Synthesis of new gels of mixed mode separation mechanism and their application in capillary electrochromatography

Abstract

Capillary electrochromatography, combining the advantages of high performance liquid chromatography and capillary electrophoresis, is one of the most successful separation techniques that can be applied for the separation of complex biological compounds such as pharmaceuticals, natural compounds and chiral molecules. Separation of neutral molecules is also a possible application of capillary electrochromatography. In size exclusion, separation is possible due to molecular mass difference and uncharged molecules are driven through the column by electroosmotic flow. To our knowledge there is no report of capillary electrochromatography for size exclusion separation of the macromolecules reported yet. Aim of this work was to create the stationary phase for the separation of neutral molecules and anions with capillary electrochromatography using contactless conductivity detector. Agarose is a linear polysaccharide polymer that can be used in analytical methods in order to separate molecules [1]. Agarose gel was modified by attaching ionic groups, creating a stationary phase that acts as a molecular sieve - because it is a porous structure that holds up a larger molecular weight analytes, which results in a faster movement of molecules having lower molecular weight through the capillary column in the electric field. The reaction was performed for 24 hours. Modified and 4% pure agarose gel were mixed and washed with electrolyte. 75 μm I.D. fused silica capillary was filled with molten agarose gel mixture using pressure and was used for the analysis of 1% dextran solutions using electrical field. Results showed the ability to perform mixed mode separation of neutral molecules and anions simultaneously. Conditions of analysis were: background electrolyte 30 mM acetic acid, pH 3.3; applied voltage 3.6 kV; total capillary length 40 cm, effective capillary length 35 cm; separation temperature 21 ºC. The results obtained will be reported in the presentation.