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.     

Continuous wave-based multiphoton excitation fluorescence for capillary electrophoresis

It was reported that a novel detection method, continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection with diode laser (DL), has been firstly proposed for capillary electrophoresis (CE). Special design of end-column detection configuration proved to be superior to on-column type, considering the detection sensitivity. Three different kinds of fluorescent tags that were widely used as molecular label in bio-analysis, such as small-molecule dye, fluorescent protein and nano particle or also referred to as quantum dot (QD), have been evaluated as samples for the constructed detection scheme. Quantitative analyses were also performed using rhodamine species as tests, which revealed dynamic linear range over two orders of magnitude, with detection limit down to zeptomole-level. Simultaneous detection of fluorescent dyestuffs with divergent excitation and emission wavelengths in a broad range showed advantage of this scheme over conventional laser-induced fluorescence (LIF) detection. Further investigations on CW-MPE fluorescence detection with diode laser for capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations of fluorescein isothiocyanate (FITC) labeled amino acids indicated good prospect of this detection approach in various micro or nano-column liquid phase separation technologies.     

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.     

Electrically driven microseparation methods for pesticides and metabolites: VI. Surfactant-mediated electrokinetic capillary chromatography of aniline pesticidic metabolites derivatized with 9-fluoroenylmethyl chloroformate and their detection by laser-induced fluorescence

In this report, we describe a surfactant-mediated electrokinetic capillary chromatography (SM-EKC) system for the separation of 9-fluoroenylmethyl chloroformate (FMOC)-derivatized anilines by capillary electrophoresis (CE). The SM-EKC system consisted of dioctyl sulfosuccinate (DOSS)/acetonitrile mixtures and was suited for the CE separation of the relatively hydrophobic FMOC-aniline analytes and other neutral compounds, e.g. alkylphenyl ketones. While the organic modifier acetonitrile (ACN) allowed the solubilization of the hydrophobic solutes and maintained the DOSS surfactant in its monomeric form by inhibiting micellization, the DOSS surfactant associated with the FMOC anilines to a varying degree thus leading to their differential migration and separation. Under these conditions, the FMOC-anilines were readily detected at the 10(-6) M level by UV at 214 nm and at the 10(-8) M level by laser-induced fluorescence (LIF) using a solid-state UV laser operating at 266 nm line as the excitation wavelength. The FMOC precolumn derivatization was also readily performed in lake water spiked with anilines at near the limit of detection (LOD) level. The lake water matrix showed no significant effects on the extent of derivatization at the LOD level as well as on the detection of the analytes due to the selectivity of the FMOC derivatization. The derivatization and detection of spiked lake water necessitated only the removal of microparticles by microfiltration prior to derivatization and detection.     

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.     

Strategies to improve the sensitivity in capillary electrophoresis for the analysis of drugs in biological fluids

Capillary electrophoresis (CE) is a useful method to quantify drugs in biological fluids. However, especially for blood or plasma samples, the sensitivity is not sufficient to quantify drugs and their metabolites as they often need to be quantified in the lower microg/L range. To overcome this limitation and to increase the sensitivity, two strategies are applied: first, to increase the amount of analyte added to the capillary and, second, to increase the sensitivity on the detector site. To improve the sensitivity on the detector site, alternative detection techniques to UV detection, e.g., laser-induced fluorescence detection (LIF) or mass spectroscopy (MS), can be applied. However, LIF detection can only be used for fluorescent analytes and the current equipment for CE-MS coupling provides only small improvements in sensitivity compared to UV detection. The detection window for UV detection can be enhanced using capillaries with an extended light path (bubble cell) or Z-shaped capillaries. Sensitivity improvements up to a factor of 10 have been reported. Increasing the amount of analyte in the capillary can be done either by chromatographic or by electrokinetic methods. Chromatographic methods such as on-capillary membrane preconcentration have been used for several analytes. However, no validated application has been reported to date. In contrast, several validated examples can be found in which electrokinetic techniques like sample stacking have been applied to achieve limits of quantification in the lower microg/L range. In conclusion, to date, electrokinetic techniques such as field-amplified sample injection offer the most promising results in achieving a sufficient sensitivity to quantify drugs in biological fluids.     

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.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.     

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.     

Enhanced detection of porphyrins by capillary electrophoresis-laser induced fluorescence

A highly sensitive technique for the analysis of urinary porphyrins using capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection is reported. Separation of mesoporphyrin IX, coproporphyrin, uroporphyrin and the penta-, hexa- and heptacarboxylic acid porphyrins was achieved in 11 min using a 10 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES, pH 10) -75 mM sodium dodecyl sulfate (SDS) buffer. Migration time and peak area repeatability were less than 1 and 5% relative standard deviation (RSD), respectively. Limits of detection of 20 pM (2 x 10(-11) M) were achieved employing the recently introduced Nichia violet diode laser for excitation at 400 nm. This represents an enhancement in sensitivity of over two orders of magnitude compared to previous reports. This high sensitivity for urinary porphyrins was demonstrated through the quantification of coproporphyrin and uroporphyrin in urine samples after up to a 100-fold dilution.     

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.     

Profiling of impurities in illicit methamphetamine by high-performance liquid chromatography and capillary electrochromatography

High performance liquid chromatography (HPLC) with photodiode array (PDA) UV and fluorescence (FL) detection, and capillary electrochromatography (CEC) with laser-induced fluorescence (LIF) detection were investigated for the analysis of acidic extracts derived from illicit methamphetamine. These compounds include major impurities from the hydriodic acid/red phosphorous reduction method, i.e., 1,3-dimethyl-2-phenylnaphthalene and 1-benzyl-3-methylnaphthalene, and other trace-level, structurally related impurities. For certain of these solutes, HPLC with conventional FL detection gave at least a 60× increase in sensitivity over UV detection. In addition, other highly fluorescent impurities were detected in methamphetamine produced via four other synthetic routes. The use of a rapid scanning FL detector (with acquisition of “on the fly” excitation or emission) provided structural information and gave “optimum” excitation and emission detection wavelengths. CEC with LIF detection using UV laser excitation provided greatly improved chromatography over HPLC, with good detection limits in the low ng/ml range. Both methodologies provide good run-to-run repeatability, and have the capability to distinguish between samples.     

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.     

An ellipsoidal mirror for detection of laser-induced fluorescence in capillary electrophoresis system: applications for labelled antibody analysis

An LIF detector was integrated into a CE system which uses a ball lens to focus the laser beam on the CE capillary. The detector employs an ellipsoid that is glued on the capillary window, to permit the collection of the fluorescence in the capillary. This 'trapped' fluorescence stays in the capillary because the angle of the silica/air interface is greater than the critical angle. The performance of this new detector setup is found to be identical to the collinear setup using the same ball lens. An application to the analysis of FITC-labeled IgG was optimized using a 14 cm effective length capillary. The LOD of an FITC-labeled IgG2 at an excitation wavelength of 488 nm was 150 pg/mL, which was 10 times better than the LOD recorded with slab gel silver staining. Using a tetramethylrhodamine (TAMRA)-labeled IgG2 and a 532 nm excitation wavelength the LOD is 50 pg/mL. The electropherograms of four different commercial FITC conjugates of IgG were studied. The presence of aggregates was observed in two samples while close kinetics of reduction was observed between free aggregates and high aggregates concentration samples. The integrated LIF detector provides an extremely powerful and convenient tool for antibody analysis and should be useful for therapeutic MAb control in pharmaceutical facilities.     

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.     

多材料3D打印技术制作用于毛细管电泳的非接触电导/激光诱导荧光二合一检测池

利用多材料3D打印技术研制了用于毛细管电泳(CE)的二合一检测池,实现了电容耦合非接触电导(C4D)与共聚焦激光诱导荧光(LIF)两种检测方法在毛细管柱上同一位置同时检测.3D打印的检测池采用了导电的复合聚乳酸(PLA)材料制作C4 D的屏蔽层,采用普通的绝缘PLA材料支撑C4 D金属管电极并隔离屏蔽层.两根金属管电极...     

Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis

The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser‐induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE‐LIF‐REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE‐LIF. The results demonstrate that the CE‐LIF‐REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.     

Whole-column fluorescence-imaged capillary electrophoresis

An axially illuminating whole-column fluorescence imaging capillary electrophoresis (CE) experimental setup was developed. A 6 cm long Teflon tube with an inside diameter (ID) of 42 microm was used as separation column. Excitation light of 488 nm from Ar+ laser was introduced to one end of the separation column by an optical fiber. The excitation light propagated inside the separation column by total internal reflection, since the refractive index of the buffer solution was larger than that of the Teflon tube. The fluorescence from the whole separation column was imaged with a charge-coupled device (CCD) camera. Fluorescent compounds such as fluorescein isothiocyanate (FITC), 5-carboxyfluorescein, and FITC-labeled protein were used to test the basic performance of the experimental setup. Experimental results illustrate that the whole-column-fluorescence imaging CE is a fast and sensitive separation method for fluorescent compounds and fluorescent-labeled proteins. Furthermore, it could be used for simple, fast, and easy comparisons of the resistance to photodegradation for various fluorescent compounds.     

Laser-induced fluorescence as a powerful detection tool for capillary electrophoretic analysis of heparin/heparan sulfate disaccharides

In quest for high sensitivities necessary for determining the disaccharide composition of heparin/heparan sulfate present in trace amounts in biologic samples, an ultrahighly sensitive capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection was developed. Heparin/heparan sulfate-derived Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone and resolved by a reversed-polarity CE method. Estimation of the limit of detection in concentration term and limit of quantitation showed that LIF detection of AMAC-derivatives of Delta-disaccharides resulted in 27-744 times higher sensitivity as compared to those detected by UV at 255 nm. These data suggest that CE-LIF is a powerful tool to quantify minute amounts of heparin/heparan sulfate disaccharides.     

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.