolithic column affording complexing groups was synthesized for automated solid-phase extraction of potentially toxic metal ions in a low-pressure sequential injection analyzer. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths were synthesized by free-radical polymerization in the coffins of fused silica-lined stainless-steel tubes (2.10 mm i.d. × 5-6 cm length). High permeability (4.33 × 10-13 m2) was achieved for monoliths polymerized for 24 h at 60 °C from a mixture of 30 wt% glycidyl methacrylate, 10 wt% ethylene glycol dimethacrylate, 35 wt% n-propanol, 20 wt% 1,4 butanediol and 5 wt% water. Azobisisobutyronitrile (1 wt% with respect to the monomers) initiated the free-radical polymerization. These generic columns were modified with iminodiacetate to create complexing functionalities on the polymer surface, being further used for online solid-phase extraction of Cu2+, Pb2+ and Cd2+ from natural, tap and drinking waters prior to their determination by stripping chronopotentiometry. The high permeability of the column allowed the loading, washing, eluting and reconditioning steps to be made at flow rate of 10 µL s-1. The limits of detection and quantification achieved by processing 1500 µL of sample were 0.51 and 1.7 µg L-1 for Cu2+, 1.4 and 4.7 µg L-1 for Pb2+, and 1.2 and 3.8 µg L-1 for Cd2+, respectively. Recoveries were between 75.5% and 117%, obtained by quantification via external calibration curves, thus eliminating the need for the laborious standard addition strategies.

An electrochemical sequential injection method to investigate the adsorption of selenite on Fe(III) polyhydroxy cations intercalated vermiculite.

A sequential injection - square wave anodic stripping voltammetry (SI-SWASV) method for determination of Se(IV) at a gold working electrode was developed to investigate the adsorption of Se(IV) onto vermiculite intercalated with Fe(III) polyhydroxy cations. The limits of detection and quantification were 0.060 and 0.20 µmol L-1, respectively (4.7 and 15.7 µg L-1). The linearity was up to 1.0 µmol L-1, and the sampling throughput was 18 analyses h-1. The proposed approach is a low-cost alternative to more expensive spectrometric methods. Adsorption onto vermiculite intercalated with Fe(III) polyhydroxy cations removed 93% of Se(IV) from a 1.0 µmol L-1 solution (250 mL) after 5 min of contact time with 625 mg of adsorbent. Adsorption isotherms (25.0 ± 0.5 °C) were fitted by the Freundlich equation resulting in 1/n = 0.51 ± 0.03 and Kf = (1.584 ± 0.002) × 103 µmol1-1/n g-1 L1/n (r2 = 0.995). Fitting by the Langmuir equation resulted in an adsorption constant of 0.026 ± 0.008 L g-1 and adsorption capacity of 47 ± 5 µmol g-1 (3.7 ± 0.4 mg g-1) (r2 = 0.97). This capacity was higher than that found for several other iron oxides, but lower than that obtained for oxide/hydroxide-based Fe(III) nanoparticles.

Semi-micro reversed-phase liquid chromatography for the separation of alkyl benzenes and proteins exploiting methacrylate- and polystyrene-based monolithic columns.

Monolithic columns were synthesized inside 1.02 mm internal diameter fused-silica lined stainless-steel tubing. Styrene and butyl, hexyl, lauryl, and glycidyl methacrylates were the functional monomers. Ethylene glycol dimethacrylate and divinylbenzene were the crosslinkers. The glycidyl methacrylate polymer was modified with gold nanoparticles and dodecanethiol (C12 ). The separation of alkylbenzenes was investigated by isocratic elution in 60:40 v/v acetonitrile/water. The columns based on polystyrene-co-divinylbenzene and poly(glycidyl methacrylate)-co-ethylene glycol dimethacrylate modified with dodecanethiol did not provide any separation of alkyl benzenes. The poly(hexyl methacrylate)-co-ethylene glycol dimethacrylate and poly(lauryl methacrylate)-co-ethylene glycol dimethacrylate columns separated the alkyl benzenes with plate heights between 30 and 60 µm (50 µL min(-1) and 60°C). Similar efficiency was achieved in the poly(butyl methacrylate)-co-ethylene glycol dimethacrylate column, but only at 10 µL min(-1) (0.22 mm s(-1) ). Backpressures varied from 0.38 MPa in the hexyl methacrylate to 13.4 MPa in lauryl methacrylate columns (50 µL min(-1) and 60°C). Separation of proteins was achieved in all columns with different efficiencies. At 100 µL min(-1) and 60°C, the lauryl methacrylate columns provided the best separation, but their low permeability prevented high flow rates. Flow rates up to 500 µL min(-1) were possible in the styrene, butyl and hexyl methacrylate columns.

Separation of proteins by cation-exchange sequential injection chromatography using a polymeric monolithic column.

Since sequential injection chromatography (SIC) emerged in 2003, it has been used for separation of small molecules in diverse samples, but separations of high molar mass compounds such as proteins have not yet been described. In the present work a poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic column was prepared by free radical polymerization inside a 2.1-mm-i.d. activated fused silica-lined stainless steel tubing and modified with iminodiacetic acid (IDA). The column was prepared from a mixture of 24% GMA, 16% EDMA, 20% cyclohexanol, and 40% 1-dodecanol (all% as w/w) containing 1% of azobisisobutyronitrile (AIBN) (in relation to monomers). Polymerization was done at 60 °C for 24 h. The polymer was modified with 1.0 M IDA (in 2 M Na2CO3, pH 10.5) at 80 °C for 16 h. Separation of myoglobin, ribonuclease A, cytochrome C, and lysozyme was achieved at pH 7.0 (20 mM KH2PO4/K2HPO4) using a salt gradient (NaCl). Myoglobin was not retained, and the other proteins were separated by a gradient of NaCl created inside the holding coil (4 m of 0.8-mm-i.d. PTFE tubing) by sequential aspiration of 750 and 700 µL of 0.2 and 0.1 M NaCl, respectively. As the flow was reversed toward the column (5 µL s(-1)) the interdispersion of these solutions created a reproducible gradient which separated the proteins in 10 min, with the following order of retention: ribonuclease A < cytochrome C < lysozyme. The elution order was consistent with a cation-exchange mechanism as the retention increased with the isoelectric points.

Influence of humic acid on adsorption of Hg(II) by vermiculite.

Geochemical mobility of Hg(II) species is strongly affected by the interactions of these compounds with naturally occurring adsorbents such as humic acids, clay minerals, oxides, etc. Interactions among these sorbents affect their affinity for Hg(II) and a full understanding of these processes is still lacking. The present work describes the influence of a humic acid (HA) sample on the adsorption of Hg(II) by vermiculite (VT). Adsorption isotherms were constructed to evaluate the affinity of Hg(II) by VT, HA, VT modified with humic acid (VT-HA), and VT-HA in presence of soluble humic acid (VT-HA + HA). All experiments were made at pH 6.0 ± 0.1 in 0.02 M NaNO3 and 25.0 ± 0.5 °C for initial Hg(II) concentrations from 1.0 to 100 µM. Determinations of Hg(II) were made by square wave voltammetry automated by sequential injection analysis, an approach that enables the determination of the free plus labile fractions of Hg(II) in HA suspensions without the need for laborious separation steps. The adsorption isotherms were fitted to Langmuir and Freundlich equations, showing that HA was the material with the higher adsorption capacity (537 ± 30 µmol g(-1)) in comparison with VT and VT-HA (44 ± 3 and 51 ± 11 µmol g(-1), respectively). Adsorption order was HA >> VT-HA + HA > VT = VT-HA. At pH 6.0 the interaction of HA with VT is weak and only 14% of C initially added to the suspension was effectively retained by the mineral. Desorption of Hg(II) in acidic medium (0.05 M HCl) was higher in binary (VT-HA) and ternary (VT-HA + HA) systems in comparison with that of VT and HA alone, suggesting that interactions between VT and HA are facilitated in acidic medium, weakening the binding to Hg(II).

Monolithic columns in plant proteomics and metabolomics.

Since "omics" techniques emerged, plant studies, from biochemistry to ecology, have become more comprehensive. Plant proteomics and metabolomics enable the construction of databases that, with the help of genomics and informatics, show the data obtained as a system. Thus, all the constituents of the system can be seen with their interactions in both space and time. For instance, perturbations in a plant ecosystem as a consequence of application of herbicides or exposure to pollutants can be predicted by using information gathered from these databases. Analytical chemistry has been involved in this scientific evolution. Proteomics and metabolomics are emerging fields that require separation, identification, and quantification of proteins, peptides, and small molecules of metabolites in complex biological samples. The success of this work relies on efficient chromatographic and electrophoretic techniques, and on mass spectrometric detection. This paper reviews recent developments in the use of monolithic columns, focusing on their applications in "top-down" and "bottom-up" approaches, including their use as supports for immobilization of proteolytic enzymes and their use in two-dimensional and multidimensional chromatography. Whereas polymeric columns have been predominantly used for separation of proteins and polypeptides, silica-based monoliths have been more extensively used for separation of small molecules of metabolites. Representative applications in proteomics and in analysis of plant metabolites are given and summarized in tables.

Complexation of Hg(II) by humic acid studied by square wave stripping voltammetry at screen-printed gold electrodes.

This paper reports the development of a sequential injection (SI) method to study the complexation of Hg(II) by humic acid (HA) using square wave anodic stripping voltammetry at a screen-printed gold electrode (SPGE). The SI system injected samples (in 0.020 mol L(-1) NaNO(3) and pH 6.0) to the flow cell during the deposition step and exchanged the medium to 0.050 mol L(-1) HCl to perform the stripping step under stopped flow conditions. For sample volume=750 µL, flow rate=15 µL s(-1) (deposition step), and square wave frequency=100 Hz, the detection limit was 20 nmol L(-1) and the sampling throughput was 17 analyses per hour. Complexation of Hg(II) was studied using a 25.0 mg L(-1) suspension of HA (72.5 µmol L(-1) of ionizable sites), and total Hg(II) concentrations from 1 to 20 µmol L(-1). The diffusion coefficient of the complex was (1.3±0.2)×10(-7) cm(2) s(-1). A complexing capacity of 537 µmol g(-1) was found. The log of the differential stability constant (logK(DEF)) decreased from 7.0 to 5.3 as the log of the degree of site occupation (logθ) increased from -1.6 to -0.5. The values of logK(DEF) were consistent with complexation of Hg(II) by carboxylic and phenolic groups.

Fluorimetric determination of intra- and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode.

This paper describes the development and application of an RP HPLC method using a C(18) monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mercaptoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution >1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 micromol/L) to 12% for Ile (0.10 micromol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facilitates the analysis by shortening the separation time and saving solvents and instrumentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns).

The profiles of nitrate reductase and carbonic anhydrase activity in batch cultivation of the marine microalgae Tetraselmis gracilis growing under different aeration conditions.

Tetraselmis gracilis, a Prasinophycean alga found in estuaries and in the open ocean, was cultivated under different conditions of aeration, which resulted in variations of inorganic carbon in the medium. Relative growth rates, nitrate reductase and carbonic anhydrase activities were daily determined and correlated to the concentration of nitrate, nitrite, phosphate, inorganic and organic carbon in the media. Nitrate reductase catalyzes the reversible carbon dioxide hydration reaction. The activity profiles of both enzymes during 10 days of cultivation under aeration with air showed an inverse relationship: the maximum in the activity of nitrate reductase coincided with the minimum of carbonic anhydrase activity. An ionizable organic carbon species with pKa in the range of metabolites of the photorespiratory path was found parallel with the increase of carbonic anhydrase activity and the decrease of nitrate reductase activity. The onset of photorespiration is probably one of the factors involved in the simultaneous regulation of these enzymatic processes. Cultures aerated with air containing 5% CO2 showed different profiles for nitrate reductase activity and nitrate uptake.

Adsorption of atrazine, hydroxyatrazine, deethylatrazine, and deisopropylatrazine onto Fe(III) polyhydroxy cations intercalated vermiculite and montmorillonite.

This paper describes the modification of the clay minerals vermiculite (VT) and montmorillonite (MT) by intercalating Fe(III) polymers of different [OH(-)]:[Fe(III)] ratios with the aim of removing atrazine (AT) and its metabolites deethylatrazine (DEA), deisopropylatrazine (DIA), and hydroxyatrazine (ATOH) from aqueous solution. An enhancement of adsorption capacity was observed for both intercalated clay minerals in comparison to the potassium-saturated materials (KVT or KMT). The results showed that different [OH(-)]:[Fe(III)] molar ratios had a small influence on the adsorption capacity, as well as in the basal spacing, BET surface area, and porosity. For the lowest initial concentrations of AT, DIA, and ATOH (0.050 mg L(-)(1)) studied, the modified VT adsorbed almost 80% of AT and DIA, while ATOH was removed at concentration levels below the detection limit of the technique, implying in at least 99.5% of sorption. Weak interaction between intercalated VT and DEA was observed, although a significant adsorption enhancement occurred in comparison to KVT. Within a 24 h interval, desorption of AT and DIA in aqueous medium reached levels close to 20% of the amount initially adsorbed, while for ATOH only 3% of the adsorbed compound was desorbed. The adsorption capacity of the Fe(III)-intercalated VT decreased after the first adsorption/desorption cycle, implying that the material is not suitable for reutilization. The intercalated MT was a powerful sorbent for AT, DEA, DIA, and ATOH, removing all of these chemicals from solution almost quantitatively (sorption greater than 99.5%), even at initial concentrations as high as 1.0 mg L(-)(1). Additionally, desorption of AT, ATOH, and DIA in water was not measurable up to the tube corresponding to the initial concentration of 1.0 mg L(-)(1), suggesting strong irreversible binding of these compounds to the intercalated MT materials. Desorption of DEA from the intercalated MT was between 5 and 30%. Unlike what was observed for VT, the intercalated MT materials were recyclable, keeping an excellent performance when reutilized.