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The use of migration times and peak areas referred to another sample component − internal standard, brings many benefits in improving reliability of capillary electrophoresis. However, it is quite commonly overlooked that despite relative migration time and peak area ratio are more stable than the absolute values upon alteration in the flow rate, some shift should always be expected. The present work offers a new look at this analytically-important issue. We have derived a simple model allowing to estimate the magnitude of error for the selected pair of molecules of known mobilities upon the given flow alteration. Then, we have confronted the theoretical predictions with the experimental results obtained for the model sample separated in various flow conditions reached by the external pressure manipulation, including several internal standards of different mobilities. A good agreement has been obtained, pointing out that the magnitude of error may be large even for the seemingly “good” internal standards. Several potentially useful means have been tested to address this issue: the use of electrophoretic mobilities and electrophoretic mobility ratios instead migration times in the qualitative analysis, and performing time-correction of peak area ratios, or alternatively, transformation of electropherograms from the time-related scale into the electrophoretic mobility-related scale in the quantitative analysis. We have also considered some additional factors. The results may be of interest for all users dealing with the development and optimization of analytical methods using capillary electrophoresis.

In this study, we developed a novel assay that simultaneously detects multiple miRNAs (microRNAs) within a single capillary by combining a tandem adenosine-tailed DNA bridge-assisted splinted ligation with denaturing capillary gel electrophoresis with laser-induced fluorescence. This proposed method not only represents a significant improvement in resolution but also allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The assay's linear range covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N=3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus (EBV) were also detected in EBV-infected nasopharyngeal carcinoma cells, while they did not appear in non-virus infected cells. Moreover, the electropherogram indicated that the screening of isomiRs (isomer of miRNA) of BART2 by CE-LIF is feasible by our proposed method. The developed electrophoresis-based method for miRNA detection is fast, amplification-free, multiplexed and cost-effective, making it potentially applicable to large-scale screening of isomiRs.

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.

Electrophoretically mediated microanalysis (EMMA) was used to monitor the on-column complexation of Pb2+ and fulvic acid (FA). Electropherograms revealed several characteristic regions, the areas of which correlate with the metal concentration. The analysis of the electropherograms suggests that at least two different complexes are formed. Therefore, the EMMA is a prospective technique for structural investigation of humic substances (HS).

DNA sequencing is performed in a multiplexed capillary electrophoresis system by UV absorption detection. Four individual electropherograms are obtained by simultaneously running the unlabeled DNA products of the four ddNTP-terminated reactions in the capillary array. The sequence of the template used in the cycle-sequencing reaction can be determined by overlaying the four electropherograms. Two internal standards are employed to adjust for the variance in migration times among the capillaries. After applying the correction algorithm, base calling can be done at a high level of confidence.

Capillary zone electrophoresis has been used for the characterization and separation of humic acids. It was found that addition of saccharides like alpha-, beta-, gamma-cyclodextrins, maltose, hydroxyethylcellulose or dextran sulfate in the background electrolyte (50 mM Na2 B4 O7, pH 9.6) yields better separation patterns and highly reproducible electropherograms. Electropherograms with higher numbers of peaks and high reproducibility were obtained with alpha- and beta-cyclodextrins or with a mixture of alpha- + gamma-cyclodextrin-modified background electrolytes. Separation was carried out with the cathode at the detector end of the column. Adsorption of humic acids to the capillary wall was diminished using an epoxy-coated capillary tube.

Migration times in a capillary zone electropherogram obtained by using the field enhanced sample stacking technique are strongly affected by the injected sample volume. That is, the migration times significantly decrease with the increase of the sample volume. To avoid inaccurate qualitative analysis due to the above phenomena, the time axis of the electropherograms was converted into an effective mobility axis using our conversion method taking account of the temperature increase in the separation tube and relaxation of the potential gradient of the separation field. After the conversion, accurate qualitative analysis was possible in spite of drastic change of the migration time, suggesting our conversion method could be successfully used for the standardization of electropherograms obtained even by using the stacking effect. The cause of the decrease of the migration time in the stacking process was briefly discussed.

A new method for standardization of electropherograms obtained by capillary zone electrophoresis was proposed, where the migration time axis was replaced by the effective mobility axis. The mobility increase due to temperature increase by Joule heating and the relaxation effect of the potential gradient were eliminated successfully by introducing a temperature coefficient for mobility expression and a delay time, respectively. The precision of the mobility evaluated by the proposed conversion methods was evaluated for a model sample. By using the conversion method, almost the same electropherograms could be obtained even from the electropherograms originally obtained by using different hardware conditions.

A chemometric methodology was used to study capillary efficiency and the separation of ten benzodiazepines in capillary electrophoresis. The resolution between two adjacent peaks on the electropherogram was estimated and the overall quality of the separation was assessed by means of a new response function. The nature (methanol or acetonitrile) and proportion of the organic modifier both in the background electrolyte and the sample buffer and the injection time were considered. The results predicted that if the sample had a lower dielectric constant than the background electrolyte buffer then a much larger injection volume could be used. The computer optimization routine was experimentally validated and the result demonstrated that the fastest electrophoretic reparation was obtained with acetonitrile (7 min instead of 9 min with methanol).

A stepwise development for the use of capillary electrophoresis and inductively coupled plasma mass spectrometry (ICP-MS) for speciation investigations is presented. The high resolution power of CE is used for the separation of metal species, whereas ICP-MS is taken for element-specific detection with low detection limits. This contribution starts with an off-line combination of both instruments. Separation and identification of species in model solutions and real samples are shown by scanning UV detection at the CE unit with subsequent metal quantification in peak related fractions, applying electrothermal vaporization ICP-MS. Finally, first separations are demonstrated, using the on-line hyphenation with a laboratory-made nebulizer. Here, standard solutions are separated and monitored by UV and ICP-MS. Stability of electrical current during nebulization was checked and a possibly interfering suction flow was estimated. After optimization sufficient electropherograms were obtained. Advantages and problems are discussed for both modes.

Capillary isoelectric focusing (cIEF) and IEF of recombinant humanized monoclonal antibody HER2 (rhuMAbHER2) show five charged isoforms with estimated p I values ranging from 8.6–9.1. The cIEF assay demonstrated good precision with relative standard deviations (R.S.D.) 0.7–3.7% and 0.4–4.2% for intra and interassay analysis, respectively. The method was linear for the area of the main peak over the concentration range 2–250 μg/ml with a Pearson correlation coefficient >0.99. The limit of detection for the main peak was determined to be 2 ppm. With both sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and SDS-polyacrylamide gel electrophoresis, the nonreduced rhuMAbHER2 migrated as a single major peak with minor peaks in the aggregate and clip regions. After reduction, the electropherogram and the slab gel showed the expected heavy chain and light chain fragments with minor peaks in the aggregate and clip regions. The SDS-CGE assay showed good precision with R.S.D. values of 0.1–7.8% and 0.1–8.1% for intra and interassay analysis, respectively. The Pearson correlation coefficient for the area of the main peak was >0.99 demonstrating linearity for the concentrating range 0.5–500 μg/ml. The limit of detection for intact rhuMAbHER2 was determined to be 0.5 ppm. The data presented demonstrates the feasibility of replacing the slab gel techniques with capillary electrophoresis in a quality control environment.

A method for the determination of apolipoprotein A-I, the major protein compartment of HDL, in human serum is described. Rapid and easy serum sample preparation, well separated Apo A-I peaks in the human serum electropherogram and good linearity of the peak area vs. concentration plot, covering the range of the clinically relevant Apo A-I serum contents, suggest the introduction of this method routinely in clinical laboratories.

Recombinant extracellular superoxide dismutase was proteolytically degraded by trypsin and the digest was thereafter separated using three different separation techniques. The size differences of the obtained fragments were exploited by size-exclusion chromatography (SEC) on a highly efficient column for peptide separation. Collected fractions representing different size groups of peptides were then separated on the basis of hydrophobicity on reversed-phase liquid chromatography (RPLC). This led to simplified RPLC-chromatograms where collected peaks were considerably more pure than if the digest was separated direct on RPLC and the identity of eluted peaks could easily be determined by amino acid analysis. Finally the digest was run on capillary electrophoresis for separation based on charge differences. For the identification of the different peaks in the electropherogram, the total digest was spiked with known peptides purified by the two other separation techniques. This technique proved to be very powerful for peak identification in an electropherogram from a digest composed of many fragments eluting closely together.