Integrated refractive index optical ring resonator detector for capillary electrophoresis

We developed a novel miniaturized and multiplexed, on-capillary, refractive index (RI) detector using liquid core optical ring resonators (LCORRs) for future development of capillary electrophoresis (CE) devices. The LCORR employs a glass capillary with a diameter of approximately 100 mum and a wall thickness of a few micrometers. The circular cross section of the capillary forms a ring resonator along which the light circulates in the form of the whispering gallery modes (WGMs). The WGM has an evanescent field extending into the capillary core and responds to the RI change due to the analyte conducted in the capillary, thus permitting label-free measurement. The resonating nature of the WGM enables repetitive light-analyte interaction, significantly enhancing the LCORR sensitivity. This LCORR architecture achieves dual use of the capillary as a sensor head and a CE fluidic channel, allowing for integrated, multiplexed, and noninvasive on-capillary detection at any location along the capillary. In this work, we used electro-osmotic flow and glycerol as a model system to demonstrate the fluid transport capability of the LCORRs. In addition, we performed flow speed measurement on the LCORR to demonstrate its flow analysis capability. Finally, using the LCORR's label-free sensing mechanism, we accurately deduced the analyte concentration in real time at a given point on the capillary. A sensitivity of 20 nm/RIU (refractive index units) was observed, leading to an RI detection limit of 10-6 RIU. The LCORR marries photonic technology with microfluidics and enables rapid on-capillary sample analysis and flow profile monitoring. The investigation in this regard will open a door to novel high-throughput CE devices and lab-on-a-chip sensors in the future.     

Capillary HPLC-NMR Coupling: High-Resolution (1)H NMR Spectroscopy in the Nanoliter Scale

Coupling HPLC and NMR is one of the most powerful techniques for simultaneous separation and structural elucidation of unknown compounds in mixtures. To date, however, minimizing the detection volume, as is required when coupling NMR with miniaturized separation techniques, has been accompanied by a dramatic loss in resolution of the NMR spectra. Here, we report on the coupling of gradient capillary HPLC with on-column, high-resolution NMR detection. On-line stopped-flow and static (1)H NMR spectra were acquired with capillary columns of 75-315 μm i.d. With detection over a length of 1.2 cm, cell volumes cover a range of 50-900 nL. An on-line-detected NMR separation of dansylated amino acids was carried out in a 315 μm i.d. fused silica capillary packed to a length of 12 cm with C(18) stationary phase. The low solvent consumption makes the use of fully deuterated solvents economically feasible. NMR spectra with resolution on the order of 3 Hz were obtained using a 50 nL detection cell to measure 1.1 nmol of dansylated γ-aminobutyric acid under static conditions in a 75 μm i.d. capillary.     

Determination of polycyclic aromatic sulfur heterocycles in fossil fuel-related samples

An analytical method is described for the separation, identification, and quantification of a number of polycyclic aromatic sulfur heterocycles (PASHs) in three fossil fuel-related samples including two Standard Reference Materials (SRMs), SRM 1597 (coal tar) and SRM 1582 (petroleum crude oil), and a decant oil. The compounds measured include the 3 possible naphtho[b]thiophenes; dibenzothiophene and selected methyl-, ethyl-, dimethyl-, and trimethyl-substituted isomers; the 3 possible benzo[b]naphthothiophenes; and the 30 methylbenzo[b]naphthothiophenes isomers. Because of the occurrence of polycyclic aromatic hydrocarbons and PASHs together with their large number of possible alkyl-substituted isomers, the analytical method described requires a number of prerequisites: effective sample cleanup, selective stationary phases, and selective methods of detection. The sample cleanup involves solid-phase extraction using aminopropylsilane cartridges with different solvent mixtures followed by normal-phase liquid chromatographic isolation of the PASHs based on the number of aromatic carbons. These aromatic ring fractions are then separated by capillary gas chromatography using two stationary phases with different selectivities, 5% phenyl-substituted methylpolysiloxane stationary phase and 50% phenyl-substituted methylpolysiloxane stationary phase, and analyzed with mass-selective detection and atomic emission detection. A liquid crystalline stationary phase was also used to separate the methylbenzo[b]naphthothiophene isomers in the crude oil sample. Advantages and limitations of each chromatographic and detection technique are discussed.     

十二烷基苯磺酸钾色谱固定相及其毛细管柱的研究

本文合成了十二烷基苯磺酸钾色谱固定相,并制备了毛细管色谱柱,研究了其柱性能和分离能力。实验表明,十二烷基苯磺酸钾色谱固定相对烷烃、芳香烃、酯类、醇类等具有较强的选择分离能力,制成的毛细管柱具有较高的柱效,有很高的实用价值。     

Refractometric Sensors for Lab-on-a-Chip Based on Optical Ring Resonators

We demonstrate refractive index measurement of liquids using two sensor system designs, both based on microring resonators. Evanescent sensors based on microrings utilize the resonating nature of the light to dramatically decrease the required size and sample consumption volume, which are requirements of lab-on-a-chip sensor systems. The first design, which utilizes an optical microsphere, exhibits a sensitivity of 30 nm/RIU and a resulting detection limit on the order of 10^-7 RIU. The second approach is a novel design called a liquid core optical ring resonator (LCORR). This concept uses a quartz capillary as the fluidics and as the ring resonator and achieves a sensitivity of 16.1 nm/RIU. The detection limit of this system is around 5times10^-6 RIU. Both of these systems have the potential to be incorporated with advanced microfluidic systems for lab-on-a-chip applications. In particular, the LCORR combines high sensitivity, performance stability, and microfluidic compatibility, making it an excellent choice for lab-on-a-chip development     

Cellulose acetate decoupler for on-column electrochemical detection in capillary electrophoresis.

A new decoupler for on-column electrochemical detection in capillary electrophoresis is presented. The decoupler is constructed by etching a series of holes through the side of the separation capillary with a CO2 laser and then coating the holes with cellulose acetate. The decoupler shows isolation of the detection circuit for separation currents up to 30 microA. Detection limits below 1 nM were achieved for four model compounds, including anions, neutrals, and cations, using the laser-etched decoupler. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.     

Open-loop operation experiments in a resonator fiber-optic gyro using the phase modulation spectroscopy technique

A detection system in the resonator fiber-optic gyro is set up by the phase modulation (PM) spectroscopy technique. The slope of the demodulated curve near the resonant point is found to affect the ultimate sensitivity of the gyro. To maximize the demodulated signal slope, the modulation frequency and index are optimized by the expansion of the Bessel function and optical field overlapping method. Using different PM frequencies for the light waves, the open-loop gyro output signal is observed. The modulation frequency in this PM technique is limited only by the cutoff frequency of the LiNbO3 phase modulators, which can reach several gigahertz. This detection technique and system can be applied to the resonator micro-optic gyro with a less than 10 cm long integrated optical ring.     

Light-scattering studies of packed stationary phases for capillary electrochromatography

Multiply scattered light through turbid media, packed particles, or compressed powders will inherently have a significantly longer optical path length than that of light which is not scattered. The concept of using the multiply scattered light potentially generated in the packed stationary phase of a capillary electrochromatography (CEC) column for enhanced detection as a result of its increased optical path length was examined. Ultraviolet (UV) light at 365 nm or laser light at 635 nm was focused to a small spot onto the packed section of a 3 microns spherisorb ODS1 CEC column (100 microns i.d.). The light was transported inside the capillary, and an image of the multiply scattered light several millimeters along the capillary was collected using a charged-couple device detector. Even if the spot size was less than 100 microns in diameter, evidence of light scattering was observed at a detection spatial off-set distance of 1-2 mm from the illumination point. When the calcium channel blocking drug felodipine was flushed through the column, the light intensity value dropped (increase in absorbance) to a greater degree at a spatial off-set (1.5 mm) than at the illumination point. The greater absorbance values at the spatial off-set were examined experimentally when felodipine was eluted from the column in the CEC mode in 6 min using MeCN/50 mM TRIS (pH 8.0) (80:20, v/v) at an applied voltage of 300 V/cm and an injection time of 2 s at 10 kV. A factor of 8.5 increase in absorbance was observed at a spatial off-set of 1 mm compared to the value obtained at the illumination point. An efficiency value of approximately 234,000 plates m-1 was obtained for this higher felodipine peak. Higher noise values, however, were also observed with this increase in absorbance. Using a spectrophotometer or an open capillary to obtain reference values for optical length, it was possible to estimate the average optical path length of light traveled through the packed stationary phase when transmitted at a spatial off-set. It was concluded that, although an increase in absorbance of 8.5 was observed at a spatial off-set, this most likely arises from the light being "redirected" and scattered in a straightforward fashion along the capillary. It was expected that if substantial multiple scattering did occur inside the packed stationary phase, a significantly larger absorbance increase would be attained. A number of proposals are thus given to explain the relatively low degree of multiple scattering in this stationary phase and suggestions offered on means to attain even higher absorbance increases at a spatial off-set. Additional potential applications are also discussed.     

Optical axis perturbation in folded planar ring resonators

A mathematical model of a four-sided folded planar ring resonator is established. The model can be modified into a triangular ring resonator, a square ring resonator, and a four-sided folded ring resonator, all of which are widely used for ring laser gyroscopes by changing certain design parameters such as incident angle Ai and side ratio H. By use of the extended matrix formulation, the optical axis perturbation, including optical axis decentration and optical axis tilt, in those planar ring resonators is analyzed in detail resulting in some novel findings. It has been determined that the longer the mirror radius, the larger the mode volume, the higher the sensitivity of optical axis decentration and the lower the sensitivity of optical axis tilt. The same mirror misalignment value, mostly the misalignment induced by optical axis decentration in the x and y components, has the conventional ratio of 1:[cos(Ai)](2) for the symmetrical points of the resonator. Details of the effect of Ai and H on the optical axis tilt have also been determined. The difference in optical axis tilt between different kinds of ring resonator is disclosed. The sensitivity of optical axis tilt was found to undergo singular rapid change along with the right edge of the second stable area. This singular behavior is useful for those resonators that have a small incident angle, such as Ai=15 degrees , because those resonators have a second stable region. These interesting findings are important for cavity design, cavity improvement, and alignment of planar ring resonators.     

Imaging solute distribution in capillary electrochromatography with laser scanning confocal microscopy

A method for the direct observation of solute molecules interacting with a C18 stationary phase under real separation conditions in capillary electrochromatography (CEC) is investigated. The experiments were performed in a capillary electrochromatographic mode; however, the method and findings are useful both in CEC and revered-phase liquid chromatography. The distribution of solute molecules in the packed capillary is directly imaged with laser scanning confocal fluorescence microscopy. Conventional imaging techniques produce images where the C18 silica beads cannot be distinctively identified as a result of the deep depth of field. The optical sectioning capability of confocal imaging overcomes this problem to afford clearly defined images of the stationary-phase packing and the surrounding mobile phase. Fluorescein molecules are preferentially distributed in the mobile phase under reversed-phase chromatographic conditions. Nile Red and rhodamine 6G molecules prefer the environments of the porous C18 beads. Intensity distributions over time for areas within the stationary-phase beads differ from distributions of areas outside the beads in the mobile phase. Images taken at different depths into the capillary probe the internal structure of the C18 beads. While the internal structures of most beads are porous, confocal images show a small fraction (2%) of the silica beads have porous shells and nonporous cores. The capability of imaging the stationary phase distinctively from the mobile phase opens the possibilities of studying the quality of stationary phase, the structure of the column packing, and the mechanisms of separation.     

On-column electrochemical detection for microchip capillary electrophoresis

The development of a cellulose acetate decoupler for on-column electrochemical detection in microchip capillary electrophoresis is presented. The capillary based laser-etched decoupler is translated to the planar format to isolate the detector circuit from the separation circuit. The decoupler is constructed by aligning a series of 20 30-microm holes through the coverplate of the microchip with the separation channel and casting a thin film of cellulose acetate within the holes. The decoupler shows excellent isolation of the detection circuit for separation currents up to 60 microA, with noise levels at or below 1 pA at a carbon fiber electrode. Detection limits of 25 nM were achieved for dopamine. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.     

Use of a mixed-mode packing and voltage tuning for peptide mixture separation in pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer detector.

A mixed-mode (reversed-phase/anion-exchange) stationary phase has been used as the capillary column packing for investigation of the separation of peptide mixtures in pressurized capillary electrochromatography (pCEC). This stationary phase contains both octadecylsilanes and dialkylamines. The amine groups of the stationary phase determine the charge density on the surface of the packing and can produce a strong and constant electroosmotic flow (EOF) at low pH. A comparison was made in terms of the capability of separating tryptic digests between the mixed-mode phase and C18 reversed phase. In addition, the constant EOF enabled the tuning of the retention and the selectivity of the separation by adjusting the mobile phase pH from 2 to 5. Furthermore, the magnitude and the polarity of the electric voltage were demonstrated to greatly influence the elution profiles of the peptides in pCEC. An ion trap storage/reflectron time-of-flight mass spectrometer was used as an on-line detector in these experiments due to its ability to provide rapid and accurate mass detection of the sample components eluting from the separation column.     

Capillary ultrahigh performance liquid chromatography with elevated temperature for sub-one minute separations of basal serotonin in submicroliter brain microdialysate samples

Improving the time resolution in microdialysis coupled to high performance liquid chromatography (HPLC) requires that the volume of the separation system be decreased. A low-volume separation permits smaller microdialysate volumes to be injected without suffering a sensitivity loss from dilution. Thus, improved time resolution can be achieved with offline analysis simply by decreasing the separations system volume. For online (near real-time) analysis, there is a further requirement. The separation speed must be at least as fast as the sampling time. Here, the combined use of high column pressures and temperatures, sub-2-μm stationary phase particles, capillary columns, and sensitive, low dead-volume detection resulted in a retention time for the neurotransmitter serotonin of less than 1 min in a 500 nL dialysate sample volume. Two sensitive detectors, photoluminescence following electron transfer (PFET) and electrochemical, were used for the detection of subnanomolar concentrations of serotonin in brain microdialysate samples. The general principles developed are applicable to a wide range of separations with the additional advantages of increases in sample throughput and decreases in mobile phase usage.     

Single-walled carbon nanotubes used as stationary phase in GC

Single-walled carbon nanotubes (SWNTs) have high surface area, high adsorption ability, and nanoscale interactions. In this study, capillary columns including SWNTs, ionic liquid (IL), and IL + SWNTs for GC were prepared. The separation results showed that SWNTs possessed a wide selectivity toward alkanes, alcohols, aromatic compounds, and ketones, and a SWNT capillary column was a very useful GC column for the separation of gas samples. Coating the IL stationary phase on the SWNT capillary column, the SWNTs were able to improve chromatographic characteristic of ionic liquid. Comparing the IL coated on three graphite carbon black capillary columns, which were prepared by dynamic coating, static coating, and chemical bonding the Carbopack C with on SWNTs capillary column, the capacity factors were much higher on the SWNT column. The SEM showed that SWNTs could be bonded to the inner surface of capillary tubing, and most of them were linked end-to-end to form a layer of network structure of skeletons resulting in a high surface area, which increased the interactions between stationary phase and analytes. This is the first single-wall carbon nanotubes bonded to the fused-silica capillary tubing. In the first approach, SWNTs assist ionic liquid with enhanced chromatographic characteristic in GC. This work indicates that SWNTs make it possible to extend the application range on the newly prepared chromatographic stationary phases for GC.     

Reversed phase liquid chromatography of organic hydrocarbons with water as the mobile phase

Reversed phase high-performance liquid chromatography (RP-HPLC) is demonstrated for hydrophobic analytes such as aromatic hydrocarbons using only water as the mobile phase. Achievement of reasonable capacity factors for these types of compounds without the need for toxic and costly organic modifiers in the mobile phase is accomplished by substantially decreasing the phase volume ratio of stationary phase relative to the mobile phase volume and by increasing the polarity of the stationary phase relative to stationary phase materials commonly used for RP-HPLC. Applying a stationary phase of trifluoropropylsiloxane, which is a common gas chromatographic stationary phase material, to nonporous glass microspheres yields a stationary phase with a phase volume ratio reduced by about 2 orders of magnitude as compared to common liquid chromatographic packing materials. As a result, a separation was obtained for hydrophobic organic analytes such as benzene, toluene, ethylbenzene, and isopropylbenzene using a water mobile phase at ambient temperature. A separation of sodium benzoate, benzaldehyde, benzene, and butyrophenone is shown in less than 3 min using a water mobile phase and UV/visible absorbance detection. Additionally, the separation of the ionic surfactant species octyl sulfate and dodecyl sulfate in water in less than 3 min, using unsuppressed conductivity detection, is achieved with a separation mechanism based on interactions with the hydrophobic portion of the surfactant. A water mobile phase offers many potential advantages over traditional mixed aqueous/organic solvent systems. In addition to saving on the cost and expense of buying and disposing of toxic solvents and waste, there is less exposure of the operator to potentially harmful solvents. Increased consistency in reproducing retention times can be expected, since there will not be any variability in solvent strength due to slight variations in mobile phase composition. A water mobile phase produces an environment that should provide an inherent advantage of increased signal-to-noise ratio for detection. Additionally, excellent predictions of the octanol/water partitioning coefficient and aqueous solubility for hydrophobic analytes are obtained from a single measurement of the capacity factor in the water mobile phase.     

An integrated fritless column for on-chip capillary electrochromatography with conventional stationary phases

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.     

An etched porous interface for on-line capillary electrophoresis-based two-dimensional separation system

The construction and evaluation of an on-column etched fused-silica porous junction for on-line coupling of capillary isoelectric focusing (CIEF) with capillary zone electrophoresis (CZE) are described. Where two separation columns were integrated on a single piece of fused-silica capillary through the etched approximately 4 to 5-mm length porous junction along the capillary. The junction is easily prepared by etching a short section of the capillary wall with HF after removing the polyimide coating. The etched section becomes a porous glass membrane that allows only small ions related to the background electrolyte to pass through when high voltage is applied across the separation capillary. The primary advantages of this novel porous junction interface over previous designs (in which the interface is usually formed by fracturing the capillary followed by connecting the two capillaries with a section of microdialysis hollow fiber membrane) are no dead volume, simplicity, and ruggedness, which is particularly well suited for an on-line coupling capillary electrophoresis-based multiple dimensional separation system. The performance of the 2D CIEF-CZE system constructed by such an etched porous junction was evaluated by the analyses of protein mixtures.     

Broadband absorbance in a liquid-core optical ring resonator

We have demonstrated the application of broadband absorption spectroscopy in a liquid-core optical ring resonator. An initial proof of concept of the broadband liquid-core optical ring resonator (BLCORR) was constructed using a thinned-wall, 250-μm-inner-diameter fused silica capillary, tapered multimode optical fibers for input and output coupling, and a light-emitting diode (LED) source. When compared with standard cuvette measurements, an apparent path length as high as 5 cm was observed for methylene blue (MB). MB is a cationic dye that exhibits strong surface interaction with bare silica. Bromothymol blue (BTB), on the other hand, has a similar absorbance spectrum but does not share this same surface activity. On comparing these two dyes, the apparent path length for MB was found to reach more than 50 times that of BTB, confirming the expectation that the sensing region being probed is largely within the evanescent field at the inner surface of the capillary. The BLCORR may also inherit, from attenuated total reflection (ATR) spectroscopy, the ability to analyze highly concentrated chromophores. Concentrations of BTB as high as 10(-2) and 10(-3) M were easily distinguished from each other at the λ(max) in the BLCORR, whereas this was not the case in a 4-mm cuvette cell. Our presented device employs commercially available materials and could incorporate well into microfluidic systems. These benefits, along with the demonstrated ability to take enhanced surface absorbance measurements in a capillary, give the BLCORR potential in a variety of applications.     

Electrochemically modulated liquid chromatography coupled on-line with electrospray mass spectrometry

Electrochemically modulated liquid chromatography (EMLC) has been coupled to an electrospray mass spectrometer. This combination takes advantage of the ability of EMLC to manipulate retention and enhance separation efficiency solely through changes in the potential applied to a conductive stationary phase, thereby minimizing complications because of possible changes in analyte ionization efficiencies when gradient elution techniques are used. Three examples are presented that demonstrate the attributes of this EMLC/electrospray mass spectrometry (ES-MS) coupling. The first two examples involve the separation of mixtures of corticosteroids or of benzodiazepines, showing the general utility of the union for eluent identification and low-level detection. The ability to identify products from on-column redox transformations is also demonstrated using the benzodiazepine mixture. The third example investigates the electrooxidation of aniline by utilizing an EMLC column as an on-line electrochemical reactor and product separator and ES-MS for detection and product identification.     

Parallel detection of intrinsic fluorescence from peptides and proteins for quantification during mass spectrometric analysis

Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ～300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa.