Sample stacking in CZE using dynamic thermal junctions I. Analytes with low dpK_a/dT crossing a single thermally induced pH junction in a BGE with high dpH/dT

Abstract

The possibility to compress analyte bands at the beginning of CE runs has many advantages. Analytes at low concentration can be analyzed with high signal-to-noise ratios by using the so-called sample stacking methods. Moreover, sample injections with very narrow initial band widths (small initial standard deviations) are sometimes useful, especially if high resolutions among the bands are required in the shortest run time. In the present work, a method of sample stacking is proposed and demonstrated. It is based on BGEs with high thermal sensitive pHs (high dpH/dT) and analytes with low dpK(a)/dT. High thermal sensitivity means that the working pK(a) of the BGE has a high dpK(a)/dT in modulus. For instance, Tris and Ethanolamine have dpH/dT=-0.028/ degrees C and -0.029/ degrees C, respectively, whereas carboxylic acids have low dpK(a)/dT values, i.e. in the -0.002/ degrees C to+0.002/ degrees C range. The action of cooling and heating sections along the capillary during the runs affects also the local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band compression is theoretically calculated using a simple model. Finally, this stacking method was demonstrated for amino acids derivatized with naphthalene-2,3-dicarboxaldehyde and fluorescamine using a temperature difference of 70 degrees C between two neighbor sections and Tris as separation buffer. In this case, the BGE has a high pH thermal coefficient whereas the carboxylic groups of the analytes have low pK(a) thermal coefficients. The application of these dynamic thermal gradients increased peak height by a factor of two (and decreased the standard deviations of peaks by a factor of two) of aspartic acid and glutamic acid derivatized with naphthalene-2,3-dicarboxaldehyde and serine derivatized with fluorescamine. The effect of thermal compression of bands was not observed when runs were accomplished using phosphate buffer at pH 7 (negative control). Phosphate has a low dpH/dT in this pH range, similar to the dK(a)/dT of analytes. It is shown that mid R:dK(a)/dT-dpH/dTmid R>>0 is one determinant factor to have significant stacking produced by dynamic thermal junctions.

Enhancing the Stability and Solubility of the Glucocorticoid Receptor Ligand-Binding Domain by High-Throughput Library Screening

“…The antagonist conformation was analyzed at a concentration of 2.5 μM in the same buffer supplemented with 50 μM mifepristone. Due to the temperature dependence of Tris buffer (ΔpK a /°C = − 0.028), 46 a pH drop during the scans (from pH 7.8 at 30°C to pH 6.6 at 70°C) has to be expected.…”

Online sample pre‐concentration via dynamic pH junction in capillary and microchip electrophoresis

“…Mandaji et al [38,39] introduced a succession of two research articles with a very exciting sample stacking strategy which relies on dynamic thermal junctions. This approach relies on significant pH changes as a result of altered temperature conditions.…”

Cyclic band compression in toroidal capillary electrophoresis delivers an unlimited number of theoretical plates with a quadratic growth in time and a constant peak capacity

“…In this case, a different temperature, T s (higher or lower than T r ), is applied synchronously for a short time (much smaller than the time required to make a round trip) during each cycle along this segment, before the temperature is changed back to T r . This causes temporary changes in the local viscosity, conductivity, electric field strength, buffer's pH , and/or analyte's p K a . The following band broadening mechanisms will be taken into account: (i) initial variance of the injected sample plug (); (ii) variance added ( d σ 2 = 2 D f dt) due to the continuous injection of the very small hydrodynamic “flow” into the microhole of the intermediate reservoir ( i + 1) when the electrostatic potential difference is applied between reservoirs i and i + 2. This band broadening mechanism is the same that would be observed when running a CE experiment with unleveled buffer reservoirs (see Eq.…”

“…During the short compression time interval, when the hot (or cold) zone is active, the local conductivity changes and this in turn causes the local electric field ( E ) to change. This would be time consuming to quantify , but fortunately the compression time interval is very short compared to τ , and the following relationship can still be used: μEτ = L . Taking all of this into account produces: …”

“…Using α = 0.5 (demonstrated in Refs. and ) and k = 1 results in a factor of 4. Nevertheless, the N a delivered in any time interval Δ t = nτ is proportional to n .…”