Latex-coated polymeric monolithic ion-exchange stationary phases. 1. Anion-exchange capillary electrochromatography and in-line sample preconcentration in capillary electrophoresis

A sulfonated methacrylate monolithic polymer has been synthesized inside fused-silica capillaries of diameters 50-533-microm i.d. and coated with 65-nm-diameter fully functionalized quaternary ammonium latex particles (AS18, Dionex Corp.) to form an anion-exchange stationary phase. This stationary phase was used for ion-exchange capillary electrochromatography of inorganic anions in a 75-microm-i.d. capillary with Tris/perchlorate electrolyte and direct UV detection at 195 nm. Seven inorganic anions (bromide, nitrate, iodide, iodate, bromate, thiocyanate, chromate) could be separated over a period of 90 s, and the elution order indicated that both ion exchange and electrophoresis contributed to the separation mechanism. Separation efficiencies of up to 1.66 x 10(5) plates m(-1) were achieved, and the monoliths were stable under pressures of up to 62 MPa. Another latex-coated monolith in a 250-microm-i.d. capillary was used for in-line preconcentration by coupling it to a separation capillary in which the EOF had been reversed using a coating of either a cationic polymer or cationic latex particles. Several capillary volumes of sample were loaded onto the preconcentration monolith, and the analytes (inorganic anions) were then eluted from the monolith with a transient isotachophoretic gradient before being separated by electrophoresis in the separation capillary. Linear calibration curves were obtained for aqueous mixtures of bromide, nitrite, nitrate, and iodide. Recoveries of all analytes except iodide were reduced significantly when the sample matrix contained high levels of chloride. The preconcentration method was applied to the determination of iodide in open ocean water and provided a limit of detection of 75 pM (9.5 ng/L) calculated at a signal-to-noise ratio of 3. The relative standard deviation for migration time and peak area for iodide were 1.1 and 2.7%, respectively (n = 6). Iodide was eluted as an efficient peak, yielding a separation efficiency of 5.13 x 10(7) plates m(-1). This focusing was reproducible for repeated analyses of seawater.     

Improved sensitivity and characterization of high-speed ion chromatography of inorganic anions

In this work, a reversed-phase monolithic column was permanently coated with didodecyldimethylammonium bromide (DDAB) to perform ultrafast separations of iodate, chloride, nitrite, bromide, nitrate, phosphate, and sulfate in as little as 30 s. Separations were performed using 6 mM o-cyanophenol (pH 7.0) at flow rates up to 10 ml/min and suppressed conductivity detection. Detection limits in the parts-per-billion range were observed for all anions studied (e.g., ranging from 30 ppb for phosphate to 4 ppb for sulfate). The reproducibility was 0.7 and 0.4% RSD for retention time and peak area, respectively. Coated columns were stable for up to 12 h of continuous use at 5 mL/min (i.e., 1-min separations).     

Simulation and optimization of retention in ion chromatography using virtual column 2 software

A new software package, Virtual Column 2, is described for the simulation and optimization of the separation of inorganic anions by ion chromatography (IC). The software uses a limited amount of experimental retention data acquired according to a correct experimental design to predict retention times for analytes over a designated search area of eluent compositions. The experimental retention data are used to solve a new retention model, called the linear solvent strength model, empirical approach (LSSM-EA), which then enables prediction of retention times for all eluent compositions in the search area. The theoretical development of LSSM-EA and the processes used for solving the equations are discussed. Virtual Column 2 can be used for eluents containing one or two competing ions, and the software contains retention databases for up to 33 analytes on the Dionex AS9A-HC, AS4A-SC, and AS14A analytical columns with carbonate-bicarbonate eluents and the Dionex AS10, AS15, and AS16 analytical columns with hydroxide eluents (results for the AS10 and AS15 columns are not discussed in the present study). Virtual Column 2 has been evaluated extensively and is shown to give predicted retention times that in most cases agree with experimentally determined data to within 5%. The software has uses in practical IC method development, education and training in IC, and refinement of existing IC methodology. A free version of this program is available by download at www.virtualcolumn.com.     

Two-Dimensional detection in ion chromatography: sequential conductometry after suppression and passive hydroxide introduction

An improved method that uses sequential suppressed and nonsuppressed IC for the sensitive detection of both common anions and very weak acid anions is described. After suppressed conductometric detection of an electrolytically generated hydroxide eluent and an electrolytic suppressor, the eluent is passed into a membrane device where KOH is passively introduced into the eluent stream using Donnan forbidden leakage. A second conductivity detector then measures the conductivity of the stream. The background conductance of the second detector is typically maintained at a relatively low level of 20-30 microS/ cm. The weak acids are converted to potassium salts that are fully ionized and are detected against a low KOH background as negative peaks. The applicability of different commercially available cation exchange membranes was studied. Device configurations investigated include planar, tubular, and a filament-filled annular helical (FFAH) device. The FFAH device provided more effective mixing of the penetrated hydroxide with the eluent stream, resulting in a noise level of < or = 7 nS/cm and a band dispersion value of less than 82 microL. Optimal design and performance data are presented.     

Simultaneous separation of cationic and anionic proteins using zwitterionic surfactants in capillary electrophoresis

The zwitterionic surfactant Rewoteric AM CAS U forms a dynamic wall coating that prevents the adsorption of cationic proteins as well as suppresses the electroosmotic flow (EOF). Addition of polarizable anions to buffers containing this zwitterionic surfactant increases the once suppressed EOF to values nearing +3 x 10(-4) cm2/(V s). The retention of the EOF allows for the separation of analytes of widely different mobilities and is demonstrated by the simultaneous separation of cationic and anionic proteins. Using a buffer containing optimal amounts of the polarizable anion perchlorate and surfactant CAS U, the proteins lysozyme, ribonuclease A, alpha-chymotrypsinogen A, and myoglobin are separated in less than 15 min. Efficiencies as high as 1.5 million plates/m and recoveries greater than 91% are observed for proteins injected in distilled water. Migration time reproducibility is approximately 1% RSD within 1 day and approximately 3% RSD from day to day. The anionic and cationic proteins can be separated over a pH range of 5.5-9, all yielding good efficiencies.     

离子色谱法对糕点中丙酸钙的测定

本文采用抑制型电导检测器建立了离子色谱法测定糕点中丙酸钙含量的方法。糕点经蒸馏、提取、过滤后,用Dionex Ion Pac ASl9(4×250mm)分离柱分离,以RFC-30淋洗液自动发生器产生的5 mmol KOH为淋洗液,流速1.0ml/min,经抑制电导检测器检测。该方法在0.10～20.0mg/L范围内线性关系良好,检出限达0.02 mg/L。该方法稳定,重现性好,保留时间相对标准偏差RSD(n=7)为0.18%,峰面积RSD(n=7)为1.15%。将该方法用于糕点实际样品的测定,回收率为91.77%,结果良好。     

Microscale continuous ion exchanger

A microscale continuous ion exchanger based on two liquid streams flowing in parallel is presented. The ion exchange reaction occurs through diffusional transfer of molecules between the ion exchanger phase and the eluent phase and is applied for conductivity suppression. Two approaches are demonstrated. In the first approach, a liquid ion exchanger (i.e. a strongly basic compound, e.g., tetraoctylammonium hydroxide, or a secondary amine, e.g., Amberlite IA-2) is dissolved in an organic solvent immiscible with the aqueous eluent. The system allows for sensitive suppressed conductivity detection of various inorganic cations. When the weakly basic secondary amine is used, conductometric detection of heavy metals is possible. In the second approach, a suspension of finely ground ion-exchange resin is used as the ion exchanger phase. In this case, the suspension need not involve an organic solvent. Theoretical models and computations are presented along with experimental results. The potential of such a system as a chip-scale post-separation suppressor/reactor is evident.     

Analysis of bile acids and their conjugates by capillary electrochromatography/electrospray ion trap mass spectrometry

Different macroporous, monolithic capillary columns were prepared to separate various bile acid mixtures through capillary electrochromatography (CEC) at high efficiency. These columns are shown to be ideally suitable for coupling to an electrospray ionization/ion trap mass spectrometer. Detection and structural identification of different bile acid derivatives in either the positive- or negative-ion mode necessitated column technologies with different polarities and the capabilities of a reversed electroosmotic flow. High column efficiencies (610,000 theoretical plates/meter for glycocholic acid in normal-phase separation) were preserved in the coupling to mass spectrometry (MS), with the detection limits of approximately 40 femtomole (for cholic acid) and identification through CEC/MS/MS.     

Ion-exchange chromatography/electrospray mass spectrometry for the identification of organic and inorganic species in topiramate tablets

An ion-exchange chromatograph/electrospray ionization mass spectrometer (IC/ESI-MS) was used successfully to identify organic and inorganic species present in topiramate tablets. An ion suppressor is placed between the column and detectors to replace sodium ions in the mobile phase with hydrogen ions supplied by the suppressor. The ensuing combination of the hydrogen ions with the mobile phase hydroxide ions produces water and thus allows simultaneous ion detection by an ion conductivity detector and a mass spectrometer. Analytes, including lactate, glycolate, chloride, formate, sulfate, and oxalate, were unambiguously identified by matching the mass spectra and retention times with those of the authentic compounds. Due to its capability of detecting positive and negative as well as neutral species, ESI-MS provides valuable information which is not available with ion conductivity detection alone. Though the coupling of ion-exchange chromatography to mass spectrometry has been reported previously, this is the first demonstration of IC/ESI-MS for the identification of unknown species in real samples. Finally, with the use of deuterium/carbon-13 labeling and MS/MS techniques, we have confirmed that oxalic acid (HOOC-COOH) is formed from formic acid (HCOOH) at the electrospray interface in the presence of the electric field. This observation not only confirms the identity of an unknown peak, but it also provides new insight into chemistry that can take place during electrospray ionization.     

Phospholipid bilayer coatings for the separation of proteins in capillary electrophoresis

The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) was investigated for its use as a wall coating for the prevention of protein adsorption in capillary electrophoresis. DLPC forms a semipermanent coating at the capillary wall, which allows excess phospholipid to be removed from the capillary prior to electrophoretic separation. A DLPC-coated capillary allowed for the separation of both cationic and anionic proteins with efficiencies as high as 1.4 million plates/m. Migration time reproducibility was less than 1.3% RSD from run to run and less than 4.0% RSD from day to day. Protein recovery was as high as 93%. Cationic and anionic proteins could be separated over a pH range of 3-10, all yielding good efficiencies (N up to 1 million plates/m). The chain length of the phospholipid affected the performance of the wall coating. The C10 analogue of DLPC (DDPC) did not form a coating on the capillary wall while the C14 analogue of DLPC (DMPC) formed a stable coating that prevented protein adsorption to the same extent as its C12 counterpart.     

Open tubular anion exchange chromatography. Controlled layered architecture of stationary phase by successive condensation polymerization

The preparation and performance of a multilayered stationary phase for open tubular anion exchange chromatography in relatively large bore (75 microm diameter) columns are described. The inner surface of a fused-silica capillary tube is coated with up to 25 successive porous polymeric layers formed by condensation polymerization of a primary amine with a diepoxide. Each layer of the anion exchange stationary phase consists of copolymer of methylamine (MA) and 1,4-butanedioldiglycidyl ether (BDDE). The polymer layers are sufficiently porous or permeable; each successive layer of the stationary phase incrementally increases the observed column capacity and chromatographic performance in the open tubular mode. Even though the column inner diameter is far from optimum for open tubular liquid chromatography, we demonstrate the baseline separation of a suite of inorganic anions (F-, Cl-, NO2-, Br-, NO3-) in a 5 m x 75 microm column coated with 25 layers of the anion exchange polymer using 1 mM KOH eluent and suppressed contactless conductometric detection at a flow rate of 1 microL/min (operating pressure of approximately 1 bar) with a plate count of >30,000. Strategies for construction of microsuppressor devices used in open tubular ion chromatography are discussed.     

Improving the compatibility of contact conductivity detection with microchip electrophoresis using a bubble cell

A new approach for improving the compatibility between contact conductivity detection and microchip electrophoresis was developed. Contact conductivity has traditionally been limited by the interaction of the separation voltage with the detection electrodes because the applied field creates a voltage difference between the electrodes, leading to unwanted electrochemical reactions. To minimize the voltage drop between the conductivity electrodes and therefore improve compatibility, a novel bubble cell detection zone was designed. The bubble cell permitted higher separation field strengths (600 V/cm) and reduced background noise by minimizing unwanted electrochemical reactions. The impact of the bubble cell on separation efficiency was measured by imaging fluorescein during electrophoresis. A bubble cell four times as wide as the separation channel led to a decrease of only 3% in separation efficiency at the point of detection. Increasing the bubble cell width caused larger decreases in separation efficiency, and a 4-fold expansion provided the best compromise between loss of separation efficiency and maintaining higher field strengths. A commercial chromatography conductivity detector (Dionex CD20) was used to evaluate the performance of contact conductivity detection with the bubble cell. Mass detection limits (S/N = 3) were as low as 89 +/- 9 amol, providing concentration detection limits as low as 71 +/- 7 nM with gated injection. The linear range was measured to be greater than 2 orders of magnitude, from 1.3 to 600 microM for sulfamate. The bubble cell improves the compatibility and applicability of contact conductivity detection in microchip electrophoresis, and similar designs may have broader application in electrochemical detection as the expanded detection zone provides increased electrode surface area and reduced analyte velocity in addition to the reduction of separation field effects.     

Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns

This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.     

离子色谱法测定肉制品中亚硝酸盐含量

建立了离子色谱法快速定量肉制品中亚硝酸盐含量的分析方法。以1.2mmol/L Na2CO3-1.8mmol/L NaHCO3为淋洗液,分析柱为Dionex AS14,检测器为化学抑制型电导检测器。检出限为0.04mg.L-1,加标回收率为96.8%～99.4%,与GB/T5009.33比色法比较相对偏差小于1%。方法简便,快速,用于肉制品中亚硝酸盐含量的测定结果满意。     

Determination of bioactive peptides using capillary zone electrophoresis/mass spectrometry.

Mixtures of bioactive peptides have been analyzed by capillary zone electrophoresis/mass spectrometry (CZE/MS) using an on-line coaxial continuous-flow fast atom bombardment interface. High separation efficiencies (up to 410,000 theoretical plates) were obtained from low femtomole levels of peptides. The analysis of basic peptides was accomplished by using aminopropyl-silylated CZE columns to minimize zone broadening due to adsorption effects. CZE/MS/MS data were acquired from femtomole levels of peptides in electrophoretic real time.     

Contactless conductivity detector for microchip capillary electrophoresis

A microfabricated electrophoresis chip with an integrated contactless conductivity detection system is described. The new contactless conductivity microchip detector is based on placing two planar sensing aluminum film electrodes on the outer side of a poly(methyl methacrylate) (PMMA) microchip (without contacting the solution) and measuring the impedance of the solution in the separation channel. The contactless route obviates problems (e.g., fouling, unwanted reactions) associated with the electrode-solution contact, offers isolation of the detection system from high separation fields, does not compromise the separation efficiency, and greatly simplifies the detector fabrication. Relevant experimental variables, such as the frequency and amplitude of the applied ac voltage or the separation voltage, were examined and optimized. The detector performance was illustrated by the separation of potassium, sodium, barium, and lithium cations and the chloride, sulfate, fluoride, acetate, and phosphate anions. The response was linear (over the 20 microM-7 mM range) and reproducible (RSD = 3.4-4.9%; n = 10), with detection limits of 2.8 and 6.4 microM (for potassium and chloride, respectively). The advantages associated with the contactless conductivity detection, along with the low cost of the integrated PMMA chip/detection system, should enhance the power and scope of microfluidic analytical devices.     

Microchip devices for high-efficiency separations

We have fabricated a 25-cm-long spiral-shaped separation channel on a glass microchip with a footprint of only 5 cm x 5 cm. Electrophoretic separation efficiencies for dichlorofluoroscein (DCF) on this chip exceeded 1,000,000 theoretical plates and were achieved in under 46 s at a detection point 22.2 cm from the injection cross. The number of theoretical plates increased linearly with the applied voltage, and at a separation field strength of 1,170 V/cm, the rate of plate generation was approximately 21,000 plates/s. The large radii of curvature of the turns minimized the analyte dispersion introduced by the channel geometry as evidenced by the fact that the effective diffusion coefficient of DCF was within a few percent of that measured on a microchip with a straight separation channel over a wide range of electric field strengths. A micellar electrokinetic chromatography separation of 19 tetramethylrhodamine-labeled amino acids was accomplished in 165 s with an average plate number of 280,000. The minimum resolution between adjacent peaks for this separation was 1.2.     

Sample cleanup by on-line chromatography for the determination of Am in sediments and soils by alpha-spectrometry

A novel sample cleanup procedure for the Am determination in environmental samples by alpha-spectrometry is described. The method is based on the use of three analytical chromatographic columns. TEVA-Spec. from Eichrom has been packed in an analytical chromatographic column to carry out the lanthanide/actinide separation. A CS5A mixed-bed column from Dionex was used to separate Am from lanthanide impurities and other actinides. A TCC-II column from Dionex was used to connect the TEVA to the CS5A and act as a preconcentrator column for the trivalent ions. The behavior of the columns was studied by coupling the chromatograph to an ICPMS detector. A chromatographic fraction has been used for sample preparation for alpha-spectrometric determination of 241Am. The analytical procedure has been validated with certified reference materials (sediment and soil) and was applied to sediment core samples from the Irish Sea and compared with the classical radiochemical separation of Am.     

Elaboration and electrical characterization of silicone-based anion-exchange materials

Ion exchange phenomena have been investigated in a polymeric anion-exchange membrane prepared from cross-linked silicone polymer grafted with cationic groups. The affinity of different anions towards the membrane was inferred from impedance measurements in a electrolyte/insulator/semiconductor (EIS) electrochemical set up, allowing the survey of the detection limit, flat band potential and capacity variations. The selectivity of ion-exchange for a series of anions having a the same electrical charge followed the Hofmeister series. It was found that the affinity of anions and the electrical potential variations were related because the anion exchange inside the bulk of the membrane was correlated with the adsorption of anions at its surface. Electrical capacity measurements gave supplementary information which was difficult to rationalize because they depended on several parameters.     

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 1. Optimization of polymerization conditions, porous properties, and chemistry of the stationary phase

Monolithic columns for chiral capillary electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of mixtures of O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine , ethylene dimethacrylate, and glycidyl methacrylate or 2-hydroxyethyl methacrylate in the presence of mixture of cyclohexanol and 1-dodecanol as a porogenic solvent. The porous properties of the monolithic columns can easily be controlled through changes in the composition of the binary porogenic solvent. Although both thermal- and UV light-initiated polymerizations afford useful capillary columns, monoliths prepared using the former approach exhibit better chromatographic properties. The ability to control pore size independently of the polymerization mixture composition enables the preparation of monoliths with varying percentages of the chiral monomer and cross-linker, as well as the optimization of their separation properties. Very good separations of model racemate (R,S)-N-3,5-dinitrobenzoylleucine were achieved using an optimized monolithic CEC column, with high efficiencies of up to 74000 plates/m for the retained peaks.