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Micellar Electrokinetic Chromatography (MEKC) 5.2.2019 CE capillary electrophoresis CEC capillary electrokinetic chromatography CZE capillary zone electrophoresis CITP capillary isotachophoresis EKC electrokinetic chromatography CIEF capillary isoelectric focusing MEKC/MECC micellar electrokinetic chromatography CGE capillary gel electrophoresis chromatography MEEKC microemulsion electrokinetic chromatography ACE Affinity capillary electrophoresis Aqueous CE Nonaqueous CZE MEKC/MECC MEEKC CIEF CITP CGE CEC ACE Capillary electromigration techniques: EKC: MEKC,MEEKC CEC CITP CIEF 1 5.2.2019 EKC: MEKC,MEEKC Theory Methods Applications Electrokinetic Chromatography (EKC) A separation technique based on a combination of electrophoresis (buffered electrolyte solutions) and interactions of the analytes with additives (e.g. cyclodextrins, surfactants), which form a dispersed phase moving at a different velocity than the analytes. In order to achieve separation either 1) the analytes or 2) the dispersed phase (=secondary phase) should have charges. Notes: 1. EKC is not totally compatible with the definition of chromatography. 2. The same technique is also called electrokinetic capillary chromatography (ECC) 2 5.2.2019 EKC: additives (e.g. cyclodextrins, surfactants) Sulphonated cyclodextrin. The structure is supposed to be formed in water. Electrokinetic chromatography is a new type of analytical separation method, which utilizes the experimental technique of capillary zone electrophoresis in combination with the principle of chromatography. A theoretical plate number of 250 000 is easily obtained in 10 min. TrAC Trends in Analytical Chemistry 8 (1989) 129-134 3 5.2.2019 The capacity factor (k´) of the analyte (solute, studied compound) can be calculated from retention times k´= (tR—t0) /(1-t R/tc)t0 Sudan III a lysochrome (fat-soluble dye) diazo dye. Cyclodextrin-inducedelectokinetic chromatography (CD-EKC) 5-12.5 mM carboxymethyl cyclodextrin in 25 mM borate, 1 mM 1-bromo-4-naphtalenesulonate (BrN), pH 8-5 EKC: Chiral separation in capillary electrophoresis The chemicals for chiral separation are used as additives in the electrolyte solution. They can be divided to 3 main groups: 1) inclusion systems (e.g. cyclodextrins and crown ethers), 2) enantiomer selective metal-ion complexes (copper(II)-L-histidine or copper(II)-aspartame) and 3) optically active surfactants (e.g. chiral mix-micelles or bile acids). Those enantiomers which interact the most intensively with cyclodetrin cavity, move with the lowers speed. Usually cyclodextrins are added to acidic electrolyte solutions (BGE). But, they can also be added into alkaline electrolyte solutions. Then the enantiomers are anions and their appearent mobility (natural movement) is from detector to capillary inlet. In this case, the enantiomer that interacts the most with cyclodextrin enters first the detector, since its migration (speed) away from the detector is hindered the most. Crown ethers (e.g. 18-crown-6 ether) are suitable for chiral separation of primary amines. 4 5.2.2019 Monomers of chiral selectors: 5 5.2.2019 Chiral selectors: Calixarene Chiral selectors: Vancomycin MW 1449 g/mol Vancomycin is an glycopeptide antibiotic 6 5.2.2019 Chiral separation in CE Epinephrine- catecholamine Choice of chiral selector for enantioseparation by capillary electrophoresis Blanco M. Valverde I. TrAC Trends in Analytical Chemistry, Volume 22, Number 7, July 2003 , pp. 428-439(12) 7 5.2.2019 Micellar Electrokinetic Chromatography (MEKC) A special case of electrokinetic chromatography, in which the secondary phase is a micellar dispersed phase (pseudo stationary phase) in the capillary. Note: The same technique is also called micellar electrokinetic capillary chromatography (MECC) Micellar Electrokinetic Capillary Chromatography Micellar Electrokinetic Capillary Chromatography (MEKC or MECC) is a mode of electrokinetic chromatography in which surfactants are added to the BGE (buffered electrolyte) solution at concentrations that form micelles. The separation principle of MEKC is based on a differential partition between the micelle (a pseudo stationary phase) and the solvent (BGE). This principle can be employed with charged or neutral solutes and may involve stationary or mobile micelles. MEKC has great utility in separating mixtures that contain Analytes moves into a micelle both neutral and ionic species, and has become a valuable method in the separation of very hydrophobic pharmaceuticals from their very polar metabolites. e.g. sodium dodecyl sulfate: polar headgroups, non-polar tails 8 5.2.2019 Sodium dodecyl sulfate celluloses www.scielo.br Bile acid 9 5.2.2019 10 5.2.2019 Micellar electrokinetic capillary chromatography, MEKC Silica capillary + + + + + Electrophoresis + _ EOF + + + + + + Silica capillary 11 5.2.2019 12 5.2.2019 www.particlesciences.com 3 MEKC of mAU corticosteroids 4 Separation of neutral 1 2 5 corticosteroids 1 -1 3 PS, 6 phosphatidyl serine -3 POPC, 1-palmitoyl-2-oleyl- sn-glycero- -5 phosphatidylcholine -7 0 2 4 6 8 10 CHES time (min) phospholipid vesicles (liposomes) in EKC Electrolyte solution: 3 mM 80/20 mol% POPC/PS in 50 mM CHES pH 9 Experimental: uncoated capillary 50/58.5 cm, 245 nm, 20 kV, inj. 5s 50 mbar, 25°C Electrophoresis 2009 13 5.2.2019 Surfactants in MEKC CMC (mM) Aggregation in water number Anionic SDS 8.2 62 (Sodium dodecyl phosphate) Cationic DTAB 14 50 CTAB (Cetyl trimethylammonium 1.3 78 bromide Non Ionic Octylglucoside --- --- n-Dodecyl-b-D-maltoside 0.16 --- Triton X-100 0.24 140 Zwitterionic CHAPS 8 10 CHAPSO 8 11 Bile Salt Cholic acid 14 2 –4 Deoxycholic acid 5 4 – 10 Taurocholic acid 10 –15 4 The most popular surfactant in EKC SDS sodium dodecyl sulphate = ja amount of the analytes (in mole) in micelle and BGE 14 5.2.2019 Environmental Toxicology and Chemistry, 35, pp. 2173–2181, 2016 Environmental Toxicology and Chemistry, 35, pp. 2173–2181, 2016 15 5.2.2019 MEKC (MECC) - principle e s n micelle analyte o Cation p s e r Anion _ _ _ _ _ _ _ _ _ Neutral _ (EOF marker) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + _ - t 1 migration time (s) _ _ _ t eo _ t2 _ _ _ _ _ _ _ _ CZE N e 2 s MEKC n (tr) o N3 p s (tmc) e r N1 (teo) migration time (s) Basic operation: High pH, SDS micelles, Positive polarity pubs.rsc.org 16 5.2.2019 Surfactant Electrophoresis Analytes Electro-osmotic flow Silica capillary Migration window analytes micelle EOF Time 0 teo tm1 tm2 tmc Migration time of micelles (in micellar electrokinetic chromatography), tmc Experimentally determined as the migration time of a compound that is completely retained in the micellar phase CZE ------------------- + _ ------------------- The neutrals move with EOF and are not separated. Separation can only be realised by introduction of a chromatographic partitioning mechanism 17 5.2.2019 MEKC ------------------- CMC + R _ ------------------- Separation in MEKC: Add anionic or cationic surfactant, forming micelles with a hydrophobic core and hydrophilic shell. Sodium dodecylsulfate (SDS) is the most common one. Micellar Electrokinetic Capillary Chromatography • The MEKC surfactants are surface active agents such as soap or synthetic detergents with polar and non-polar regions. • At low concentration, the surfactants are evenly distributed • At high concentration the surfactants form micelles. The most hydrophobic molecules will stay in the hydrophobic region on the surfactant micelle. • Less hydrophobic molecules will partition less strongly into the micelle. • Small polar molecules in the electrolyte move faster than molecules associated with the surfatant micelles. • The voltage causes the negatively charged micelles to flow slower than the bulk flow (endo-osmotic flow). 18 5.2.2019 MEKC ------------------- N CMC 3 + R _ N N1 -------------------2 Neutrals distribute (partition) between hydrophobic micelles (stationary phase) and background electrolyte (mobile phase). But, the ‘stationary’ phase is not stationary (pseudostationary!) MEKC Partitioning of Analytes • More the analyte is incorporated into micelle, the higher the electrophoretic mobility or more likely it will travel less slowly toward the cathode Landers, James P. Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques pg. 109-130. Boca Raton, FL: CRC Press, Taylor & Francis group, 2008. The electropherogram is described in units of Intensity vs. Time (min) 19 5.2.2019 MEKC with anionic micelles Partitioning of neutral and ionic analytes § If the system contains both neutral and ionic species with micellar solution, the elution order is as follows; I.More negatively charged anionic species II.Less negatively charged anionic species III.Neutral species (hydrophilic before hydrophobic) IV.Less positively charged species V.More positively charged species • Why is this? MEKC Partitioning of neutral and ionic analytes • In CE the migration order of ionic species is cations first and anions last, but order is reversed in MEKC with anionic micelles due to the micellar interaction • The negatively charged species are repelled by the anionic micelle while the positively charged species are attracted to the micelle; because the micelle elutes later than the buffer (EOF), so do the positively charged species which are attracted to it • The more negatively charged species - more repelled by micelle than the less negatively charged species (elute earlier) • The more positively charged species - more attracted (strong electrostatic interaction) to the micelle than the less positively charged species (elute later with the micelle) • The neutral species migrate
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