Electrically driven capillary size exclusion chromatography

Venema, E.; Kraak, J.C.; Poppe, H.; Tijssen, R.

doi:10.1007/bf02467702

Cited by 40 publications

(28 citation statements)

References 17 publications

(18 reference statements)

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“…Size-based separation mechanisms have not been fully exploited in CEC, excluding capillary gel electrophoresis, which relies on sieving rather than size exclusion [47]. The only true size exclusion-based separations in a pure CEC mode, for MW neutral polymers, were reported by Venema et al and Stol et al, using bare silica columns [48][49][50] and by our group using polymeric stationary phases [51]. Peters et al [52] had demonstrated the separation of polystyrene standards on a methacrylate-based monolithic column.…”

Section: Introductionmentioning

confidence: 99%

Size‐exclusion capillary electrochromatographic separation of polysaccharides using polymeric stationary phases

2003

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We report the successful size-based separations of large, neutral polysaccharides using capillary electrochromatography (CEC). As the polysaccharides possessed little chromophore for photometric detection, two separate approaches were taken. In the first approach, indirect detection was combined with size-exclusion chromatography using a sulfonated polystyrene/divinylbenzene stationary phase. The separations were performed using a 300 Å pore size stationary phase under aqueous conditions. Nonsize based interactions were minimal using this material, resulting in an effective calibration range of molecular masses 180 to 112 000 g?mol 21 for pullulans. In the second approach, the polysaccharides were derivatized with phenylisocyanate and were subsequently separated on columns made using a combination of high capacity ionexchanger and a neutral polystyrene/divinylbenzene material of various pore sizes. The sulfonated ion-exchange phase provided the electroosmotic flow, while the mixed pore size material provided the extended calibration range. The linear range for this primarily nonaqueous system using tetrahydrofuran was determined to be from molecular masses 738 to 404 000 g?mol 21 of the original, untagged pullulan. This approach overcame the limited solubility issue associated with analysis of some polysaccharides. Analysis of pullulan and amylose samples by CEC correlated well with results obtained by conventional high-performance liquid chromatography (HPLC). The sizeexclusion electrochromatographic separations provide an alternative mode for determining the relative molecular weights of polysaccharides with reduced sample and solvent consumption, as well as analysis times.

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Section: Introductionmentioning

confidence: 99%

Size‐exclusion capillary electrochromatographic separation of polysaccharides using polymeric stationary phases

2003

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“…, and zone electrophoresis of inorganic anions [7] and drugs (sometimes in a binary mixture with ,6% acetic acid) [8][9][10][11][12]. It has been used in chiral separations [13][14][15][16], in capillary electrochromatography of retinyl esters (in a mixture with 1% methanol) [17], in capillary size exclusion electrochromatography [18][19][20], in capillary gel electrophoresis of neutral polymers (mostly in mixtures with 1-6% water) [21], and in other applications [22][23][24][25][26]. Its influence on the electroosmotic flow (EOF) has been studied in both uncoated [7,[27][28][29][30] …”

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confidence: 99%

Capillary electrophoresis in N,N‐dimethylformamide

Porras

Kenndler

2005

Electrophoresis

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N,N-Dimethylformamide (DMF) is a dipolar protophilic solvent with physicochemical properties that makes it suitable as solvent for capillary electrophoresis (CE). It is prerequisite for the proper application of CE to adjust and to change the pH of the background electrolyte (BGE) in a defined manner. This was done in the present work using benzoic acid-benzoate by selecting different concentration ratios of acid and salt, and calculating the theoretical pH from the activity-corrected Henderson-Hasselbalch equation. The mobilities of the analytes (chloro- and nitro-substituted phenolates) were found to follow reasonably well the typical sigmoid mobility versus pH curve as predicted by theory. The actual mobilities and pK(a) values (at 25 degrees C) of the analytes were derived from these curves. pK(a) values were in the range of 11.1-11.7, being thus 3-4.4 units higher than in water. This pK(a) shift is caused by the destabilization of the analyte anion and the better stability (solubility) of the molecular analyte acid in DMF, which overcome the higher basicity of DMF compared to water. Absolute mobilities were calculated from the actual mobilities; they were between 32x10(-9) and 42x10(-9) m(2)/Vxs. Slight deviations of the measured mobilities from the theoretical mobility versus pH curve were discussed on the bases of ion pairing and heteroconjugation and homoconjugation of either buffer components or buffer components and analytes. Heteroconjugation was used as a mechanism for the electrically driven separation of neutral analyte molecules in a BGE where salicylate acted as complex forming ion. Rough estimation of the complexation constants for the phenolic analytes gave values in the range of 100-200 L/mol. Addition of water to the solvent decreased the effect of heteroconjugation, but it was still present up to the surprisingly high concentration of 20% water. Electrophoretically relevant parameters like ionic mobilities and pK(a) values, and conjugation and ion pairing are dependent on the water content of the solvent. The water uptake of DMF was measured when exposed to humidity of ambient air. The resulted behavior of the water uptake was found rather similar to that for acetonitrile and methanol.

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“…Figure 3 shows the van Deemter plot of a 2-mm-long SEEC column for FITC-insulin using either 50 or 10% ACN in the running buffer. This figure clearly shows that, at 10% ACN, the velocity is high enough for the mass transfer terms of the van Deemter equation to dominate [38], while these contributions are negligible at 50% ACN. Additionally, the high ACN content may reduce hydrophobic interactions, contributing to an overall decrease in H at 50% ACN.…”

Section: Seecmentioning

confidence: 91%

“…Pressure imbalances that arise from the difference in EOF between the open-channel segments and the packed column can disrupt flow patterns [33]. To reduce these imbalances and perform SEEC separation of proteins, charge needs to be introduced by dynamically modifying the beads with a charged surfactant [34][35][36][37][38]. Through the use of both neutral 0.01% Tween 20 to reduce nonspecific adsorption [2,39] and 0.25 mM (0.008%) SDS [40],] protein separation was possible, the currents were stable and the performance was reproducible.…”

Section: Seecmentioning

confidence: 99%

Capillary electrochromatography with packed bead beds in microfluidic devices

et al. 2009

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Microchip-based bead-packed columns for electrochromatography are described for several types of stationary phases. Chromatography columns 2 mm in length were used for the separation of proteins and peptides by size- and ion-exchange modes of separation, respectively. In size-exclusion electrochromatograpgy, FITC-IgG and FITC-insulin were baseline resolved in less than 10 s, with efficiencies of up to 139,000 plates/m for FITC-insulin. In strong cation-exchange electrochromatography, a mixture of three fluorescently labeled peptides was baseline resolved in less than 40 s, with efficiencies up to 400,000 plates/m. The RSD for the analytes retention times were<3% in both size-exclusion and ion-exchange modes of separations. The use of a 1-mm-long reverse-phase column for the semiquantitative evaluation of pharmaceutical formulations in drug solubility tests illustrates the use of this microfluidic chip-based electrochromatographic approach to drug development.

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Electrically driven capillary size exclusion chromatography

Cited by 40 publications

References 17 publications

Size‐exclusion capillary electrochromatographic separation of polysaccharides using polymeric stationary phases

Size‐exclusion capillary electrochromatographic separation of polysaccharides using polymeric stationary phases

Capillary electrophoresis in N,N‐dimethylformamide

Capillary electrochromatography with packed bead beds in microfluidic devices

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