On-column detection of multiphoton-excited fluorescence in CE using hyphenated cylindrical-square capillaries

Multiphoton-excited fluorescence (MPEF) is a complementary and useful mode of LIF detection in CE with advantages of ultra-low mass detectability and spectral excitability, but it is currently quite limited by its end-column configuration. In this article, we demonstrate a novel strategy of on-column schemes that can greatly facilitate MPEF detection in CE. FITC-labeled amine species were used as the model samples for the evaluation and comparison of those detection scenarios. By using the square capillary instead of the conventional cylindrical one, the on-column MPEF could be readily achieved, with detection sensitivity of 0.72 microM that was comparable with the end-column mode. However, this strategy unfavorably reduced separation efficiency. The theoretical plate number on averaging all the sample peaks was significantly decreased from 283,000 to 19,000/m. To minimize such an influence, a short square capillary acting as an on-column MPEF detection cell was then mounted to a long cylindrical capillary responsible for the CE separation. Results indicated that both high separation efficiency (240,000/m) and better detectability (0.42 microM) were realized simultaneously by using this binary-capillary configuration. Quantitative analysis was performed under the optimized detector configuration and revealed a linear dynamic range of 2 orders of magnitude, with mass detection limit down to the mid-yottomole level.     

Separation and determination of amino acids by micellar electrokinetic chromatography coupling with novel multiphoton excited fluorescence detection

In this article, it was demonstrated that separation and determination of 20 amino acids were accomplished by micellar electrokinetic chromatography (MEKC) coupling with novel multiphoton excited fluorescence (MPEF) detection method. Different from MPEF achieved by expensive fs laser, continuous wave (CW) diode laser of ultra-low cost was uniquely employed in our MPEF system. Amino acids were fluorescently labeled with fluorescein isothiocyanate (FITC), and were subjected to sodium dodecyl sulfate (SDS)-based MEKC separation and CW-based MPEF detection. The result was compared with that by single photon excited fluorescence (SPEF), which indicated that MPEF had the advantages of better mass detectability and higher separation selectivity over SPEF. Quantitative analysis was performed and revealed linear dynamic range of over 2 orders of magnitude, with mass detection limit down to ymole level. To evaluate the reliability, this method was successfully applied for analyzing a commercial nutrition supplement liquid.     

Sensitivity enhancement of fluorescence detection in CE by coupling and conducting excitation light with tapered optical fiber

This paper reports the enhancement of sensitivity of detection for in‐column fiber optic‐induced fluorescence detection system in CE by tapered optical fiber (TOF). Two types of optical fiber, TOF and conventional cylindrical optical fiber (COF), were employed to construct the CE (TOF‐CE and COF‐CE) and were compared for sensitivity to riboflavin (RF). The fluorescence intensities from a RF sample with excitation light sources and fibers at various coupling angles were investigated. The fluorescence signal from TOF‐CE was ca. ten times that of COF‐CE. In addition, the detection performance of four excitation light source‐fiber configurations including Laser‐TOF, Laser‐COF, LED‐TOF, and LED‐COF were compared. The LODs for RF were 0.21, 0.82, 0.80, and 7.5 nM, respectively, for the four excitation light source–fiber configurations. The results demonstrate that the sensitivity obtained by LED‐TOF is close to that of Laser‐COF. Both Laser‐TOF and LED‐TOF can greatly improve the sensitivity of detection in CE. TOF has the major attribute of collecting and focusing the excitation light intensity. Thus, the sensitivity obtained by LED‐TOF without focusing lens is just same as that of LED‐COF with a focusing lens. This demonstrates that the CE system can be further simplified by eliminating the focusing lens for excitation light. LED‐TOF‐CE and LED‐COF‐CE system were applied to the separation and determination of RF in real sample (green tea), respectively. The tapered fiber optic‐induced fluorescence detection system in CE is an ideal tool for trace analysis.     

In-column fiber-optic laser-induced fluorescence detection for CE

A highly sensitive in-column fiber-optic LIF detector for CE has been constructed and evaluated. In this detection system, a 457-nm diode-pumped solid-state blue laser was used as the excitation light source and an optical fiber (40 mum od) was used to transmit the excitation light. One end of the optical fiber was inserted into the separation capillary and was in situ positioned at the detection window. The other end of the fiber was protruded from the capillary to capture the excitation light beam from the blue laser. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a yellow color filter before reaching the photomultiplier tube. The present CE-fluorescence detection is a simple and compact optical system. It reduces the laser scattering effect from the capillary and fiber as compared to the conventional LIF detection for CE. Its utility was successfully demonstrated by the separation and determination of D-penicillamine labeled with naphthalene-2,3-dicarboxaldehyde. The detection limit was 0.8 nM (S/N = 3). The present detection scheme has been proven to be attractive for sensitive fluorescence detection for CE.     

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.     

UV- and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis

UV- and visible-excited fluorescence detection strategies were compared for nucleic acids separated by capillary electrophoresis (CE). A dual-polymer sieving matrix consisting of hydroxypropylmethylcellulose and poly(vinylpyrrolidone) was used to separate DNA fragments from a 100-base pair ladder and RNA from individual cells. Two nucleic acid dyes, SYBR Gold and SYBR Green I, were evaluated for their performance at both UV (275 nm) and visible (488 nm) excitation wavelengths. While SYBR Gold-bound RNA from single cells yielded a substantially reduced UV-excited signal compared to that with visible excitation (as expected), the sensitivity of SYBR Gold-bound double-stranded DNA was comparable for UV and Vis excitation wavelengths. This study reveals the first demonstration of using SYBR Gold dyes for DNA detection following separation with CE and also the first example of SYBR-based detection of RNA sampled and separated from individual cells.     

Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

A lamp‐based fluorescence detection (Flu) system for CE was extended with a wavelength‐resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe‐Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut‐off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal‐intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE‐WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged‐polymer coated capillary. Using the 3‐D data set, signal averaging over time and emission‐wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.     

Optical fiber light-emitting diode-induced fluorescence detection for capillary electrophoresis

A highly sensitive optical fiber light-emitting diode (LED)-induced fluorescence detector for CE has been constructed and evaluated. In this detector, a violet or blue LED was used as the excitation source and an optical fiber with 40 microm OD was used to transmit the excitation light. The upper end of the fiber was inserted into the separation capillary and was situated right at the detection window. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a cutoff filter before reaching the photomultiplier tube. Output signals were recorded and processed with a computer using in-house written software. The present CE/fluorescence detector deploys a simple and inexpensive optical system that requires only an LED as the light source. Its utility was successfully demonstrated by the separation and determination of amino acids (AAs) labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and FITC. Low detection limits were obtained ranging from 17 to 23 nM for NDA-tagged AAs and 8 to 12 nM for FITC-labeled AAs (S/N=3). By virtue of such valuable features as low cost, convenience, and miniaturization, the presented detection scheme was proven to be attractive for sensitive fluorescence detection in CE.     

A simple light-emitted diode-induced fluorescence detector using optical fibers and a charged coupled device for direct and indirect capillary electrophoresis methods

We constructed a simple fluorescence detector for both direct and indirect CE methods using a blue light-emitted diode (470 nm) as excitation source, a bifurcated optical fiber as a waveguide, and a CCD camera as a detector. The connection of all the components is fairly easy even for nonexperts and the use of a CCD camera improves the applicability of this detector compared to the others using PMTs because it permits the recording of 2-D electropherograms or phosphorescence measurements. This detector provides a compact, low cost, and rapid system for the determination of native fluorescence compounds which have high quantum yields by CE with direct fluorescence detection, showing an LOD of 2.6 x 10(-6) M for fluorescein; the determination of fluorescence derivative compounds by CE with direct fluorescence detection, showing an LOD of 1.6 x 10(-7) M for FITC-labeled 1,6-diaminohexane; and nonfluorescence compounds by CE with indirect fluorescence detection with an LOD of 2.7 x 10(-6) M for gallic acid.     

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.     

A simple and compact fluorescence detection system for capillary electrophoresis and its application to food analysis

A novel fluorescence detection system for CE was described and evaluated. Two miniature laser pointers were used as the excitation source. A Y‐style optical fiber was used to transmit the excitation light and a four‐branch optical fiber was used to collect the fluorescence. The optical fiber and optical filter were imported into a photomultiplier tube without any extra fixing device. A simplified PDMS detection cell was designed with guide channels through which the optical fibers were easily aligned to the detection window of separation capillary. According to different requirements, laser pointers and different filters were selected by simple switching and replacement. The fluorescence from four different directions was collected at the same detecting point. Thus, the sensitivity was enhanced without peak broadening. The fluorescence detection system was simple, compact, low‐cost, and highly sensitive, with its functionality demonstrated by the separation and determination of red dyes and fluorescent whitening agents. The detection limit of rhodamine 6G was 7.7 nM (S/N = 3). The system was further applied to determine illegal food dyes. The CE system is potentially eligible for food safety analysis.     

Comparative study between a commercial and a homemade capillary electrophoresis instrument for the simultaneous determination of aminated compounds by induced fluorescence detection

The performance of two capillary electrophoresis (CE) instruments, one commercial and one homemade device, were compared for the determination of derivatised aminated compounds with fluorescein isothiocyanate (FITC). The commercial CE system first uses an argon ion laser as excitation source; the homemade CE device uses an inexpensive blue-light-emitting diode (LED) as the light source and a charge-coupled device (CCD) as the detection system. After fine optimisation of several separation parameters in both devices, a co-electroosmotic flow CE methodology was achieved in coated capillary tubing with 0.001% hexadimetrine bromide (HDB), and 50 mmol L⁻¹ sodium borate at pH 9.3 with 20% 2-propanol for the determination of several amines and aminoacids. Analytical performances, applicability in beer samples and other aspects such as cost or potential for miniaturization have been compared for both devices.     

Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT

A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.     

Miniaturized liquid core waveguide-based fluorimetric detection cell for capillary separation methods: application in CE of amino acids

A miniaturized post-column fluorimetric detection cell for capillary separation methods based on optical fibers and liquid core waveguides (LCWs) is described. The main part of the detection cell is a fused-silica capillary coated with Teflon AF serving as an LCW. The optical fibers are used both for coupling the excitation source with the detection domain in the LCW and for the axial fluorescence collection from the LCW end. The latter fiber is connected with a compact CCD spectrometer that serves for the rejection of the scattered excitation light and for the fluorescence signal detection. The proposed design offers a compact fluorescence detector for various microcolumn separation techniques without optical elements such as filters or objectives. Moreover, its construction and optical adjustment are very simple and the whole system is highly miniaturized. The function of the detection cell is demonstrated by CE of amino acids labelled by fluorescein-based tags. Separations of different standard amino acid mixtures and plasma samples are presented. The comparison of plasma amino acid levels of individuals being in good health with those of patients with inherited metabolic disorders is also shown.     

Simultaneous light emitting diode-induced fluorescence and contactless conductivity detection for capillary electrophoresis.

A combined detection system of simultaneous contactless conductometric and fluorescent detection for capillary electrophoresis (CE) has been designed and evaluated. The two processes share a common detection cell. A blue light-emitting diode (LED) was used as the excitation source and an optical fiber was used to collect the emitting fluorescence for fluorescent detection (FD). Inorganic ions, fluorescein isothiocyanate (FITC)-labeled amino acids and small molecule peptides were separated and detected by the combined detector, and the detection limits (LODs) of sub-microM level were achieved.     

LIF detection of peptides and proteins in CE

CE- and microchip-based separations coupled with LIF are powerful tools for the separation, detection and determination of biomolecules. CE with certain configurations has the potential to detect a small number of molecules or even a single molecule, thanks to the high spatial coherence of the laser source which permits the excitation of very small sample volumes with high efficiency. This review article discusses the use of LIF detection for the analysis of peptides and proteins in CE. The most common laser sources, basic instrumentation, derivatization modes and set-ups are briefly presented and special attention is paid to the different fluorogenic agents used for pre-, on- and postcapillary derivatization of the functional groups of these compounds. A table summarizing major applications of these derivatization reactions to the analysis of peptides and proteins in CE-LIF and a bibliography with 184 references are provided which covers papers published to the end of 2005.     

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser‐induced fluorescence detection

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK_a determination of small molecule analytes is demonstrated with the multilane microchip.     

Automated method for analysis of tryptophan and tyrosine metabolites using capillary electrophoresis with native fluorescence detection

Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) detection offers the ability to characterize low levels of selected analyte classes, depending on the excitation and emission wavelengths used. Here a new automated CE-LINF system that provides deep ultraviolet (DUV) excitation (224 nm) and variable emission wavelength detection was evaluated for the analysis of small molecule tryptophan- and tyrosine-related metabolites. The optimized instrument design includes several features that increase throughput, lower instrument cost and maintenance, and decrease complexity when compared with earlier systems using DUV excitation. Sensitivity is enhanced by using an ellipsoid detection cell to increase the fluorescence collection efficiency. The limits of detection ranged from 4 to 30 nmol/L for serotonin and tyrosine, respectively. The system demonstrated excellent linearity over several orders of magnitude of concentration and intraday precision from 1–11 % relative standard deviation (RSD). The instrument’s performance was validated via tryptophan and serotonin characterization using tissue extracts from the mammalian brain stem, with RSDs of less than 10 % for both metabolites. The flexibility and sensitivity offered by DUV laser excitation and tunable emission enables a broad range of small-volume measurements.     

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

An LIF detector was integrated into a CE system based on silver mirror coating detection window and small‐angle optical deflection from collinear configuration. For this detection scheme, the incident light beam was focused on capillary through the edge of a lens, resulting in a small deflection angle that deviated 18° from the collinear configuration. Meanwhile, the excitation light and emitted fluorescence were effectively reflected by silver mirror coating at the detection window. The fluorescence was collected through the center of the same lens and delivered to a PMT in the vertical direction. In contrast to conventional collinear LIF detection systems, the fluorescence intensity was greatly enhanced and the background level was significantly eliminated. FITC and FITC‐labeled amino acids were used as model analytes to evaluate the performance with respect to design factors of this system. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems. All the results indicate that the proposed detection scheme will be promising for development of sensitive and low‐cost CE system.