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Towards adaptive method for peak migration time correction: discretization period in electropherograms
Abstract
Capillary electrophoresis often causes unrepeatable peak migration times in the electropherogram due to changes of electroosmosis, yet in some cases this separation technique does not have a replacement alternative. Some attempts to overcome this issue have been performed introducing internal standards into the sample and compensating peak shifting in time. However, existing vector calculation-based methods are computationally intensive for portable instrumentation and usually limited to post-processing applications with 1 or 2 markers. In this work, an original approach of compensating peak migration time shift via signal discretization period correction is proposed. Using the proposed method, the number of reference points or markers that are used for compensation is extended. This method is effective in compensating migration time of peaks in real samples, where high sample injection volumes are used. Using 4 reference peaks in compensation, the method was capable of reducing the relative standard deviation of migration time of the peaks in the electropherograms more than 15 times. Corrected signal discretization periods indicated very high correlations with recorded separation currents, what can be perspective developing an adaptive peak migration time compensation method in capillary electrophoresis.
The review covering the development of capillary electrophoresis with capacitively coupled contactless conductivity detection from 2020 to 2024 is the latest in a series going back to 2004. The article considers applications employing conventional capillaries and planar lab‐on‐chip devices as well as fundamental and technical developments of the detector and complete electrophoresis instrumentation.
In capillary electrophoresis (CE), analyte identification is primarily based on migration time, which is a function of the analyte's electrophoretic mobility and the electro-osmotic flow (EOF). The migration time can be impacted by the presence of parasitic flow from changes in temperature or pressure during the run. Presented here is a high-voltage-compatible flow sensor capable of monitoring the volumetric flow inside the capillary during a separation with nL/min resolution. The direct measurement of both flow and time allows for compensation of flow instabilities. By expressing the electropherogram in terms of signal versus electromigration velocity instead of time, it is possible to improve the run-to-run reproducibility up to 25×.
The reproducibility in migration time is essential in the use of capillary electrophoresis to identify components in a complex sample or compare constituent differences for different botanical drug samples. A powerful one-marker technology for correcting migration time was proposed in this paper. This technology is practically simple compared with the multi-marker technologies developed in literature, due to only one marker required. Paeoniae Radix which is a crude drug used in many traditional prescriptions in China and Japan was selected as an example to verify the effectiveness of the powerful one-marker technology in analyzing complex botanical samples. Relative standard deviations in migration times treated with the powerful one-marker technology were below 0.5% for Paeoniae Radix analyzed during three different days. The technology was successfully applied to classify Paeoniae Radix from nine different growing regions. Based on their corrected electropherograms, Paeoniae Radix was classified into three groups which were in agreement with their real origins.
The practical and theoretical problems associated with interpretation of binding isotherms obtained by the investigation of weak analyte-ligand interactions using mobility shift affinity CE were investigated with special emphasis on various correction methods to compensate for media effects due to high additive concentrations. The interaction between 2-hydroxypropyl-alpha-CD and two bile salts (glycocholate and glycodeoxycholate) was studied applying correction factors based on viscosity, CE based conductance and separately measured conductivities. Accurate measurement of the stability constants proved to be difficult; several points of general nature relating to the investigation of the weak analyte-ligand interactions were identified. These included undesired dilution of the BGE due to the high amounts of additive, avoidance of temperature effects by maintaining a constant power during the separations and limited validity of Walden's rule. The relative sizes of the buffer constituents, the nonelectrolyte ligand additive, and the analytes as well as noninteracting marker substances should be considered in order to minimize errors in the correction factors when significant deviation from Walden's rule is observed. Under such conditions, the mobilities should preferably be corrected by the use of a noninteracting marker; and the most generally applicable corrections may be based on conductance measurements.
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