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.     

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.     

Pulsed lasers versus continuous light sources in capillary electrophoresis and fluorescence detection studies: Photodegradation pathways and models

Pulsed lasers are widely used in capillary electrophoresis (CE) studies to provide laser induced fluorescence (LIF) detection. Unfortunately pulsed lasers do not give linear calibration curves over a wide range of concentrations. While this does not prevent their use in CE/LIF studies, the non-linear behavior must be understood. Using 7-hydroxycoumarin (7-HC) (10–5000 nM), Tamra (10–5000 nM) and tryptophan (1–200 μM) as dyes, we observe that continuous lasers and LEDs result in linear calibration curves, while pulsed lasers give polynomial ones. The effect is seen with both visible light (530 nm) and with UV light (355 nm, 266 nm). In this work we point out the formation of byproducts induced by pulsed laser upon irradiation of 7-HC. Their separation by CE using two Zeta LIF detectors clearly shows that this process is related to the first laser detection. All of these photodegradation products can be identified by an ESI-/MS investigation and correspond to at least two 7HC dimers. By using the photodegradation model proposed by Heywood and Farnsworth (2010) and by taking into account the 7-HC results and the fact that in our system we do not have a constant concentration of fluorophore, it is possible to propose a new photochemical model of fluorescence in LIF detection. The model, like the experiment, shows that it is difficult to obtain linear quantitation curves with pulsed lasers while UV-LEDs used in continuous mode have this advantage. They are a good alternative to UV pulsed lasers. An application involving the separation and linear quantification of oligosaccharides labeled with 2-aminobezoic acid is presented using HILIC and LED (365 nm) induced fluorescence.     

A comparative study of LED-induced fluorescence and laser-induced fluorescence in SDS-CGE: application to the analysis of antibodies

LEDs present an alternative to lasers in LIF detection with CE, resulting in LED-induced fluorescence (LEDIF). LEDs are much less expensive, consume less energy and are more stable. In addition, LED light sources allow a greater range of wavelengths to better match the maximum wavelength for the fluorescence of the dye. Antibodies were largely studied in SDS capillary gel electrophoresis (SDS-CGE) and LIF detection with different dyes to label the proteins. In this work, our goal is to show that LEDs can advantageously replace lasers. We used 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA.SE), 3-(2-furoyl)-quinoline-2 carboxaldehyde (FQ), and naphthalene-2,3-dialdehyde (NDA) to label IgG and we compared the LIF sensitivity with that obtained from LEDIF. We measured that the LOD values of LEDIF are identical to that obtained with the wavelength equivalent laser, and for 5-TAMRA.SE analysis, LOD values are about six times better than when the classical 488 nm laser was used.     

Solid-state UV laser-induced fluorescence detection in capillary electrophoresis

Two solid-state UV lasers were applied to the laser-induced fluorescence (LIF) detection of various groups of compounds after separation by capillary electrophoresis. These lasers are thermoelectric-cooled, highly compact, and inexpensive. Such lasers provide few mW of quasi-continuous wave (CW) power which are sufficient and stable for LIF detection. Native fluorescence detection of tryptophan-containing proteins and peptides and related indoles was achieved at the nM level with the laser operating at 266 nm. Detection of fluorescamine-labeled amino acids and peptides was also possible at the nM level with the laser operating at 355 nm. Amino acids at a concentration as low as 10 ng/mL could be labeled with fluorescamine. Solid-state UV-LIF detection of the tryptic digest of cytochrome c after fluorescamine derivatization was demonstrated.     

High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis.

A highly sensitive laser-induced (LIF) detection scheme for native, tryptophan- or tyrosine-containing proteins in capillary electrophoresis (CE) has been demonstrated. The 275.4 nm line from an argon-ion laser is used to excite native protein fluorescence. A limit of detection (LOD) (S/N = 2) of 1 x 10(-10) M for conalbumin represents a 140-fold improvement over earlier reports. With stacking at injection, the LOD is 3 x 10(-12) M. Linear dynamic ranges of at least 5 and 4 orders of magnitude for, respectively, tryptophan and bovine serum albumin are found. The practical performance and blueprint of an easily constructed, rugged, compact and user-friendly LIF detector for CE are shown.     

Determination of lead in water using laser ablation–laser induced fluorescence

The detection sensitivity of laser-induced breakdown spectroscopy (LIBS) is improved by coupling it with a laser-induced fluorescence method. A waterjet sample containing 500 ppm of Pb as an analyte was ablated by a 266 nm, frequency-quadrupled Q-switchedNd:YAG laser at an energy of ~ 260 μJ. After a short delay the resulting plume was re-excited with a 283.306 nm, nanosecond pulse dye laser at energies ranging from 45 to 100 nJ. The limit of detection (LOD) of lead in water was determined both by the single-pulse LIBS technique and Laser Ablation coupled with Laser-Induced Fluorecence (LA–LIF) method. It was found to be 75 ppm in the case of single-pulse LIBS and 4.3 ppm for LA–LIF. When the resonant pulse was detuned from the transition wavelength the LA–LIF signal disappeared demonstrating the resonant selectivity of this technique.     

Direct Measurement of SO2 in Air by Laser Induced Fluorescence Spectrometry Using a Nontunable Laser Source

Studies have been performed to evaluate a direct laser induced fluorescence (LIF) technique for measurements of atmospheric sulfur dioxide (SO₂). The technique is novel in that it uses a nontunable laser source that is spectrally coincident with absorption of the SO₂ molecule near 223 nm that allows sensitive measurements at environmentally relevant concentrations. In this report, the spectral characteristics and analytical capabilities of the nontunable LIF approach have been evaluated and preliminary measurements of ambient SO₂ are reported. The current limit of detection is 0.5 ppb and compares well to other analytical spectroscopy methods used for atmospheric measurements of SO₂. The results indicate strong feasibility for the nontunable LIF approach for SO₂ measurements and suggest ways for method improvement.     

Highly sensitive and direct detection of DNA fragments using a laser-induced capillary vibration effect.

A pulsed laser-induced stationary wave capillary vibration detection method was applied to the sensitive detection of capillary gel electrophoresis, and the direct detection of non-labeled nucleic acids, such as DNA sequencing products, was demonstrated. An excimer laser operating at 248 nm was used as a CVL excitation source, and polynucleotides were sensitively detected without derivatization. From an investigation on the endurance of several matrixes to pulsed laser irradiation, a polyacrylamide without a cross-linker (0%C) was found to have adequate endurance, and it exhibited no serious damage during an analysis. A cytosine-terminated sequence reaction product was detected with a sensitivity close to that of laser-induced fluorometry (LIF). These results suggest the feasibility of the highly sensitive detection of ultramicro amounts of biological materials without a pre- or post-column derivatization, which has usually been required in sensitive detection procedures, such as LIF. Furthermore, the feasibility of a novel DNA sequencing method is also suggested.     

Laser-Induced fluorescence detection in conventional-size liquid chromatography with a frequency-doubled argon-ion laser

Laser-induced fluorescence (LIF) detection in conventional-size column liquid chromatography is achieved at 257 nm with a frequency-doubled argon-ion laser. Short-wavelength excitation offers two important advantages: firstly, a wide variety of analytes can be excited, and secondly, the Raman scatter of the eluent does not interfere with the fluorescence of the analytes. A standard mixture of polynuclear aromatic hydrocarbons was studied, both with LIF detection and with a commercially available sensitive conventional fluorescence detector. The improvement in the detection limits ranges from about a factory of 4 to 30; the LIF detection limits are typically at the 50 ng l^?1 level, which corresponds to an injected amount of 0.5 pg.     

Development and Application of a Hydride Generation Laser Induced Fluorescence Method for Measurements of Bismuth

A hydride generation laser induced fluorescence (HG LIF) approach has been investigated for trace level measurements of bismuth. The technique uses a tunable dye laser operating at 306.7 nm as the excitation source and bismuth fluorescence is measured at 472 nm. The optimized HCl and NaBH₄ concentrations for bismuth measurements were 1.2 M and 2.0%, respectively. The current technique has a limit of detection of 0.03 ppb and 0.01 ppb for blank measurements performed with the laser tuned on and off the bismuth excitation wavelength, respectively. Measurements of bismuth in different sample matrices have demonstrated the effectiveness of thiourea and ascorbic acid as masking agents for measuring samples containing interfering ions. Measurements of bismuth have been performed in reference materials, a bismuth-containing medication, and tea leaves. The results demonstrate that the HG LIF approach has feasibility for measuring bismuth in various samples at environmentally relevant concentrations.     

Optical-fiber sensor based on the second-harmonic emission of a near-infrared semiconductor laser as light source

Second-harmonic emission from a near-infrared semiconductor laser is used as the light source for fluorimetry. The efficiency of the second-harmonic generation is 2.5×10^?6; at 390 nm, the power achieved is 50 nW when a 20-mW semiconductor laser (780 nm) is used. For fluorimetric determination of 7-diethylamino-4-methylcoumarin, the calibration plot is linear in the range 0–8×10^?7 M, the detection limit being 5×10^-∞ M (S/N=3 ). The generated ultraviolet emission is used with benzo (ghi)perylene in an optical-fiber sensor for oxygen (0–15%).     

Nonaqueous capillary electrophoresis coupled with laser-induced native fluorescence detection for the analysis of berberine, palmatine, and jatrorrhizine in Chinese herbal medicines

LIF detection is one of the most sensitive detection methods for CE. However, its application is limited because the analyte is usually required to be derivatized with a fluorescent label. As a result, LIF is seldom used to analyze active ingredients in plants. In this work, we introduce a rapid, simple, and sensitive method of nonaqueous CE (NACE) coupled with laser-induced native fluorescence detection for the simultaneous analysis of berberine, palmatine, and jatrorrhizine. This method skillfully utilizes the native fluorescence of these alkaloids and requires no troublesome fluorescent derivatization. As these alkaloids can fluoresce to some degree, they were simply detected by a commercially available 488 nm Ar+ laser. The native fluorescence of the analytes was greatly enhanced by nonaqueous media. Compared with the reported UV detection method, much lower LOD was achieved (6.0 ng/mL for berberine, 7.5 ng/mL for palmatine, and 380 ng/mL for jatrorrhizine). This method was successfully applied to analyze berberine, palmatine, and jatrorrhizine in two Chinese herbal medicines, Rhizoma coptidis and Caulis mahoniae.     

High-performance liquid chromatographic detector based on near-infrared semiconductor laser fluorimetry

Three polymethine dyes, which fluoresce at 780–820 nm, are separated by reversed-phase chromatography and detected fluorimetrically with a semiconductor laser for excitation. The detection limit for anhydro-3,3,3′,3′-tetramethyl-1,1′-bis(4-sulfomethyl)-4,5,4′,5′-dibenzoindotricarbocyanine hydroxide (sodium salt) is 0.3 pg, which is almost three orders of magnitude better than the value obtained by conventional fluorimetry.     

Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresis

Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.     

Some factors affecting enantiomeric impurity determination by capillary electrophoresis using ultraviolet and laser-induced fluorescence detection.

The key factors influencing enantiomer trace determination were investigated; these include resolution capillary diameter, limit of detection, linear range and type of detection. Chiral reagents, (+)- and (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC), were employed as probes to demonstrate the influence of the variables. In order to find the best resolution, separation variables were optimized in both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) modes by the application of factorial design experiments. A highly efficient chiral separation of the (+/-)-FLEC, derivatized with nonchiral amino acids, was achieved when using gamma-cyclodextrin as the chiral selector. The benefits of using a small diameter capillary for direct determination of both (+) and (-)-FLEC impurity (0.05-0.1% area/area) were demonstrated using UV detection and applying a sample stacking condition. A frequency-doubled argon ion laser (244 nm) was used as light source for laser-induced fluorescence (LIF) detection. Excitation light was provided by means of an optical fiber directed into the Hewlett Packard 3D capillary cartridge. The signals from UV and LIF were monitored simultaneously. The application of LIF detection greatly improved sensitivity and linear range. Further, as a consequence of the increased sensitivity, sample loading could be decreased, which led to an improvement of separation efficiency. Direct determination of 0.005% impurity could be achieved within the linear range.     

Laser-induced fluorescence detection at 266 nm in capillary electrophoresis. Polycyclic aromatic hydrocarbon metabolites in biota

The separation of five phenolic polycyclic aromatic hydrocarbon metabolites (hydroxy-PAHs) has been performed by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using a 30 mM borate buffer (pH 9.0) containing 60 mM sodium dodecyl sulfate and varying concentrations of gamma-cyclodextrin (gamma-CD). A concentration of 12.5 mM gamma-CD was found to provide a baseline separation of the five hydroxy-PAHs. We applied conventional fluorescence and laser-induced fluorescence (LIF) detection, using a new, small-size, quadrupled Nd-YAG laser emitting at 266 nm. The best limits of detection, in the low ng/ml range, were achieved using LIF detection. For all analytes, linearity was observed up to ca. 100 ng/ml. As an application, conjugated pyrene metabolites in hepatopancreas samples from the terrestrial isopods Oniscus asellus and Porcellio scaber were separated and detected. Finally, flatfish bile samples from individuals exposed to polluted sediment or crude oil, which were part of an interlaboratory study, were analyzed by CD-MEKC with conventional fluorescence and LIF detection to determine the 1-hydroxypyrene concentrations.     

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.     

超声射流下12C16O+离子A2∏1/2,3/2←X2∑+激光诱导荧光激发谱

用一束波长为230.1 nm的激光, 通过(2+1)共振增强多光子电离(REMPI)过程激发超声射流冷却的CO分子制备处于基电子态X2∑+的CO+离子, 随后引入另一束可调谐激光将CO+离子激发至A2∏1/2,3/2态, 利用光电倍增管(PMT)检测发射的荧光信号强度随激发光波长的变化, 分别在487-493 nm和453-459 nm波长范围内获得了CO+离子A2∏1/2,3/2←X2∑+电子态跃迁(0,0)和(1,0)带的激光诱导荧光(LIF)激发谱.     

Boron‐chelating fluorescent probe (BOPB) in the red region combined with CE‐LIF for the detection of NO in mice liver

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8‐(3’,4’‐Diamino phenyl)‐3,5‐(2‐hydroxyphenyl)‐dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE‐LIF detection. Therefore, BOPB was used in CE‐LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB‐T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H₃BO₃–NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10^?9–5.0 × 10^?7 M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization‐based CE‐LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.