Elimination of N,O-bis(trimethylsilyl)trifluoroacetamide interference by base treatment in derivatization gas chromatography mass spectrometry determination of parts per billion of alcohols in a food additive.

A novel base treatment followed by liquid-liquid extraction was developed to remove the interference of excess derivatization reagent BSTFA [N,O-Bis(trimethylsilyl)trifluoroacetamide] and its byproducts for trace determination of 1-chloro-2-propanol and 2-chloro-1-propanol in a food additive. The corresponding trimethylsilyl derivatives were analyzed by gas chromatography mass spectrometry (GC/MS) detection in selective ion monitoring mode. Due to a large volume splitless injection needed for achieving the required sensitivity, excess BSTFA in the derivatization sample solution interfered with the trimethylsilyl derivatives of the analytes of interest, making their quantitation not attainable. Efforts were made to decompose BSTFA while keeping the trimethylsilyl derivatives intact. Water or aqueous sulfuric acid treatment converted BSTFA into mainly N-trimethylsilyltrifluoroacetamide, which partitions between aqueous and organic layers. In contrast, aqueous sodium hydroxide decomposed BSTFA into trifluoroacetic acid, which went entirely into the aqueous layer. No BSTFA or its byproduct N-trimethylsilyltrifluoroacetamide or trifluroacetamide was found in the organic layer where the derivatized alcohols existed, which in turn completely eliminated their interference, enabling accurate and precise determination of parts per billion of the short-chain alcohols in the food additive. Contrary to the conventional wisdom that a trimethylsilyl derivative is susceptible to hydrolysis, the derivatized short-chain alcohols were found stable even in the presence of 0.17N aqueous sodium hydroxide as the improved GC/MS method was validated successfully, with a satisfactory linearity response in the concentration range of 10-400ng/g (regression coefficient greater than 0.999), good method precision (<4%), good recovery (90-98%), and excellent limit of detection (3ng/g) and limit of quantitation (10ng/g).

Selectivity tuning via temperature pulsing using low thermal mass liquid chromatography and monolithic columns.

Low thermal mass LC was applied to the capillary LC separation of a complex insecticide mixture by increasing temperature and decreasing gradients, as well as fast selected temperature pulses to increase resolution of overlapped components. The technology was applied using a new generation of capillary monolithic stationary phases. Considerable peak shifts and selectivity changes were observed for given temperature conditions. The concept of temperature pulsing during an elution profile shows promise for increasing resolution in difficult separations and can provide a relatively simple means to solve coelution problems.

Enhancing concentration and mass sensitivities for liquid chromatography trace analysis of clopyralid in drinking water.

A theoretical treatment was developed and validated that relates analyte concentration and mass sensitivities to injection volume, retention factor, particle diameter, column length, column inner diameter and detection wavelength in liquid chromatography, and sample volume and extracted volume in solid-phase extraction (SPE). The principles were applied to improve sensitivity for trace analysis of clopyralid in drinking water. It was demonstrated that a concentration limit of detection of 0.02 ppb (µg/L) for clopyralid could be achieved with the use of simple UV detection and 100 mL of a spiked drinking water sample. This enabled reliable quantitation of clopyralid at the targeted 0.1 ppb level. Using a buffered solution as the elution solvent (potassium acetate buffer, pH 4.5, containing 10% of methanol) in the SPE procedures was found superior to using 100% methanol, as it provided better extraction recovery (70-90%) and precision (5% for a concentration at 0.1 ppb level). In addition, the eluted sample was in a weaker solvent than the mobile phase, permitting the direct injection of the extracted sample, which enabled a faster cycle time of the overall analysis. Excluding the preparation of calibration standards, the analysis of a single sample, including acidification, extraction, elution and LC run, could be completed in 1 h. The method was used successfully for the determination of clopyralid in over 200 clopyralid monoethanolamine-fortified drinking water samples, which were treated with various water treatment resins.

Preparation of polymeric monoliths by copolymerization of acrylate monomers with amine functionalities for anion-exchange capillary liquid chromatography of proteins.

Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20-30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.

Low thermal mass liquid chromatography.

A novel technique, low thermal mass liquid chromatography (LTMLC), is introduced in this study. The use of an LTM assembly that utilizes the principle of resistive wire heating and a temperature sensor to accurately deliver unprecedented heating (up to 1800 degrees C/min) or cooling (100 to approximately 200 degrees C/min) rates is reported. With the use of packed microcolumns (<0.5 mm i.d.), essentially instantaneous heat transfer from the assembly to the mobile phase was obtained. A systematic investigation was conducted to study the performance of the LTMLC technique. Both isocratic and gradient mobile phase conditions were used. For temperature control, isothermal, temperature-increasing, and temperature-decreasing gradients were applied. Three model mixtures, two of which containing neutral and acidic analytes and the other containing neutral, acidic, and basic analytes, were used to study the effect of temperature on elution time, resolution, column efficiency, and selectivity. It was found that the LTMLC experimental setup delivered reliable temperature control, as evidenced by linear van't Hoff plots for neutral and acidic compounds. The effect of temperature on the elution of basic analytes yielded nonlinear van't Hoff plots, explaining the dramatic selectivity changes observed for bases with changes in column temperature. Column efficiency generally increased with the increase in column temperature in the range of 25 to approximately 75 degrees C and decreased in the range of 75 to approximately 150 degrees C at a fixed column flow rate (3 microL/min), when extra column band broadening was taken into account. The increase in efficiency upon the increase in column temperature in the low temperature range was mainly due to the decreased mass transfer term resulting from increased analyte diffusivity. However, under even higher temperatures, the longitudinal diffusion dominated band broadening, explaining the decrease in column efficiency upon a further increase in column temperature. Resolution and selectivity decreased at elevated temperature for neutral and acidic compounds. For mixtures that contain bases, improved resolution was obtained by simultaneously tuning temperature and solvent programming. In addition to heating ability, LTMLC also demonstrated reliable cooling capability, allowing performance of oscillated or cycled temperature programming for fine-tuning the separation of critical band pairs for the first time. Finally, ultrafast reproducible LTMLC was also demonstrated, showing the potential of utilization of this technology for fast and ultrafast separations.

Polymer monoliths with low hydrophobicity for strong cation-exchange capillary liquid chromatography of peptides and proteins.

Two polymer monoliths were designed and synthesized from commercially available monomers with an attempt to decrease hydrophobicity for strong cation-exchange chromatography. One was prepared from the copolymerization of sulfoethyl methacrylate and poly(ethylene glycol) diacrylate, and the other was synthesized from vinylsulfonic acid and poly(ethylene glycol) diacrylate. Both of the monoliths were synthesized inside 75-microm i.d., UV-transparent fused-silica capillaries by photopolymerization. The hydrophobicities of the two monoliths were systematically evaluated using standard synthetic undecapeptides under ion-exchange conditions and propyl paraben under reversed-phase conditions. The poly(sulfoethyl methacrylate) monolith demonstrated similar hydrophobicity as a monolith prepared from copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid and poly(ethylene glycol) diacrylate, and 40% acetonitrile was required to suppress any hydrophobic interactions with peptides under ion-exchange conditions. However, with the use of vinylsulfonic acid as the functional monomer, a monolith with very low hydrophobicity was obtained, making it suitable for strong cation-exchange liquid chromatography of both peptides and proteins. It was found that monolith hydrophobicity could be adjusted by selection of monomers that differ in hydrocarbon content and type of vinyl group. Finally, excellent separations of model protein standards and high-density lipoproteins were achieved using the poly(vinylsulfonic acid) monolith. Five subclasses of high-density lipoproteins were resolved using a simple linear NaCl gradient.

Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis.

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.

Efficient polymer monolith for strong cation-exchange capillary liquid chromatography of peptides.

A stable poly[2-acrylamido-2-methyl-1-propanesulfonic acid-co-poly(ethylene glycol) diacrylate] monolith was synthesized inside a 75-microm-i.d. capillary by photoinitiated copolymerization with water, methanol, and ethyl ether as porogens. The resulting monolith was evaluated for strong cation-exchange capillary liquid chromatography of both synthetic and natural peptides. Although the monolith possessed relatively strong hydrophobicity due to the use of 2-acrylamido-2-methyl-1-propanesulfonic acid as one monomer, the monolith had a high dynamic binding capacity of 157 microequiv of peptide/mL, or 332 mg of cytochrome c/mL. Exceptionally high resolution resulting from extremely narrow peaks was obtained, resulting in a peak capacity of 179 when using a shallow salt elution gradient. Although a second, naturally formed gradient might contribute to the sharp peaks obtained, high efficiency was mainly due to the use of poly(ethylene glycol) diacrylate as a biocompatible cross-linker.

Design and evaluation of a coupled monolithic pre-concentrator-capillary zone electrophoresis system for the extraction of immunoglobulin G from human serum.

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively pre-concentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, the development of an on-line protein G monolithic pre-concentrator-CE device is proposed. To generate active groups for protein immobilization, glycidyl methacrylate (GMA) was used to prepare polymer monoliths. A 1.5-2 cm monolith was cast inside a 75 microm I.D. fused silica capillary that had previously been coated with alternating layers of negatively (dextran) and positively (polybrene) charged polymers. Protein G was covalently bound to GMA. Monoliths from different formulations were prepared and evaluated for binding capacity to optimize the monolith formulation for protein preconcentration. The physical properties of the column considered best for preconcentration were determined by mercury intrusion porosimetry. The total pore area was 4.8m(2)/g, the average pore diameter was 3.3 microm and the porosity was 82%. The monolith had a low flow resistance and was macroscopically homogeneous. The effectiveness of the monolith to rapidly pre-concentrate proteins at flow rates as high as 10 microL/min was demonstrated using a 1.8 microM IgG solution. This system proved effective for on-line sample extraction, clean-up, preconcentration, and CE of IgG in human serum. IgG from diluted (500 and 65,000 times) human serum samples was successfully analyzed using this system. The approach can be applied to the on-line preconcentration and analysis of any protein for which an antibody is available.

Preparation and evaluation of poly(polyethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monolith for protein analysis.

A poly(polyethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monolith was prepared by UV-initiated polymerization. Methanol and ethyl ether were selected as porogens from a variety of organic solvents to achieve the desirable characteristics of the monolith. The preparation of the monolith could be achieved within 10 min. The monolith was macroscopically homogeneous, had low flow resistance, and did not swell or shrink significantly in tetrahydrofuran. Inverse size-exclusion data indicate that the monolith had a total porosity of 75.4% and an internal porosity of 9.1%. The monolith could be used for size-exclusion separation of peptides, although it could not separate proteins with molecular masses between 10 and 100 K due to its unique pore size distribution. It was found to resist adsorption of proteins in capillary liquid chromatography when using 100 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl. Complete recovery of both acidic and basic proteins was achieved. The monolith can be used for applications in which inert materials are required for protein analysis.