Quantitative and qualitative precision improvements by effective mobility-scale data transformation in capillary electrophoresis analysis

By transforming the time-based x-axis of electropherograms in capillary zone electrophoresis (CZE) into the corresponding effective mobility-scale, we propose a simple and robust data representation for a better qualitative and quantitative capillary electrophoresis (CE) analysis. The time scale of the raw electrophoretic data (detection signal versus time) is transformed into an effective electrophoretic mobility scale (mu eff-scale) with account of the electroosmotic flow (EOF) peak or of an internal standard of known effective mobility. With the new scaling (detection signals versus effective mobility), the obtained electropherograms are more representative of the velocity-based electrophoretic separation and the comparison of complete electropherograms is directly possible. This is of importance when tracking peaks in real samples where alteration in EOF stability can occur or when comparing electrophoretic runs from different experimental setups (independence in column length and voltage). Beside the qualitative possibilities, a quantitative improvement is achieved in the mu eff-scale with significant better peak area reproducibility and equal to more precision in quantitative analysis than with the primary time-scale integration.     

Separation and determination of active components in Schisandra chinensis Baill. and its medicinal preparations by non-aqueous capillary electrophoresis

A simple, economical and effective non-aqueous capillary electrophoresis separation and detection method was developed for the quantification of deoxyschizandrin and gamma-schizandrin in Schisandra chinensis Baill. and its medicinal preparations for the first time. After optimization of separation conditions, a buffer of 140 mmol/L sodium cholate in methanol was selected for separating the two analytes, but baseline separation of SA and SB in real samples was not obtained. Therefore second-order derivative electropherograms were applied for resolving overlapping peaks. Regression equations revealed good linear relationships (correlation coefficients 0.9975--0.9988) between peak heights in second-order derivative electropherograms and concentrations of the two analytes. The relative standard deviations (RSD) of the migration times and the peak height of the two constituents were in the ranges 0.62--0.79% and 0.25--2.17% (intra-day) and 1.43--2.06 and 4.08--5.72% (inter-day), respectively. The recoveries of the two constituents ranged from 93.2 to 103.0%. The results indicated that baseline separation of the analytes was sometimes hard to obtain in real samples and second-order derivative electropherograms were applicable for the resolution and analysis of overlapping peaks.     

Separation and positive identification of compounds in complex sample mixtures using on-line,LC-UV/VIS and LC-MS,direct coupling techniques

Summary In many analytical separation problems the retention time is not sufficient to identify the compounds of interest. This is especially true with complex mixtures where additional analytical data are necessary to confirm positively the identity of the separated components. An analytical high-pressure liquid chromatograph has been coupled to a UV/Visible spectrophotometer and a mass spectrometer and used for the separation and subsequent identification of compounds in different plant extracts. The results show that the direct combination of HPLC with on-line detection techniques can be successfully used in the analysis of complex mixtures giving positive identification of components and an overall reduction in analysis time.     

CdS纳米粒子与半胱氨酸相互作用的研究

合成了粒径均匀和分散性好的CdS纳米粒子.通过改变CdS纳米粒子及半胱氨酸的浓度、体系的pH值及CdCl2和CH3CSNH2摩尔比等实验条件跟踪监测了CdS纳米粒子光谱性质的变化,探讨了CdS纳米粒子与半胱氨酸之间的相互作用及化学反应机理.     

Capillary electrophoretic determination of the protease Savinase in cultivation broth.

The highly basic washing enzyme Savinase and various analogues were analysed by micellar electrokinetic chromatography (MEKC) and electrophoresis. Broth samples were withdrawn during the cultivation of Savinase by recombinant microorganisms. Savinase peak areas obtained by MEKC-electrophoretic analysis were normalized with respect to migration time and compared with traditional enzyme activity measurements. The electropherograms indicated thermal degradation of the Savinase molecule at high field strengths. Baseline separation of Savinase and two analogues was achieved.     

单细胞中D-天冬氨酸和D-谷氨酸的毛细管电泳分析

建立了一种毛细管电泳-激光诱导荧光检测单个神经细胞中D-天冬氨酸和D-谷氨酸的方法.以萘二甲醛(NDA)为衍生试剂,β-环糊精(β-CD)和脱氧胆酸钠(SDC)为拆分体系,对16对氨基酸对映体的NDA衍生物进行了手性拆分研究.考察了蛋白氨基酸和一些胺对检测D-天冬氨酸和D-谷氨酸的影响,并确立了最佳分离条件,从而建立了单细胞中D-天冬氨酸和D-谷氨酸的检测方法,并对海兔的单个神经细胞中D-天冬氨酸和D-谷氨酸进行了检测.     

A general approach to the analysis of errors and failure modes in the base-calling function in automated fluorescent DNA sequencing

We describe the analysis of errors and failure modes in the base-calling function in automated DNA sequencing, on instruments in which fluorescently-labeled Sanger dideoxy-sequencing ladders are detected via their times of migration past a fixed detector. A general approach entails the joint use of: (i) well-defined control samples such as M13mp18, and (ii) mathematical simulation of sequencing electropherograms, with the deliberate introduction of different types of distortion and noise. An algorithm, the electrophoretic trace simulator (ETS), is used to calculate electrophoresis traces corresponding to the output data stream of an automated fluorescent DNA sequencer. The ETS accepts a user-defined sequence of nucleotide bases (A, C, G, T) as input, and employs user-adjustable functions to compute the following critical parameters of an electropherogram: peak intensity, peak spacing, peak shape as a function of base number; background, noise, and spectral cross-talk correction (for a sequencer using multiple dyes). We use a combination of M13mp18 controls and simulated electropherograms to analyze two problems of considerable practical importance: (i) variation in electrophoretic migration rates between different lanes of a gel, and (ii) variation in signal intensity due to user-dependent loading artifacts. The issue of base-calling errors and failure modes, for electropherograms that contain noise and distortion, is addressed.     

Exploring STR signal in the single‐ and multicopy number regimes: Deductions from an in silico model of the entire DNA laboratory process

Short tandem repeat (STR) profiling from DNA samples has long been the bedrock of human identification. The laboratory process is composed of multiple procedures that include quantification, sample dilution, PCR, electrophoresis, and fragment analysis. The end product is a short tandem repeat electropherogram comprised of signal from allele, artifacts, and instrument noise. In order to optimize or alter laboratory protocols, a large number of validation samples must be created at significant expense. As a tool to support that process and to enable the exploration of complex scenarios without costly sample creation, a mechanistic stochastic model that incorporates each of the aforementioned processing features is described herein. The model allows rapid in silico simulation of electropherograms from multicontributor samples and enables detailed investigations of involved scenarios. An implementation of the model that is parameterized by extensive laboratory data is publically available. To illustrate its utility, the model was employed in order to evaluate the effects of sample dilutions, injection time, and cycle number on peak height, and the nature of stutter ratios at low template. We verify the model's findings by comparison with experimentally generated data.     

Pattern recognition in capillary electrophoresis data using dynamic programming in the wavelet domain

A novel approach for CE data analysis based on pattern recognition techniques in the wavelet domain is presented. Low-resolution, denoised electropherograms are obtained by applying several preprocessing algorithms including denoising, baseline correction, and detection of the region of interest in the wavelet domain. The resultant signals are mapped into character sequences using first derivative information and multilevel peak height quantization. Next, a local alignment algorithm is applied on the coded sequences for peak pattern recognition. We also propose 2-D and 3-D representations of the found patterns for fast visual evaluation of the variability of chemical substances concentration in the analyzed samples. The proposed approach is tested on the analysis of intracerebral microdialysate data obtained by CE and LIF detection, achieving a correct detection rate of about 85% with a processing time of less than 0.3 s per 25,000-point electropherogram. Using a local alignment algorithm on low-resolution denoised electropherograms might have a great impact on high-throughput CE since the proposed methodology will substitute automatic fast pattern recognition analysis for slow, human based time-consuming visual pattern recognition methods.     

Speciation of dimethylarsinyl-riboside derivatives (arsenosugars) in marine reference materials by HPLC-ICP-MS

Anion and cation exchange HPLC-ICP-MS was used to separate and detect mixtures of four dimethylarsinyl-riboside derivatives (arsenosugars), in the presence of eight other arsenic species naturally occurring in the marine environment. The separations achieved showed that two arsenosugars 11 and 13 (cf. Table 2) were present in shellfish certified reference materials (CRMs) and in a lobster hepatopancreas CRM. The concentration of the two arsenosugars in the shellfish samples amounts to 18% of the total arsenic as compared to arsenobetaine at 9–13% and dimethylarsinate at 4–9% of the total arsenic. Additionally, a chromatographic peak with the same retention time as that of 2-dimethylarsinylacetic acid was detected in the shellfish samples. Further support of the identity of this peak was gained after spiking the sample extracts with the standard substance which resulted in a single, but larger peak. The indication that this novel arsenical is present in shellfish, and the recently reported finding of arsenocholine in seafood supports a proposed marine biosynthetic pathway of arsenic that includes both of these compounds as the immediate precursors of arsenobetaine, the end-product of the marine arsenic metabolism.     

High‐throughput microfluidic device for single cell analysis using multiple integrated soft lithographic pumps

The ability to accurately control fluid transport in microfluidic devices is key for developing high‐throughput methods for single cell analysis. Making small, reproducible changes to flow rates, however, to optimize lysis and injection using pumps external to the microfluidic device are challenging and time‐consuming. To improve the throughput and increase the number of cells analyzed, we have integrated previously reported micropumps into a microfluidic device that can increase the cell analysis rate to ∼1000 cells/h and operate for over an hour continuously. In order to increase the flow rates sufficiently to handle cells at a higher throughput, three sets of pumps were multiplexed. These pumps are simple, low‐cost, durable, easy to fabricate, and biocompatible. They provide precise control of the flow rate up to 9.2 nL/s. These devices were used to automatically transport, lyse, and electrophoretically separate T‐Lymphocyte cells loaded with Oregon green and 6‐carboxyfluorescein. Peak overlap statistics predicted the number of fully resolved single‐cell electropherograms seen. In addition, there was no change in the average fluorescent dye peak areas indicating that the cells remained intact and the dyes did not leak out of the cells over the 1 h analysis time. The cell lysate peak area distribution followed that expected of an asynchronous steady‐state population of immortalized cells.     

Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.     

A simple and rapid electrophoretic method to characterize simple phenols, lignans, complex phenols, phenolic acids, and flavonoids in extra-virgin olive oil

We have devised a simple and rapid capillary electrophoretic method which provides the analyst with a useful tool for the characterization of the polyphenolic fraction of extra-virgin olive oil. This method that uses a capillary with 50 microm id and a total length of 47 cm (40 cm to the detector) with a detection window of 100 x 200 microm, and a buffer solution containing 45 mM of sodium tetraborate pH 9.3 offers valuable information about all the families of compounds present in the polar fraction of the olive oil. The detection was carried out by UV absorption at 200, 240, 280, and 330 nm in order to facilitate the identification of the compounds. Concretely, the method permits the identification of simple phenols, lignans, complex phenols (isomeric forms of secoiridoids), phenolic acids, and flavonoids in the SPE-Diol extracts from extra-virgin olive oil in a short time (less than 10 min) and provides a satisfactory resolution. Peak identification was done by comparing both migration time and spectral data obtained from olive oil samples and standards (commercial or isolated (by HPLC-MS) standards), with spiked methanol-water extracts of olive oil with HPLC-collected compounds and commercially available standards at several concentration levels, studying the information of the electropherograms obtained at several wavelengths and also using the information previously reported.     

Strategies for the determination of cefazolin in plasma and microdialysis samples by short-end capillary zone electrophoresis.

Capillary zone electrophoresis was employed to determine cefazolin, a first-generation cephalosporin antibiotic, in plasma and microdialysis samples from patients. To shorten the analysis, the samples were injected from the short end of the capillary, resulting in a separation time of < 3 min. Due to a high ionic strength of the biological matrices it was necessary to optimize the stacking conditions. For microdialysis samples a 1:10 dilution with water before injection was sufficient to obtain good peak shape. For plasma samples a protein removal step was required to obtain clean electropherograms and a good peak shape. Acetonitrile was used as precipitant resulting in an enhanced sample stacking in comparison to water dilution. The disadvantage of using acetonitrile was severe evaporation loss making quantitation impossible. A self-sealing film was used to seal each individual sample vial to suppress evaporation during long-term sequences. The calibration curves for spiked plasma and cefazolin in Ringer's solutions were linear in the range from 2-500 and 2.5-100 microg/mL, respectively. Limits of detection were 1.0 and 2.0 microg/mL in plasma and microdialysis samples, respectively. The assay was successfully applied to plasma and microdialysis samples obtained in vivo from the interstial space fluid of subcutaneous adipose and muscle tissue of patients undergoing cardiac surgery.     

Laser-induced dispersed fluorescence detection of polycyclic aromatic compounds in soil extracts separated by capillary electrochromatography

Polycyclic aromatic hydrocarbons (PAHs) and nitrogen containing aromatic compounds (NCACs) are characterized in soil extracts and laboratory standards by capillary electrochromatography (CEC) with laser-induced dispersed fluorescence (LIDF) detection using a liquid-nitrogen cooled charge-coupled device detector. The LIDF detection technique provides information on compound identity and, when coupled with the high separation efficiencies of the CEC technique, proves useful in the analysis of complex mixtures. Differences in fluorescence spectra also provide a means of identifying co-eluting compounds by using deconvolution algorithms. Detection limits range from 0.5 to 96x10(-10) M for selected PAHs and 0.9-3.7x10(-10) M for selected NCACs. Soil extracts are also injected onto the CEC column to evaluate chromatographic method performance with respect to complex samples and the ability to withstand exposure to environmental samples.     

Unraveling virus identity by detection of depleted probes with capillary electrophoresis

With the emergence of new viral infections and pandemics, there is a need to develop faster methods to unravel the virus identities in a large number of clinical samples. This report describes a virus identification method featuring high throughput, high resolution, and high sensitivity detection of viruses. Identification of virus is based on liquid hybridization of different lengths of virus-specific probes to their corresponding viruses. The probes bound to target sequences are removed by a biotin–streptavidin pull-down mechanism and the supernatant is analyzed by capillary electrophoresis. The probes depleted from the sample appear as diminished peaks in the electropherograms and the remaining probes serve as calibrators to align peaks in different capillaries. The virus identities are unraveled by a signal processing and peak detection algorithm developed in-house. Nine viruses were used in the study to demonstrate how the system works to unravel the virus identity in single and double virus infections. With properly designed probes, the system is able to distinguish closely related viruses. The system takes advantage of the high resolution feature of capillary electrophoresis to resolve probes that differ by length. The method may facilitate virus identity screen from more candidate viruses with an automated 4-color DNA sequencer.     

Separation and determination of active components in Radix Salviae miltiorrhizae and its medicinal preparations by nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoresis (NACE) method was developed for simultaneous assay of three bioactive components (1: cryptotanshinone; 2: tanshinone IIA, and 3: tanshinone I) in Radix Salviae miltiorrhizae and in its herbal preparations for the first time. After optimization of separation conditions, a buffer of 250 mmol L(-1) ammonium acetate containing 30% acetonitrile and 1.0% acetic acid (V:V) in methanol was selected for separating the three analytes, but baseline separation of tanshinon I and tanshinone IIA was not obtained. Therefore second-order derivative electropherograms were applied for resolving overlapping peaks. Regression equations revealed good linear relationships (correlation coefficients 0.9943-0.9991) between peak heights in second-order derivative electropherograms and concentrations of the three analytes. The relative standard deviations (RSD) of the migration times and the peak height of the three constituents were in the range of 0.81 -0.88% and 0.34-1.13% (intra-day), 1.57-1.86% and 3.05-5.52% (inter-day), respectively. The recoveries of three constituents ranged from 90.2 to 108.5%. The results indicated that baseline separation of the analytes was sometimes hard to obtain and second-order derivative electropherograms were applicable for the resolving and analysis of overlapping peaks.     

Metabolic fingerprinting of Schistosoma mansoni infection in mice urine with capillary electrophoresis

Schistosoma mansoni infection in mice has been fingerprinted using CE to study the capabilities of this technique as a diagnostic tool for this parasitic disease. Two modes of separation were used in generating the electrophoretic data, with each untreated urine sample the following methods were applied: (i) a fused-silica capillary, operating with an applied potential of 18 kV, in micellar EKC (MEKC) and (ii) a polyacrylamide-coated capillary, operating with an applied potential of -20 kV under zonal CZE conditions. By combining normal and reverse polarities in the data treatment we have extracted more information from the samples, which is a better approach for CE metabolomics. The traditional problems associated with variability in electrophoretic peak migration times for analytes were countered by using a dynamic programming algorithm for the electropherograms alignment. Principal component analyses of these aligned electropherograms and partial least square discriminant analysis (PLS-DA) data are shown to provide a valuable means of rapid and sample classification. This approach may become an important tool for the identification of biomarkers, diagnosis and disease surveillance.     

Distinguishing malignant from normal stomach tissues and its in vivo, in situ measurement in operating process using FTIR Fiber-Optic techniques

The application of molecular spectroscopy inthe biological fields is more and more extensive.Recently vibrational spectroscopy, including FTIRATR Optic Fibers[1—3], FT-IR microspectroscopy[4—6],FT-Raman[7,8] and near IR spectroscopic[9] methods,was used…     

High-throughput automated post-processing of separation data

The development of an efficient method for high-throughput analysis of multiple electropherograms or chromatograms collected in series is presented. The method, encoded in a computer program designated "Cutter", utilizes batch processing for determining chromatographic figures of merit (CFOM) including peak centroid times, heights, areas, signal-to-noise ratios (S/N), variance (sigma2), skew, excess, and plate number (N) across a set of separations collected serially. The software was validated using simulated data with varying S/N, skew, and excess. The accuracy of the analysis was comparable to or improved over commercial software with area calculation relative errors (RE) below 5% for simulated data with S/N = 5. File sets containing 1300 electropherograms were analyzed in 5 min, representing a nearly 200-fold reduction in analysis time from other methods. Incorporated within the program is a novel method for automated peak deconvolution using an Empirically Transformed Gaussian function. Area measurements of deconvoluted peaks were within 3% of the true value of a simulated data set with S/N = 5 and resolution (R(S)) = 1 for equivalent peaks, and within 10% when the ratio of the overlapped peak heights was 10:1.