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CE–MS is increasingly gaining momentum as an analytical tool in metabolomics, due to its ability to obtain information about the most polar elements in biological samples. This has been helped by improvements of robustness in peak identification by means of mobility‐scale representations of the electropherograms (mobilograms). As a necessary step toward facilitating the use of CE–MS for untargeted metabolomics data, the authors previously developed and introduced ROMANCE, a software automating mobilogram generation for large untargeted datasets through a simple and self‐contained user interface. Herein, we introduce a new version of ROMANCE including new features such as compatibility with other types of data (targeted MS data and 2D UV‐Vis absorption‐like electropherograms), and the much needed additional flexibility in the transformation parameters (including field ramping and the use of secondary markers), more measurement conditions (depending on detection and integration modes), and most importantly tackling the issue of quantitative peak conversion. First, we present a review of the current theoretical framework with regard to peak characterization, and we develop new formulas for multiple marker peak area corrections, for anticipating peak position precision, and for assessing peak shape distortion. Then, the new version of the software is presented and validated experimentally. We contrast the multiple marker mobility transformations with previous results, finding increased peak position precision, and finally we showcase an application to actual untargeted metabolomics data.

One of the main problems of the remote complex sample analysis instrumentation is that such systems are susceptible to temperature fluctuations. Temperature regulation is energetically ineffective, and it is not used in most of the field portable analytical systems. Separations performed in a changing temperature environment provide electropherograms with considerable baseline fluctuations, resulting in significant errors in detection and integration of the peaks. This paper describes electropherogram baseline compensation that is suitable for the capillary electrophoresis-contactless conductivity detection analytical method. The baseline compensation utilizes linear or polynomial data processing methods, and can be programmed in-line using simple microcontroller, or on-line and off-line in data acquisition software. This method is targeted for field portable and autonomous analytical systems that are utilized in a fluctuating environment.

In this work, we present a novel probabilistic peak detection algorithm based on a Bayesian framework for forensic DNA analysis. The proposed method aims at an exhaustive use of raw electropherogram data from a laser-induced fluorescence multi-CE system. As the raw data are informative up to a single data point, the conventional threshold-based approaches discard relevant forensic information early in the data analysis pipeline. Our proposed method assigns a posterior probability reflecting the data point's relevance with respect to peak detection criteria. Peaks of low intensity generated from a truly existing allele can thus constitute evidential value instead of fully discarding them and contemplating a potential allele drop-out. This way of working utilizes the information available within each individual data point and thus avoids making early (binary) decisions on the data analysis that can lead to error propagation. The proposed method was tested and compared to the application of a set threshold as is current practice in forensic STR DNA profiling. The new method was found to yield a significant improvement in the number of alleles identified, regardless of the peak heights and deviation from Gaussian shape.

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.

Acupuncture sample injection is a simple method to deliver well-defined nanoliter-scale sample plugs in PDMS microfluidic channels. This acupuncture injection method in microchip CE has several advantages, including minimization of sample consumption, the capability of serial injections of different sample solutions into the same microchannel, and the capability of injecting sample plugs into any desired position of a microchannel. Herein, we demonstrate that the simple and cost-effective acupuncture sample injection method can be used for PDMS microchip-based field amplified sample stacking in the most simplified straight channel by applying a single potential. We achieved the increase in electropherogram signals for the case of sample stacking. Furthermore, we present that microchip CGE of ΦX174 DNA-HaeⅢ digest can be performed with the acupuncture injection method on a glass microchip while minimizing sample loss and voltage control hardware.

Point-of-care systems based on microchip capillary electrophoresis require single-use, disposable microchips prefilled with all necessary solutions so an untrained operator only needs to apply the sample and perform the analysis. While microchip fabrication can be (and has been) standardized, some manufacturing differences between microchips are unavoidable. To improve analyte precision without increasing device costs or introducing additional error sources, we recently proposed the use of integrated internal standards (ISTDs): ions added to the BGE in small concentrations which form system peaks in the electropherogram that can be used as a measurement reference. Here, we further expand this initial proof-of-principle test to study a clinically-relevant application of K ion concentrations in human blood; however, using a mock blood solution instead of real samples to avoid interference from other obstacles (e.g. cell lysis). Cs as an integrated ISTD improves repeatability of K ion migration times from 6.97% to 0.89% and the linear calibration correlation coefficient (R 2 ) for K quantification from 0.851 to 0.967. Peak area repeatability improves from 11.6–13.3% to 4.75–5.04% at each K concentration above the LOQ. These results further validate the feasibility of using integrated ISTDs to improve imprecision in disposable microchip CE devices by demonstrating their application for physiological samples.

Loop mediated isothermal amplification (LAMP) is a nucleic acid amplification technique performed under isothermal conditions. The output of this amplification technique includes multiple different sizes of deoxyribonucleic acid (DNA) structures which are identified by a banding pattern on gel electrophoresis plots. Although this is a specific amplification technique, the complexity of the primer design and amplification still lead to the issue of obtaining false‐positive results, especially when a positive reading is determined solely by whether there is any banding pattern in the gel electrophoresis plot. Here, we first performed extensive LAMP experiments and evaluated the DNA structures using microchip electrophoresis. We then developed a mathematical model derived from the various components that make up an entire LAMP structure to predict the full LAMP structure size in base pairs. This model can be implemented by users to make predictions for specific, DNA size dependent, banding patterns on their gel electrophoresis plots. Each prediction is specific to the target sequence and primers used and therefore reduces incorrect diagnosis errors through identifying true‐positive and false‐positive results. This model was accurately tested with multiple primer sets in house and was also translatable to different DNA and RNA types in previously published literature. The mathematical model can ultimately be used to reduce false‐positive LAMP diagnosis errors for applications ranging from tuberculosis diagnostics to E. coli to numerous other infectious diseases.

To achieve an on-line coupling of the sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) with microchip gel electrophoresis (MCGE), a sample solution, a background solution for LVSEP and a sieving solution for MCGE were loaded in a T-form channel and three reservoirs on PDMS microchips. By utilizing the difference in the flow resistance of the two channels, a low-viscosity sample and a viscous polymer solution were easily introduced into the LVSEP and MCGE channels, respectively. Fluorescence imaging of the sequential LVSEP-MCGE processes clearly demonstrated that a faster stacking of anionic fluorescein and successive introduction into the MCGE channel can be carried out on the T-channel chip. To evaluate the preconcentration performance, a conventional MCZE analysis of fluorescein on the cross-channel chip was compared with LVSEP-MCGE on the short T-channel chip, and as a result that the value of sensitive enhancement factor (SEF) was estimated to be 370. The repeatability of the peak height was good with the RSD value of 3.2%, indicating the robustness of the enrichment performance. In the successive LVSEP-MCGE analysis of φX174/HaeIII digest, the DNA fragments were well enriched to a sharp peak in the LVSEP channel, and they were separated in the MCGE channel, whose electropherogram was well-resembled with that in the conventional MCGE. The values of SEF for the DNA fragments were calculated to be ranging from 74 to 108. Thus, the successive LVSEP-MCGE analysis was effective for both preconcentrating and separating DNA fragments.

A commercial system that is comprised of a CE coupled to an ESI triple quadrupole mass spectrometer was equipped with two capacitively coupled contactless conductivity detectors (C(4) Ds). The first C(4) D was positioned inside the original cartridge, and the second C(4) D was positioned as close as possible to the ESI probe entrance by using a 3D-printed support. The C(4) Ds electropherograms were matched to the ESI-MS electropherogram by correcting their timescales by the factor LT /LD , where LT and LD are the total capillary length and the length until the C(4) D, respectively. A general approach for method development supporting the simultaneous conductivity and MS detection is discussed, while application examples are introduced. These examples include the use of C(4) D as a simple device that dismiss the use of an EOF marker, a low-selectivity detector that continuously provide information about unexpected features of the sample, and even a detector that can be more sensitive than ESI-MS. The C(4) D used in this setup proved to have a smaller contribution to the peak broadening than ESI-MS, which allowed that a C(4) D, positioned at 12 cm from the inlet of an 80-cm-long capillary, could be used to foresee position and shape of the peaks being formed 6.8 times slower at the ESI-MS electropherogram.

A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty-four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single-base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father-child-mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10(-22) and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.

The linear theory of electromigration, including the first-order nonlinear approximation, is generalized to systems with any equilibria fast enough to be considered instantaneous in comparison with the timescale of peak movement. For example, this theory is practically applied in the electrokinetic chromatography (EKC) mode of the CZE. The model enables the calculation of positions and shapes of analyte and system peaks without restricting the number of selectors, the complexation stoichiometry, or simultaneous acid–base equilibria. The latest version of our PeakMaster software, PeakMaster 6—Next Generation, implements the theory in a user-friendly way. It is a free and open-source software that performs all calculations and shows the properties of the background electrolyte and the expected electropherogram within a few seconds. In this paper, we mathematically derive the model, discuss its applicability to EKC systems, and introduce the PeakMaster 6 software. Fil: Malý, Michal. Charles University; República Checa Fil: Dovhunová, Magda. Charles University; República Checa Fil: Dvořák, Martin. Charles University; República Checa Fil: Gerlero, Gabriel Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Hruška, Vlastimil. Agilent Technologies Deutschland GmbH & Co. KG; Alemania Fil: Dubský, Pavel. Charles University; República Checa

MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

The purpose of this study was to develop a comprehensive, rapid and practical capillary electrophoresis (CE) method for quality control (QC) of Guan-Xin-Ning (GXN) injection based on fingerprint analysis and simultaneous separation and determination of seven constituents. In fingerprint analysis, a capillary zone electrophoresis (CZE) method with a running buffer of 30 mM borate solution (pH 9.3) was established. Meanwhile, ten batches of samples were used to establish the fingerprint electropherogram and 34 common peaks were obtained within 20 min. The RSD of relative migration times (RMT) and relative peak areas (RPA) were less than 5%. In order to further evaluate the quality of GXN injection, a micellar electrokinetic chromatography (MEKC) method was developed for simultaneous separation and determination of bioactive constituents. Seven components reached baseline separation with a running buffer containing 35 mM SDS and 45 mM borate solution (pH 9.3). A good linearity was obtained with correlation coefficients from 0.9906 to 0.9997. The LOD and LOQ ranged from 0.12 to 1.50 μg/mL and from 0.40 to 4.90 μg/mL, respectively. The recoveries ranged between 99.0 and 104.4%. Therefore, it was concluded that the proposed method can be used for full-scale quality analysis of GXN injection.

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product- and time-consuming. CE-MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off-line CE-UV/MALDI-MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI-MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI-MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE-UV/MALDI-MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.

This work reports an investigation for the analysis of different paper samples using CE with laser-induced detection. Papers from four different manufactures (white-copy paper) and four different paper sources (white and recycled-copy papers, adhesive yellow paper notes and restaurant serviettes) were pulverized by scratching with a surgical scalpel prior to their derivatization with a fluorescent labeling agent, 8-aminopyrene-1,3,6-trisulfonic acid. Methodological conditions were evaluated, specifically the derivatization conditions with the aim to achieve the best S/N signals and the separation conditions in order to obtain optimum values of sensitivity and reproducibility. The best conditions, in terms of fastest, and easiest sample preparation procedure, minimal sample consumption, as well as the use of the simplest and fastest CE-procedure for obtaining the best analytical parameters, were applied to the analysis of the different paper samples. The registered electropherograms were pretreated (normalized and aligned) and subjected to multivariate analysis (principal component analysis). A successful discrimination among paper samples without entanglements was achieved. To the best of our knowledge, this work presents the first approach to achieve a successful differentiation among visually similar white-copy paper samples produced by different manufactures and paper from different paper sources through their direct analysis by CE-LIF and subsequent comparative study of the complete cellulose electropherogram by chemometric tools.

Over the last few years, there has been an increase in the reports of drug-facilitated crimes. The list of drugs associated with these crimes is extensive and benzodiazepines constitute one of the groups of substances more commonly used. The sedative properties, which characterize benzodiazepines, are enhanced when such drugs are combined with alcohol, being more attractive for committing these types of crimes. In this work, a capillary electrophoresis method was applied to the analysis of 63 different samples of club drinks spiked with benzodiazepine tablets. The resulting electropherograms were processed and analyzed with the chemometric multivariate techniques: principal component analysis (PCA) and soft independent modeling of class analogies (SIMCA) classification. The PCA results allowed a clear differentiation of each drug class in a 3D plot. In addition, the SIMCA classification model (5% significance level) showed that eight out of nine test samples were automatically assigned by software to their proper sample class. The conflicting sample was correctly classified in the Coomans' plot (95% confidence). This novel approach based on the comparison of electrophoretic profiles of spiked drinks by chemometric tools allows determining the benzodiazepine used for drink spiking without the use of drug standards. Moreover, it provides an opportunity for the forensic laboratories to incorporate the identification capability provided by the electrophoretic fingerprinting of benzodiazepine solutions in existing or new databases.

Recent emergence in the use of monoclonal antibody therapeutics and other glycoprotein biopharmaceuticals requires high-throughput, robust, and automated techniques for their glycosylation analysis. Capillary electrophoresis is one of the high-performance methods of choice; however, while the necessary instrumentation is well developed, the related bioinformatics tools are lacked behind. In this paper, we introduce an integrated toolset dubbed as GUcal, to automatically calculate the glucose unit (GU) values for all sample components of interest in an electropherogram with a concomitant database search for structural assignment. The database comprises CE GUs and suggested structures of N-glycans released from human IgG. The app is freely available online (www.lendulet.uni-pannon.hu/gucal) and readily facilitates CE-based glycan analysis.

Here, we report a novel method consisting of capillary electrophoretic separation followed by two-color LIF detection with postcolumn derivatization. The method can be used to discriminate glycoproteins in a protein mixture containing both glycosylated and unglycosylated proteins. The detector permitted simultaneous measurements of two electropherograms obtained by 450 nm (diode laser) and 532 nm (Nd:YAG laser) lasers excited native proteins following postcolumn derivatization with naphthalene-2,3-dicarboxaldehyde and concanavalin A (Con A) labeled with tetramethylrhodamine (rhodamine-labeled Con A), respectively. So, a protein can be assigned as glycosylated if it shows a peak at the same migration time in both electropherograms. According to the proposed principle, in a single run we discriminated a glycosylated protein (thyroglobulin) from an unglycosylated protein (albumin) in the presence of rhodamine-labeled Con A. Because the methodology permits the simultaneous detection of native proteins and their complexes with a fluorescently labeled probe, it should have broad applicability to binding assays.

During the last decade, enormous progress regarding knowledge about composition and properties of human milk (HM) has been made. Besides nutrition, the three macro-nutrients: proteins, lipids, and carbohydrates combine a large variety of properties and functions. Especially, complex oligosaccharides emerge as important dietary factors during early life with multiple functions. The characterization of these HM oligosaccharides (HMOS) within the total carbohydrate fraction is prerequisite to understand the relationship between milk composition and biological effects. Therefore, extended studies of large donor cohorts and thus, new high-throughput glycoanalytical methods are needed. The developed method comprises sample preparation, as well as analysis of HMOS by multiplexed CGE with LIF detection (xCGE-LIF). Via a respective database the generated "fingerprints" (normalized electropherograms) could be used for structural elucidation of HMOS. The method was tested on HM samples from five different donors, partly sampled as a series of lactation time points. HMOS could be easily identified and quantified. Consequently, secretor and Lewis status of the donors could be determined, milk typing could be performed and quantitative changes could be monitored along lactation time course. The developed xCGE-LIF based "real" high-throughput HMOS analysis method enables qualitative and quantitative high-performance profiling of the total carbohydrate fraction composition of large sets of samples.

A simple data reconstruction technique in CE-UV-ESI-MS (where UV stands for ultraviolet) is presented to overcome the drift in mobilities caused by various factors compromising the reproducibility of such data, for example Joule heating effects and the variation in thermostatic control along the capillary, drift in EOF and the suction effect caused by the nebulizing gas in coaxial CE-MS interfaces. We present here a method to transform the traditional time-based electropherogram into the corresponding temperature-correlated mobility scale allowing tracking of analytes independent from capillary dimensions, electric field strengths, temperature control, and distance between the detectors. The main principle of this alignment is based on including the current in the mobility calculations and relating this to the initial electrical resistance of the buffer-filled capillary. The temperature-correlated mobility calculation eliminates the peak shifts due to the viscosity changes, improves the precision of peak identification using the observed temperature-correlated mobilities, and allows a direct comparison of signals from different detection combinations. The method allows peaks from normal CE-UV separations to be correlated with the corresponding peak obtained by MS detection in CE-MS even for differences in capillary dimensions and thermostatic control.

Analytical platforms that characterize charge heterogeneity in therapeutic proteins, such as mAbs, are important tools that can be used to define quality attributes. CZE separates protein moieties close to their native state and is a valuable physicochemical analytical method that can be used in parallel with other orthogonal methods for characterization and comparability. In this study, custom conditions for the analysis of charge heterogeneity of two mAbs were developed with regard to critical parameters in the BGE, running conditions, and sample treatment. The method application was tested for up to four mAbs and one mAb fragment. The electropherograms showed specific profiles and contrasting levels of basic and acidic isoforms with respect to the main isoform. Issues that surround this method, such as peak tailing and capillary lifetime, are summarized. Using this method, the identities of rituximab and trastuzumab were confirmed, based on the correspondence between the biosimilars and reference products, noninterference of the sample matrix, and the ability to separate spiked samples of related mAbs. The RSD of the isoform content and migration time for the method repeatability were less than 2 and 1%, respectively.

Glycosaminoglycan (GAG) analysis represents a challenging frontier despite the advent of many high-resolution technologies because of their unparalleled structural complexity. We previously developed a resolving agent-aided capillary electrophoretic approach for fingerprinting low-molecular-weight heparins (LMWHs) to profile their microscopic differences and assess batch-to-batch variability. In this report, we study the application of this approach for fingerprinting other GAGs and analyze the basis for the fingerprints observed in CE. Although the resolving agents, linear polyalkylamines, could resolve the broad featureless electropherogram of LMWH into a large number of distinct, highly reproducible peaks, longer GAGs such as chondroitin sulfate, dermatan sulfate, and heparin responded in a highly individualistic manner. Full-length heparin interacted with linear polyalkylamines very strongly followed by dermatan sulfate, whereas chondroitin sulfate remained essentially unaffected. Oversulfated chondroitin sulfate could be easily identified from full-length heparin. Scatchard analysis of the binding profile of enoxaparin with three linear polyalkylamines displayed a biphasic binding profile suggesting two distinctly different types of interactions. Some LMWH chains were found to interact with linear polyalkylamines with affinities as high as 10 nM, whereas others displayed nearly 5000-fold weaker affinities. These observations provide fundamental insight into the basis for fingerprinting of LMWHs by linear polyalkylamine-based resolving agents, which could be utilized in the design of advanced resolving agents for compositional profiling, direct sequencing, and chemoinformatics studies.

We demonstrated a proof-of-concept for novel minisatellite tandem repeat typing, called PCR-free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a ligation reaction instead of PCR thermal cycling, magnetic bead-based solid-phase capture for purification, and an elongated sample stacking microcapillary electrophoresis (μCE) for sensitive digital coding of repeat number. We designed a 16-bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin-coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min.

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE-SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)-poly (propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer as a sieving matrix for CE-SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO-PPO-PEO copolymers, 255-bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO-PPO-PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan ™ gel. Due to enhanced dynamic coating and sieving ability, PEO-PPO-PEO copolymer displayed fourfold enhancement of resolving power in the CE-SSCP to separate same-sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high-resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO-PPO-PEO triblock copolymer an excellent matrix in the CE-SSCP analysis on the microdevice.

This paper describes the hyphenation of cIEF and MALDI-TOF MS via a fractionation or spotting device. After focusing in cIEF the compounds are hydrodynamically mobilized and deposited on a MALDI target plate using a sheath liquid interface, which provides the catholyte solution and the electrical ground. From previous experiments, sample conditions that resulted in a high resolution in cIEF and acceptable protein signal intensity in MS were selected [Silvertand et al., Electrophoresis, 2008, 29, 1985-1996]. Besides the mixture of test proteins, the sample solution contains 1% Pharmalyte, 0.3% hydroxyethyl cellulose and 0.1% Tween 20 and is used for both optimization as well as characterization of the cIEF-MALDI-TOF MS system. Hyphenation problems encountered are mainly due to transfer of the liquid from the needle to the MALDI target plate and are solved by choosing the proper sheath catholyte (200 mM NH4OH in 50% methanol with 0.1% Tween20). MS electropherograms were reconstructed by plotting the intensities of the m/z values corresponding to the proteins versus migration time (related to spot number). Reproducibility, peak width and signal intensity for different focusing and spotting (fractionation) times were calculated using these reconstructed MS electropherograms as well as the UV electropherograms. The best results were obtained with focusing time of 75 min (no under- or overfocusing) and a spotting time of 5 s (highest protein signal intensity in MS). The applicability of the system is demonstrated by the analysis of a biopharmaceutical (glucagon) and its deamidation product.

Fingerprinting together with statistical analysis is often employed to compare samples in metabonomic studies of a disease. Correlation algorithms can aid by extracting information based on the variation patterns of key metabolites. This information can be linked to metabolite identification or to specific up/down-regulated biochemical pathways. Matlab-based software employing the Pearson's correlation algorithm was applied to urine electropherograms from 20 mice infected with the schistosoma parasite. The fingerprints were the sum of electropherograms analysed with normal and reverse polarity, in two different modes MEKC and CZE and with two different capillaries (uncoated and polyacrylamide coated) to provide a broad picture of the samples. Hippurate, a metabolite that was depleted in the infected group and is present in both polarities, was chosen as a test variable; it correlated with itself to a p value of

Serum protein electrophoresis is routinely used to identify pathologies involving dysproteinemia. The electropherogram mainly represents the most abundant serum proteins, one of which is the polymorphic haptoglobin (Hpt), characterized by a molecular heterogeneity with three major phenotypes (Hpt 1-1, 2-1, and 2-2). To improve the interpretation of electropherogram and possibly to extend its applicability, we aimed to explore the relationship between Hpt phenotypes (determined by immunoblotting) and protein profiles. Serum samples were separated by CZE with PROTEIN 6 and high-resolution methods. The PROTEIN 6 analysis showed significant associations between α2 zone profiles and Hpt phenotypes (chi-square = 154.06, p

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.

In this work, the electrophoretic mobility (epm) measurement of individual cells was investigated by a simple on-chip electrophoresis system with lif multipoint detection. the system enabled the characterization of cell electrophoresis behavior as well as the fluorescence signal from individual cells simultaneously. the measurement yielded the electropherograms of a large number of cells labeled with dye, in which the migration time and migration distance could be obtained easily. the epm has been demonstrated to be different between the k562 cells and k562 cells treated with anticancer drug arsenic trioxide (as(2)o(3)). the k562 cells were found to exhibit a lower epm compared to the cells after drug addition with different concentration. in this preliminary study, over 300 cells could be analyzed within 2 h, demonstrated a much higher analysis throughput compared with traditional methods. the established system is simple and fast, which is expected to be a promising method for evaluating cell surface properties and to be useful in clinical and pharmaceutical applications.

Metabolic fingerprinting of biofluids such as urine can be used to detect and analyse differences between individuals. However, before pattern recognition methods can be utilised for classification, preprocessing techniques for the denoising, baseline removal, normalisation and alignment of electropherograms must be applied. Here a MEKC method using diode array detection has been used for high-resolution separation of both charged and neutral metabolites. Novel and generic algorithms have been developed for use prior to multivariate data analysis. Alignment is achieved by combining the use of reference peaks with a method that uses information from multiple wavelengths to align electropherograms to a reference signal. This metabolic fingerprinting approach by MEKC has been applied for the first time to urine samples from autistic and control children in a nontargeted and unbiased search for markers for autism. Although no biomarkers for autism could be determined using MEKC data here, the general approach presented could also be applied to the processing of other data collected by CE with UV-Vis detection.

Multiplex_QA is a data analysis tool for visualizing short- and long-term changes in the performance of multiplexed electrophoretic assays, particularly the commercial short tandem repeat (STR) kits used by the human forensic identity community. A number of quality metrics are calculated from the signal collected for the internal size standard included in nearly all multiplex assays. These quality metrics are related to the signal intensity, symmetry, retention, resolution, and noise of data collected by capillary electrophoresis systems. Interlocking graphical displays enable the identification of changes in the quality metrics with time, evaluation of relationships among the metrics, and detailed examination of electropherographic features of particularly interesting analyses. While primarily intended for exploring which metrics are most useful for documenting data quality, the current version of the tool is sufficiently robust for use by forensic scientists with an interest in data analysis and access to a fast desktop computer.

Protein-protein interactions were analyzed by zone electrophoresis of premixed equilibrium mixtures of a fluorescence-labeled protein at a constant concentration and unlabeled protein at a variety of concentrations using a 96-CE instrument equipped with a LIF detector. The interactions between labeled-con A versus succinylated ovalbumin, labeled-trypsin versus four proteinaceous trypsin inhibitors and labeled-insulin versus seven anti-insulin monoclonal antibodies were analyzed using a dual buffer system, in which a 60 mM borate-Na buffer (pH 9.35) was used as electrophoresis buffer and 60 mM MOPS-Na (pH 7.35) containing 0.1% Tween 20 was used as a sample buffer. The dual buffer system allowed fast and reproducible analyses of interactions at a physiological pH using uncoated fused-silica capillaries. The change in the mobility moment, the first statistical moment of an electropherogram on the mobility axis (Shimura, K., Uchiyama, N., Enomoto, M., Matsumoto, H., Kasai, K., Anal. Chem. 2005, 77, 564-572), of the labeled proteins were analyzed as a function of the concentration of unlabeled proteins. The dissociation constants for seven antibodies ranging from sub nanomolar to micromolar was determined based on the results of one cycle of parallel electrophoresis runs, which completed in 30 min using 20 pmol (120 ng) of labeled insulin and 5 pmol (750 ng) each of the mAb.

This paper focused on a self-developed microfluidic array system with microfabricated capillary array electrophoresis (mu-CAE) chip for parallel chip electrophoresis of biomolecules. The microfluidic array layout consists of two common reservoirs coupled to four separation channels connected to sample injection channel on the soda-lime glass substrate. The excitation scheme for distributing a 20 mW laser beam to separation channels in an array is achieved. Under the control of program, the sample injection and separation in multichannel can be achieved through six high-voltage modules' output. A CCD camera was used to monitor electrophoretic separations simultaneously in four channels with LIF detection, and the electropherograms can be plotted directly without reconstruction by additional software. Parallel multichannel electrophoresis of series biomolecules including amino acids, proteins, and nucleic acids was performed on this system and the results showed fine reproducibility.

A new type of migrating zone boundary in electrophoresis is presented theoretically and evidenced experimentally. This type of the boundary (called hybrid boundary) shows simultaneously a steady-state part with self-sharpening properties and an unsteady-state part with time-dependent electromigration dispersion. It is shown that a sample zone may possess a hybrid boundary both as its front and rear one simultaneously. In such a case, the evolution of a sample zone injected originally as a rectangular pulse exhibits very complex transient shapes before it reaches the well-known fronting or tailing triangular shape. Depending on the stage at which detection of such a sample zone occurs, variable and peculiar-shape peaks may appear in the electropherogram. Based on theoretical predictions, experimental examples of the above-mentioned phenomena are presented in this contribution for direct and indirect UV absorbance detection of sample zones. Excellent agreement of theoretical predictions with the experiments has been found. The knowledge of hybrid boundaries is of key significance for correct interpretation of records of CE analyses in practice.

A mathematical model developed for aqueous solutions and adapted to methanol as solvent was applied to predict the electromigration characteristics of analytes and background electrolytes in capillary zone electrophoresis. These characteristics are the effective mobility, and the tendency of the analyte to undergo peak-broadening due to electromigration dispersion. The input parameters for calculation like limiting mobilities and dissociation constants were experimentally determined or taken from the literature. By the aid of the model, the molar response for conductivity detection was calculated as well as the transfer ratio when indirect UV detection was used. They allow depicting the electropherogram by computer simulation. An additional important program output is the prediction of the occurrence of system- or eigenpeaks that mimic peaks of analytes or electroosmotic flow markers. The measured electropherograms were in agreement with those theoretically predicted. Deviations were attributed to ion pairing in methanolic solutions, which was not implemented in the model.

When working with capillary zone electrophoresis (CZE), the analyst has to be aware that the separation system is not homogeneous anymore as soon as a sample is brought into the background electrolyte (BGE). Upon injection, the analyte creates a disturbance in the concentration of the BGE, and the system retains a kind of memory for this inhomogeneity, which is propagated with time and leads to so-called system zones (or system eigenzones) migrating in an electric field with a certain eigenmobility. If recordable by the detector, they appear in the electropherogram as system peaks (or system eigenpeaks). However, although their appearance can not be forecasted and explained easily, they are inherent for the separation system. The progress in the theory of electromigration (accompanied by development of computer software) allows to treat the phenomenon of system zones and system peaks now also in very complex BGE systems, consisting of several multivalent weak electrolytes, and at all pH ranges. It also allows to predict the existence of BGEs having no stationary injection zone (or water zone, EO zone, gap, dip). Our paper reviews the theoretical background of the origin of the system zones (system peaks, system eigenpeaks), discusses the validity of the Kohlrausch regulating function, and gives practical hints for preparing BGEs with good separation ability not deteriorated by the occurrence of system peaks and by excessive peak-broadening.

We are introducing a computer implementation of the mathematical model of zone electrophoresis (CZE) described in Stedry, M., Jaros, M., Hruska, V., Gas, B., Electrophoresis 2004, 25, 3071-3079 program PeakMaster. The computer model calculates eigenmobilities, which are the eigenvalues of the matrix tied to the linearized continuity equations, and which are responsible for the presence of system eigenzones (system zones, system peaks). The model also calculates other parameters of the background electrolyte (BGE)-pH, conductivity, buffer capacity, ionic strength, etc., and parameters of the separated analytes--effective mobility, transfer ratio, molar conductivity detection response, and relative velocity slope. This allows the assessment of the indirect detection, conductivity detection and peak broadening (peak distortion) due to electromigration dispersion. The computer model requires the input of the BGE composition, the list of analytes to be separated, and the system instrumental configuration. The output parameters of the model are directly comparable with experiments; the model also simulates electropherograms in a user-friendly way. We demonstrate a successful application of PeakMaster for inspection of BGEs having no stationary injection zone.

A nanoliter enzyme microreactor was developed for on-line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post-column derivatization and laser-induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on-line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies300 000 plates/m were obtained for most peaks in the electropherogram of an on-line peptide mapping experiment of denatured alpha-lactalbumin under optimal conditions.

A new, fast and efficient procedure is described for the simultaneous quantitative analysis of various non-UV absorbing species in a sample, by capillary zone electrophoresis with indirect UV absorbance detection. The procedure is based on the concept of the conversion factor (CF). The CF of an analyte is defined as the ratio of the measured temporal peak area and the product of its migration time and transfer ratio (TR). Thus defined, the CF is of general validity for all analytes separated and detected in a given background electrolyte (BGE), since it has the same value for the same amounts of various analytes. If a sample is enriched with a known concentration of a standard component and analyzed by CZE, the CF of this standard component can be calculated and then the concentrations of all other analytes can be determined, without the use of any calibration graph. The individual TRs can be determined a priori from tabulated ionic mobilities and pK values of the analytes and of the constituents of the BGE or, for strong analytes, by using experimental data from the electropherogram of the analysis itself. The practical procedure of the analysis includes enrichment of the sample with a known quantity of a suitable standard and a single CZE run of the resulting mixture. The injected volume does not need to be known and thus the procedure also eliminates the injection error. The proposed procedure has been verified experimentally and reproducible and accurate values were obtained by using four different CZE apparatus for the analyses of standard mixtures of cations in three different BGEs.

Signal denoising and baseline correction using discrete wavelet transform (DWT) are described for microchip capillary electrophoresis (MCE). DWT was performed on an electropherogram describing a separation of nine tetramethylrohodamine-5-isothiocyanate labeled amino acids, following MCE with laser-induced fluorescence detection, using Daubechies 5 wavelet at a decomposition level of 6. The denoising efficiency was compared with, and proved to be superior to, other commonly used denoising techniques such as Fourier transform, Savitzky-Golay smoothing and moving average, in terms of noise removal and peak preservation by directly visual inspection. Novel strategies for baseline correction were proposed, with a special interest in baseline drift that frequently occurred in chromatographic and electrophoretic separations.

We represent the first extensive study on DNA capillary electrophoresis using various poly(vinyl alcohol) (PVAL)-related polymers as separation media. As a separation medium, PVAL homopolymer has shown poor resolutions probably due to its very strong hydrogen-bonding characteristics, resulting in extensive self-aggregation. On the other hand, poly(vinyl alcohol-co-vinyl acetate) and poly(vinyl alcohol-co-1-vinyl-2-pyrrolidone) (PVAL-VP), both with a degree of polymerization of approximately 3.0x10(3), have been found to give excellent electropherograms with good resolutions for the analysis of double-stranded (ds)DNA. PVAL-VP, with hydrolytic stability in high and low pH regions, has also yielded fair electropherograms for single-stranded (ss)DNA under neutral and alkaline conditions, although further investigation is essential in order to increase the resolutions necessary for DNA sequencing analysis. The separations obtained under alkaline conditions have shown significantly shorter retention times, one-third of that for the current commercial separation media, due to the higher ionization of phosphate groups in the DNA chains.

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.

Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (μCE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the μCE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF-7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the μCE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF-7 and SW620 cells in a volume of 200 μL, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the μCE analysis.

The suppression of evaporation of water from small volumes of sample solutions or reagents for capillary electrophoresis by the use of a mineral-oil overlay was investigated in affinophoresis applications, in which the affinity constant of a mutant protein of recombinant human galectin-1 to a lactose affinophore, a triply negative charged ion having a lactoside as an affinity ligand, was determined. When an injection was carried out from a minimum of 20 microL of an aqueous solution beneath the oil overlay, no oil contamination inside the capillary was observed, provided the capillary was cleanly cut so that the end was flat, and the polyimide coating had been removed for a distance of about 2 mm from the end. Affinophoresis was carried out using 20 microL of an affinophore solution covered with an oil overlay. The abnormalities in the electropherograms as the result of the evaporation of the water from the solution during storage prior to use in an automatic operation of a capillary electrophoresis instrument were suppressed, with respect to the formation of a base line gap, an increase in the detection time of a marker ion and an increase in the initial current. A solution in a vial could be used repeatedly for a longer period of time when overlaid with mineral oil than in the absence of an overlay. The use of a mineral-oil overlay is a simple but very efficient technique for solving the problem of the evaporation of water from small volumes of aqueous solutions for use in capillary electrophoresis.

We present a novel method for the automated detection of fragments showing dissimilar expression in mRNA differential display. The analysis is based on aligning the numerical electrophoretic lane data in respect of a given distance function defined on a set of fragments, or signal peaks in general. We presume that significant dissimilarities between peaks result in extreme score values computed for aligned peak pairs. Whereas in sequence comparison, an overall sequence similarity score is conventionally used, the current method defines a special dissimilarity score for searching the peak pairs showing the largest relative differences between the lanes. The output of the analysis is a highly reduced list of peak pairs, along with a set of associated features extracted from the lanes. Only the peaks of this list need to be visually confirmed instead of the vast amount of peaks in the original electrophoretic results. The results obtained by the algorithm correlate well with results of visual evaluation of the same electropherograms. The current algorithm may be applied to the study of complex expression patterns in multiple lanes and, in general, to automated recognition of variously defined patterns of quantitative electrophoretic data.

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.

The allocation of proteins to stimulons and regulons is an essential step towards the understanding of the global regulation of the expression of entire genomes. The computer-aided evaluation and matching of two-dimensional protein gels loaded with radioactively labeled proteins from exponentially growing or stressed cells is a useful but time-consuming procedure for the description of stimulons and regulons. This paper describes the dual-channel image analysis that offers the opportunity to visualize the content and synthesis rate of a whole set of bacterial proteins on a single electropherogram. By pulse-labeling with L-[35S]methionine, the protein synthesis pattern (red color) can be directly compared with the protein level pattern (green color). Because matching of other gels can be avoided, this new technique is useful for the rapid search for proteins that belong to different stimulons or regulons. This approach was tested for the identification of proteins of heat stress or oxidative stress stimulons. Proteins that were induced by heat or oxidative stress colored red while proteins whose synthesis was switched off by the stress factor colored green. Proteins that were continuously synthesized before and after the imposition of stress retained their yellow color. The advantages and possible pitfalls of the technique are discussed.

Non-steady-state electrophoretic processes can be estimated by a repeated application of a steady-state model based on the electroneutrality equation, the modified version of Ohm's law, and the mass balances of the co- and counterions. With such a mathematical model, all parameters in sample zones in capillary zone electrophoresis (CZE) can be calculated. The relationships between the calculated parameters for sample zones in CZE, such as the pH, concentrations of co- and counterions, and the ratio E1m1/E2m2 versus the mobilities of both anionic and cationic analytes can be visualized in a SystChart, a set of eight panels, for a given background electrolyte (BGE). All properties of a zone, such as the fronting/tailing character and the question of peaks/dips can be read from such a SystChart. Applying n coions, n-1 discontinuities are present in such a SystChart, indicating the presence of system peaks applying that BGE. For BGEs with one coion, no system peaks (discontinuities) exist at moderate pH values. SystCharts calculated for BGEs with a low pH do show discontinuities, however, which indicates that system peaks are present in electropherograms applying BGEs at low pH. Experimentally, it is shown that system peaks are indeed present in electropherograms applying BGEs with one coion at low pH and the mobilities of the system peaks generally increase with decreasing pH. Hydrogen ions seem to act as a second coionic species. Of course, these system peaks are only visible in the UV signal if the BGE has UV-absorbing properties.

We have demonstrated in a systematic manner that the capillary electrophoresis (CE) method of genotyping the human D1S80 locus is an effective replacement for the commonly used gel electrophoresis method. The CE method is fast, with a total run time of less than 22 min per sample. Separation has been optimized so that resolution for all pairs of alleles from 14 through 41 is greater than 1, providing unambiguous identification. The method utilizes run buffer containing 0.30% hydroxyethyl cellulose as the sieving polymer in a 50 microm internal diameter (ID) column coated with a 0.1 microm DB-17 film. Laser-induced fluorescence (LIF) with YO-PRO-1 intercalating dye is used for detection. Internal standards of 300 and 1000 bp bracket the D1S80 region in the electropherogram. Two typing procedures were evaluated: matching of the normalized migration times of sample alleles to ladder alleles; and software alignment of sample and ladder electropherograms using the internal standard peaks as references. Using the first procedure, 91.5% of the D1S80 genotypes were unambiguous, while 100% were unambiguous using the second procedure. Seventy-nine routine samples were analyzed in a side-by-side comparison of the CE and slab gel methods, with complete agreement of the results. Twenty-two samples were selected from a large database previously analyzed by the slab gel method to demonstrate that all alleles from 14 through 41 were typed correctly, including samples containing adjacent alleles. Additionally, 43 samples containing alleles greater than the 41 repeat number were typed correctly.