An integrated light emitting diode-induced fluorescence detector for capillary electrophoresis

An integrated light emitting diode (LED)-induced fluorescence detector was described and evaluated. The LED and its related components including lens and interference filter, the optical fiber used to collect fluorescence, and the capillary column are integrated into a substrate block, which eliminates the need of align procedure of the fiber and the capillary. Forty-fold enhancement of sensitivity was obtained compared with our previous work and the detection limit for fluorescein was 5 nM. Application of the detector for the analysis of FITC-labeled Ephedrine extract was demonstrated.     

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.     

Enhancement of signal-to-noise level by synchronized dual wavelength modulation for light emitting diode fluorimetry in a liquid-core-waveguide microfluidic capillary electrophoresis system

The signal-to-noise level of light emitting diode (LED) fluorimetry using a liquid-core-waveguide (LCW)-based microfluidic capillary electrophoresis system was significantly enhanced using a synchronized dual wavelength modulation (SDWM) approach. A blue LED was used as excitation source and a red LED as reference source for background-noise compensation in a microfluidic capillary electrophoresis (CE) system. A Teflon AF-coated silica capillary served as both the separation channel and LCW for light transfer, and blue and red LEDs were used as excitation and reference sources, respectively, both radially illuminating the detection point of the separation channel. The two LEDs were synchronously modulated at the same frequency, but with 180°-phase shift, alternatingly driven by a same constant current source. The LCW transferred the fluorescence emission, as well as the excitation and reference lights that strayed through the optical system to a photomultiplier tube; a lock-in amplifier demodulated the combined signal, significantly reducing its noise level. To test the system, fluorescein isothiocyanate (FITC)-labeled amino acids were separated by capillary electrophoresis and detected by SDWM and single wavelength modulation, respectively. Five-fold improvement in S/N ratio was achieved by dual wavelength modulation, compared with single wavelength modulation; and over 100-fold improvement in S/N ratio was achieved compared with a similar LCW-CE system reported previously using non-modulated LED excitation. A detection limit (S/N = 3) of 10 nM FITC-labeled arginine was obtained in this work. The effects of modulation frequency on S/N level and on the rejection of noise caused by LED-driver current and detector were also studied.     

Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds

We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-mercaptoethanol (OPA/beta-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 microM. Detection limits for OPA/beta-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/beta-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively.     

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

A continuous-wave 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection of proteins in CE. The operating current and temperature of the LED were optimized in order to achieve high luminescence power. It was found that a forward current of 30 mA and a temperature of approximately 5 degrees C gave the best S/N. By using a set of two ball lenses to focus light from the LED, we achieved a spot of approximately 200 mum with a power of 0.1-0.2 mW on the detection window. Fluorescence was collected with a ball lens at 90 degrees angle through a bandpass filter onto a photomultiplier tube. In CZE an LOD of 20 nM for conalbumin was reached. In capillary gel electrophoresis all eight proteins from a commercial standard kit were detected with high S/N. For a 10 microg/mL total protein mixture, S/N was better than 3 for all proteins in solution. Further improvement in LOD should be possible on utilization of an LED with higher luminescence power.     

Indirect photometric detection of anions in nonaqueous capillary electrophoresis employing Orange G as probe and a light-emitting diode-based detector

A method based on indirect photometric detection (IPD) in CE employing a blue LED (473 nm) as a light source and the highly absorbing (478 nm) anionic dye, Orange G, as the probe ion was developed for the sensitive analysis of inorganic and organic anions. The use of nonaqueous solvents was examined as a simple way to reduce the adsorption of the dye onto the capillary wall and to thereby improve the baseline stability. The benefits of this approach were confirmed by experiments using BGEs in methanol (MeOH) and DMSO in which superior baselines were obtained relative to those achieved using aqueous electrolyte systems. A range of commercial LEDs was tested to improve the detection performance, with a difference of 25% in sensitivity being observed between the best and worst performing LED. The final system (4 mM Orange G, 0.05% w/v hydroxypropylcellulose (HPC), 20 mM triethanolamine (TEA) in pure MeOH) exhibited stable baselines and very low LODs (0.10-0.18 microM) for a test mixture comprising nine inorganic and organic anions. These values represent a two- to six-fold improvement over previous studies and the proposed method provides the most sensitive IPD method for the determination of anions using CE published to date. RSDs for ten replicates were in the ranges of 0.42-0.62% for migration time, 1.41-3.46% for peak area and 3.20-5.78% for peak height.     

Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal‐to‐noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE‐Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO‐α and EPO‐β) based on the recorded glycoform pattern. The developed CE‐Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.     

Separation of tyrosine enantiomer derivatives by capillary electrophoresis with light-emitting diode-induced fluorescence detection

Capillary electrophoresis (CE) coupled with fiber-optic light-emitting diode-induced fluorescence detection has been developed for the separation of tyrosine (Tyr) enantiomers. R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole was used as a chiral fluorescence tagged reagent for derivatization of Tyr. The effect of pH, running buffer concentration and applied voltage on enantioselectivity has been investigated. The optimum CE conditions are 15 mmol/L borate running buffer (pH 10.5) and 14-kV applied voltage. Good reproducibility was obtained with coefficient of variation (n = 7) of migration time and peak area less than 0.2 and 2.0%, respectively. The limits of detection of d- and l-Tyr derivatives were 2.9 and 2.2 μmol/L (S/N = 3), respectively. The proposed method has been successfully applied to the determination of Tyr in a commercial amino acid oral solution.     

Automated analysis of individual particles using a commercial capillary electrophoresis system

Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 +/- 6 particles, each followed by an electrophoretic separation at -300 V cm(-1) with data acquired at 100 Hz, showed 28 +/- 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 +/- 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.     

Sensitive determination of carbohydrates labelled with p-nitroaniline by capillary electrophoresis with photometric detection using a 406 nm light-emitting diode

p-Nitroaniline was explored as a derivatising reagent for UV absorbance detection of carbohydrates after separation by CE. This derivatising agent has three advantages: first, it has excellent water solubility; second, it has high molar absorptivity; and third, it is possible to obtain sensitive detection using a UV or blue light-emitting diode (LED) as the light source. The labelling reaction took less than 30 min to complete with high reaction yield. The separation process was modelled and optimised using an artificial neural network. Nine carbohydrates were separated by a CE system within 16 min using a 0.17 M boric acid buffer at pH 9.7. On-column LED detection at 406 nm allowed the detection of carbohydrates with good detection limits (<1.1 microM or 8.8 fmol) and reproducible quantification in the concentration range of 2.6-200 microM. This method was applied successfully to the determination of component carbohydrates in some food samples.     

发光二极管诱导荧光检测器的研制

探讨了以亮度发光二极管为诱导荧光检测激发光源的可行性，考察了直流驱动和脉冲驱动发光二极管(LED)对输出光强的影响以及LED塑料保护层厚度对输出光强的影响。发现脉冲驱动比直流驱动能提高光强3倍，无塑料保护层相对有保护层可提高光强2．5倍。采用毛细管电泳柱上检测方式对检测系统进行了评价，最小检出浓度为0．18μmol／L。结果表明该装置可以满足普通分析需求。     

Determination of quinine in beverages by online coupling capillary isotachophoresis to capillary zone electrophoresis with UV spectrophotometric detection

The present study illustrates the possibilities of capillary isotachophoresis (CITP) online coupled with capillary zone electrophoresis (CZE) and hyphenated with fiber-based spectrophotometric diode array detection (DAD) for the direct, highly reliable, and ultrasensitive determination of quinine (QUI) in real multicomponent ionic matrices (beverages). Here, the CITP provided an effective online sample pretreatment (preseparation and preconcentration) prior to the CZE separation. Due to the CITP sample preconcentration, a simple UV-visible absorbance spectrophotometric detection was sufficient for obtaining very low concentration limits of detection (~2.3 ng/mL). Enhanced separation selectivity due to the combination of different separation mechanisms (CITP vs. CZE) enabled to obtain a pure analyte zone, suitable for its detection and quantitation in the directly injected real samples. The spectrophotometric DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analyte zone and preliminary data indicate structurally related compounds via characteristic spectra recorded in the interval of 200-600 nm. The proposed CITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, and selectivity) and successfully applied to the control of QUI and potential QUI impurities in commercial beverages. This method is proposed as a routine automatized method for the highly reliable quality food control.     

Determination of D,L‐serine in midbrain of Parkinson's disease mouse by capillary electrophoresis with in‐column light‐emitting diode induced fluorescence detection

A capillary electrophoresis method with in‐column light‐emitting diode induced fluorescence detection is described for simultaneous determination of D ,L ‐serine in the midbrain of a Parkinson's disease mouse. D ,L ‐Serine was derivatized with fluorescein isothiocyanate, and chiral separation and determination of D ,L ‐serine derivatives were performed on a laboratory‐built capillary electrophoresis system with in‐column light‐emitting diode induced fluorescence detector using γ‐cyclodextrin as chiral selector. Using this method, the levels of D ‐ and L ‐serine in the midbrains of Parkinson's disease mice were determined. When compared to controls, the levels of D ‐ and L ‐serine showed significant differences. The result suggested that the biosynthesis and the transportation of endogenous D ,L ‐serine may participate in Parkinson's disease pathogenesis.     

Azithromycin as a new chiral selector in capillary electrophoresis

In capillary electrophoresis (CE), separation of enantiomers of a chiral compound can be achieved through the chiral interactions and/or complex formation between the chiral selector and the enantiomeric analytes on leaving their diastereomeric forms with different stability constants and hence different mobilities. A great number of chiral selectors have been employed in CE and among them macrocyclic antibiotics exhibited excellent enantioselective properties towards a wide number of racemic compounds. The use of azithromycin (AZM) as a chiral selector has not been reported previously. This work reports the use of AZM as a chiral selector for the enantiomeric separations of five chiral drugs and one amino acid (tryptophan) in CE. The enantioseparation is carried out using polar organic mixtures of acetonitrile (ACN), methanol (MeOH), acetic acid and triethylamine as run buffer. The influences of the chiral selector concentration, ACN/MeOH ratio, applied voltage and capillary temperature on enantioseparation are investigated. The results show that AZM is a viable chiral selector in CE for the enantioseparation of the type of chiral drugs investigated.     

毛细管区带电泳法测定消毒剂中三氯新

建立了消毒剂中三氯新的毛细管电泳分析方法。探讨了缓冲介质和电泳参数对三氯新测定的影响。以15mmol/LNa2HPO4(pH6.0)-乙腈(V(Na2HPO4)∶V(乙腈)=50∶50)为电泳缓冲液,三氯新在12kV电压下电泳,于254nm检测波长处测定,6min可以完成分析。本方法的检出限为0.04mg/L,线性范围0.04～2.00mg/mL(r=0.997),加标回收率在90.9%～108.2%范围内,测定值的相对标准偏差分别为峰高7.7%,迁移时间5.5%。将本法与高效液相色谱法进行比较,样品测定结果的相对误差小于10%。将所建立的方法已用于消毒剂样品中三氯新的测定。     

Online photolytic optical gating of caged fluorophores in capillary zone electrophoresis utilizing an ultraviolet light‐emitting diode

Photolytic optical gating (POG) facilitates rapid, on‐line and highly sensitive analyses, though POG utilizes UV lasers for sample injection. We present a low‐cost, more portable alternative, employing an ultraviolet light‐emitting diode (UV‐LED) array to inject caged fluorescent dyes via photolysis. Utilizing the UV‐LED array, labeled amino acids were injected with nanomolar limits of detection (270 ± 30 nM and 250 ± 30 nM for arginine and citrulline, respectively). When normalized for the difference in light intensity, the UV‐LED array provides comparable sensitivity to POG utilizing UV lasers. Additionally, the UV‐LED array yielded sufficient beam quality and stability to facilitate coupling with a Hadamard transform, resulting in increased sensitivity. This work shows, for the first time, the use of an UV‐LED for online POG with comparable sensitivity to conventional laser sources but at a lower cost.     

Ionic liquids used in and analyzed by capillary and microchip electrophoresis

This review, covering reports published from 2001 to December 2008, shows how ionic liquids (ILs) have made significant contributions in the improvement of capillary and microchip electrophoresis (CE and μCE) for the separation and detection of analytes such as phenols and aromatic acids, metal ions, medicines, enantiomers, biological materials, etc. Furthermore, CE methods applied in the sensitive and accurate determination of physico-chemical properties of ILs have been summarized. Accordingly, research vacancies and future development trends in these areas are discussed.     

Fibre coupled micro-light emitting diode array light source with integrated band-pass filter for fluorescence detection in miniaturised analytical systems

In this work, a new type of miniaturized fibre-coupled solid-state light source is demonstrated as an excitation source for fluorescence detection in capillary electrophoresis. It is based on a parabolically shaped micro-light emitting diode (μ-LED) array with a custom band-pass optical interference filter (IF) deposited at the back of the LED substrate. The GaN μ-LED array consisted of 270 individual μ-LED elements with a peak emission at 470 nm, each about 14 μm in diameter and operated as a single unit. Light was extracted through the transparent substrate material, and coupled to an optical fibre (OF, 400 μm in diameter, numerical aperture NA = 0.37), to form an integrated μ-LED-IF-OF light source component. This packaged μ-LED-IF-OF light source emitted approximately 225 μW of optical power at a bias current of 20 mA. The bandpass IF filter was designed to reduce undesirable LED light emissions in the wavelength range above 490 nm. Devices with and without IF were compared in terms of the optical power output, spectral characteristics as well as LOD values. While the IF consisted of only 7.5 pairs (15 layers) of SiO₂/HfO₂ layers, it resulted in an improvement of the baseline noise as well as the detection limit measured using fluorescein as test analyte, both by approximately one order of magnitude, with a LOD of 1 × 10⁻⁸ mol L⁻¹ obtained under optimised conditions. The μ-LED-IF-OF light source was then demonstrated for use in capillary electrophoresis with fluorimetric detection. The limits of detection obtained by this device were compared to those obtained with a commercial fibre coupled LED device.     

N-methylformamide as a separation medium in capillary electrophoresis

Summary The organic solvent N-methylformamide (NMF) has been used as a separation medium in capillary electrophoresis. The advantageous properties of this compound are its high dielectric constant, high solubilizing power and low conductivity, as well as its amphiprotic character. It was shown that, unlike for most organic solvents, the electroosmotic flow is substantial. It was found to be possible to utilize NMF without added electrolyte. Field strengths exceeding 1000 volts/cm could be employed, while a low current was maintained and it was thus possible to obtain rapid analyses. Also, the properties of NMF allowed the analysis of substances with a low solubility in aqueous media. These features are exemplified by separation of carboxylic acids and pharmaceuticals. Excellent reproducibility of migration and no sign of electrical breakdown were observed, even under high field strength conditions.Presented at the Sixth International Symposium on High Performance Capillary Electrophoresis, Jan. 31–Feb. 9, 1994, San Diego, USA, Poster #P-113.