2013-05-13 1 New Developments in Capillary Electrophoresis with focus on Bioanalysis Lecture 11 Christian Nilsson Today • Capillary Electrochromatography – Open tubular columns – Packed columns – Monolithic columns Capillary Electrochromatography (CEC) • Liquid phase separation in a column containing a stationary phase media conatining ionizable groups • The flow is driven by electroosmosis • The electric field across the capillary can affect the retention of the analytes Packed column CEC • The procedures for fabricationg packed columns came from HPLC – Retaining frits – Solvent slurry packing • More complicated to pack capillaries than standard analytical columns Packed column CEC • Smith and Evans were early to demonstrate the extreme efficiency that can be achieved by CEC – 8 millions plate per meter • However, the results were difficult to reproduce • Highly polar basic compounds were anlysed using strong cation exchanger Chromatographia 1995, 41, 197-203 Packed column CEC Chromatographia 1995, 41, 197-203 2013-05-13 2 Packed column CEC • Typical packing procedure (1) – Attach a inline end end-frit and pack the column by pumping the slurry of beads and solvent through the capillary with high pressure – Flush the packed column with water to replace the solvent – Prepare the outlet end-frit at the desired distance from the capillary end by sintering the beads using heat over 550°C Packed column CEC • Typical packing procedure (2) – Remove the inline end-frit and flush out the extra material using reversed flow direction – Create the inlet end-frit by sintering the packing material – Remove the polyimide coating to create the detection window – Cut off excess capillary at the inlet side – Equiblirate the column with desired mobile phase Packed column CEC Packed column CEC • A major challenge is the fabrication of the retaining frits • The charged particles can move in the electric field Open tubular CEC • Developed by Pretorius already in 1974 • The stationary phase is attached to the capillary wall • Disadvantages with conventional OT-CEC – Low phase ratio of the stationary phase because of the small surface area – Relatively long distance for the analytes to reach the stationary phase material Open tubular CEC • Organic moieties bound to the capillary inner wall – The chromatographic effect is small – However, EOF and adsorption properties can be changed 2013-05-13 3 Open tubular CEC • Use of carboxymethyl-chitosan as stationary phase – A hydrophilic polysaccharide J. Chromatogr. A, 2010, 1217, 8346-8351 OT-CEC Chitosan as stationary phase J. Chromatogr. A, 2010, 1217, 8346-8351 OT-CEC • Effect of chitosan concentration on EOF J. Chromatogr. A, 2010, 1217, 8346-8351 OT-CEC • The EOF can be normal or reversed depending on the pH J. Chromatogr. A, 2010, 1217, 8346-8351 OT-CEC J. Chromatogr. A, 2010, 1217, 8346-8351 Chitosan-coated capillary Bare capillary OT-CEC Separation of basic proteins 2013-05-13 4 Open tubular CEC • Use of etched capillaries – Etching to increase the surface area • Chemical modification of etched capillaries • The etching procedure will affect the EOF – Nitrogen and fluoride from the etching reagent are incorporated into the surface – The surface that is generated is more biocompatible (with less tendency for adsorption) – The nitrogen alter the electroosmotic behavior of the capillary . At low pH the EOF will be reversed OT-CEC Open tubular CEC • Etching procedure (50 μm ID capillary) (1) – Fill the capillary with concentrated HCl, heat overnight (80°C) • To remove impurities from the capillary wall – Flush with water, acetone and diethyl ether – Dry capillary with nitrogen – Etching with ammonium hydrogen difluoride (5%) in methanol, wait one hour, remove methnol by nitrogen J. Chromatogr. A 1996, 736, 255-264 Open tubular CEC • Etching procedure (50 μm ID capillary) (2) – Methnol is used to assure uniform distribution of the ethcing agent during drying. – Heating to 300-400°C for 3-4 hours in a GC oven – The temperature and time determines the morphology of the surface J. Chromatogr. A 1996, 736, 255-264 Open tubular CEC J. Chromatogr. 2000, 887, 31-41 SEM of etched silica capillaries OT-CEC • Chemically modified etched capillaries – The surface area can be enhanced 1000 times – Silica material can extend 5 μm into the capillary and thereby decrease the distance for the analyte to reach the stationary phase – Can be coupled with MS • Rinse the column after formation, to avoid leakage when coupled to MS Anal. Chem. 2007, 79, 4942-4949 2013-05-13 5 OT-CEC • Protein separation in etched and non-etched capillaries OT-CEC • Separation of proteins and peptides • Porous-layer open tubular (PLOT) column • A crosslinked polymer layer on the wall of the capillary • Polymerization of vinylbenzyl chloride and divinylbenzene in presence of 2-octanol J. Chromatogr. A, 1999, 858, 91-101 OT-CEC • Functionalisation of PLOT column J. Chromatogr. A, 1999, 858, 91-101 OT-CEC Use of PLOT column OT-CEC • SEM of PLOT column J. Chromatogr. A, 1999, 858, 91-101 Monolithic CEC columns • Composed of in situ prepared polymers • Porous properties and surface chemistry can be controlled • Used in LC as well as CEC • First used by Hjertén in 1989 J. Chromatogr. 1989, 473, 273-275 2013-05-13 6 Monolithic CEC • Monolithic columns solve most of the problems with the other types of CEC (open tubular and packed column • No retaining frits • No movement of particles • Easier to prepare • Possible to form molecularly imprinted monoliths by the same procedure Monolithic CEC • Early methods to produce monolithic columns involved the fixation of an already packed capillary column • A heated wire can be drawn along the capillary to achieve sintering of the beads • The capillary can be heated to to 360°C in presence of sodium bicarbonate solution • The interstitial volume can be filled with sol gel structures Monolithic CEC • The described methods solve the problem with column stability • However, retaining frits still needed to be fabricated • Instead the monolithic CEC column can be fabricated by in situ polymerization Monolithic CEC • The length of the column can easily be adjusted – UV initiated polymerization can be used instead of thermial initiated polymerization. A mask can be used. • The polymerization mixture can be prepared using a wide variety of monomers allowing an almost unlimited choice of polymer morphology and surface chemistry Monolithic CEC • EOF is not as dependent on the pore size as a mechanically pumped flow • By using monoliths with small pores improving the analyte mass transfer • Will not give problem with large pressure drop Monolithic CEC • CEC as one dimension in 2D separation • CIEF followed by CEC for separation of proteins Journal of Proteome research 2006, 5, 2001-2008 2013-05-13 7 Monolithic CEC Journal of Proteome research 2006, 5, 2001-2008 Monolithic CEC Journal of Proteome research 2006, 5, 2001-2008 Monolithic CEC Journal of Proteome research 2006, 5, 2001-2008 Monolithic CEC • CEC in affinity mode • Immobilize lectins for separation of glycoproteins • For recognition of N-glycans • Lectins: Sugar binding proteins. Sits on the cell surface and is responsible for recognition Electrophoresis 2006, 27, 1020-1030 Monolithic CEC Electrophoresis 2006, 27, 1020-1030 Lectin-based CEC Glycoprotein Monolithic CEC • Modification of the surface chemistry of a monolith • A monolith can be prepared with an ”optimal” morphology/ pore structure • In a second step the surface can be modified to achieve the requested interaction properties and a suitable EOF 2013-05-13 8 Monolithic CEC • Protein separation in monolithic CEC • As with conventional CE proteins tend to adsorb to the surface • One common way to solve the adsorption problems is to use an acidic buffer to ensure that the proteins are positively charged and interact less with the positively charged surface Anal. Chem. 2004, 1044, 3-22