CRNT PRB ANLYT CHEM
CRNT PRB ANLYT CHEM CHEM 520
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This 34 page Class Notes was uploaded by Carmela Kilback on Wednesday September 9, 2015. The Class Notes belongs to CHEM 520 at University of Washington taught by Staff in Fall. Since its upload, it has received 49 views. For similar materials see /class/192582/chem-520-university-of-washington in Chemistry at University of Washington.
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Date Created: 09/09/15
ElectroOsmotic Flow EOF Capillary Electophoresis CE Capillary electrophoresis is electrophoresis performed in a capillary tube or channel Electrophoresis refers to the migration of charged species and neutrals in an electrolyte When an electric current is passed The migration of species is mainly driven by eletroosmotic ow EOF and electrophoretic ow It involves momentum heat and mass transport under the in uence of an electric eld httpWWWceandcecxomceitheoryhtm SiOH 9 SiO39 H pKa lt 2 Electrical Double Layer Potential 1 S tern Layer LIIS Double Layer e quot r a Almaquot Cathode x o o 39 l e 7 39 r35 39v Displacement from surface X G G G o e 3 The rest of G G G G 9 solution dragged Bulk buffer 6 by diffuse layer lt9 9 G lt2 39 Mobile layer Diffuse layer moves at V0S x Stern layer Fixed layer Capillary wall Electrical Double Layer The diffuse layer moves and drags the rest of solution with it at a velocity V OS vOS ocE VOS Z UOSEV when an electric eld is applied Electrical permeability Zeta potential Kw Velocity a 39 y N 0 VOS g E H Electric Field Electroosmotic coef cient W0 05 E 5 u Viscosity NOTE is not a function of x radial position in fact can reach plateau at 14nm Electrical Double Layer Charge density of ions pex 21611 x Valence of each ion Charge of e No of ions of type I per unit volume 0 IliX depends on competition between 2 forces l electrostatic interactions with wall 2 Brownian motion 7 Described by Boltzmann distribution 711 x Z quotwe kT zieVx pex Zzie nwe H 139 Electrical Double Layer DebyeHuckel approximation So eX lX truncate after 1st term of power series expansion 0 pex Z zienw I MH kT n z2e2 x pex ZZZmm ZT the rst term is ZERO because of electrical neutrality zero potential at X 00 712262 x pexZ 10 Electrical Double Layer 52 Cons1der Po1sson s equat1on a amp x 8 52W 2 BC llOatX0 8x2 K W06 l0 atxzoo Solution p606 grzw ge ml Properly Ofeleclrolyle solution Property of capillary wall Electrical Double Layer More on Poisson s equation 2 2 Consider the eld fVrwsurface S Flux J EdS S analogous to Flux I vdS Where V is velocity By divergence theoretil j EdS j V EdV S V 3 3 3 6w 6w Lev K x By 62 K x By 62 Nextcons1derVoE EVyxgtVoEV2w r62 62 62 lwv2w j EdS j Vzde S V K xz Byz 622 Electrical Double Layer More on Poisson s equation Now consider I EdS S Flux JEdS E dS S S Electric field is constant 2 over the surface of a sphere E 47 9 Surface area of a sphere is 4 7272 Coulomb s law describes the electric eld of a point at some distance away from a point charge a A 2 54727quot2 This law can be extended to any amount of charge enclosed with a surface S So the ux becomes Ip V Flux IEa S Q V Gauss s Law S 8 8 Electrical Double Layer V2de EdS dV 1D 2 v2 amp a W I09 w 2 8 6x 8 Derivation of Poisson s Equation E 6Ex Ax 39Fl aE AxAAz EAAz E aE AxAAz E x ax uX ax y xy xax y AZ AX xAX Fluxx aiAMyAz 0x Flux aEy AxAyAz F1uXz EMAyAz y 8y 62 6E aEy aEz F1UXTomz a 6y JFEJAV Total volume element AV F1uXT0ml j VEdV dV V V 8 2 VEamp buwzw lvzwzpe 3 MD 8 8 6x 8 Electrical Double Layer Alternatively the ux can be obtained by Q Flux 8 Q pedV Charge density Fluxsze dV V 8 Flux 2 MS S OR IVoEdV I I Flux is conserved over the control volume by Divergence Thm Electrical Double Layer Qualitative sense of magnitude of 110 By DebyeHiickel approximation Z16 wx lt kT Consider only the case of monovalence ie z1 zOltkTe kTT25 Ce 257 millivolt Physical signi cance of K pg x aft06 1 K391 has unit of length 2 At xK391 we have 6 1x we l quotW Vo e K391 is called the double layer thickness It is 96nm for a 10395 M salt and 3nm for a 10392 M salt Electrical Double Layer Express volume charge density pe as surface charge density 6 Total charge on wall total charge in solution I 0 memx 2 Recall pg x g d 2 I dzwd Effectively it means ie we have 039 8 x 0 dxz 1 lla Oakng 00 dlJ dlJ I D dxzjgdw with BC 308Jd 8 Z 0 0 0 dx dx 71 gt d 3039 K gl0 dx O at x oo IadxeclllJ 3025K110 11ng at x0 From preV1ous page we had peoc 6K2woe Volume charge density pa in terms of surface charge density 0 EOF velocity pro le Navier Stokes Equation at low Re qu 2 VP F For EOF VPO no pressure qu 0CE 3 quu pexE Consider 1D case 3212200 pexE of it BC 0 at xoo vzxO at x0 E5 W0 E8W 122 vZ 0lt1 e39 gt u E810 Egg0 6 I I e39 e39xKil gt 0whenx gt K391 but K 1 m 0nm EOF velocity pro le E8v0 Vz luosE u If we are interested in charge density on surface 5 0 airy0 8 0 0 0 VZZ E E OR luosz u 8K uK uK NOTE VZ has not X dependence v2 2 g and thus the pro le must be at 1 eguOS 61 X 10398 mZV for pH71 Volumetric ow rate of EOF is slow Typical value z 353 nlsec EOF velocity pro le Some intuitions about EOF For a 50 um capillary With water as uid of interest and under an electric eld strength of E30 kVm 1 The volumetric ow rate due to EOF Qos V0s S QOS HOS E s z uOSE z 353XlO12m3sec 2 For Poiseuille Flow 7rAPR4 P 8M To achieve some ow rate 353 X 103912 m3sec 23x104 NmZm34psim 30kVm 34 psim for 50 um capillary Capillary Electrophoresis In capillary electrophoresis CE EOF is uid ow and has nothing to do with separation of analytes Now consider electrophoresis ie movement of charged species in solution under the in uence of an electric eld Nothing to do with EOF A charged species under in uence of an electric eld experience a force FE e E Charge Charge of e In previous lectures an object moving in solution experience a drag force and for a spherical object it is F1376 wa viscosity radius velocity Capillary Electrophoresis At steady state it is reached in about 10394 sec after the eld is turned on Consider FEFD zeE or zeE6nan 3 WWW Velocity of an ion i or a charged species v zie F 1 67wri39 gt nielectrophoretic mobility zie charge NOTE W mass CE separation is achieved by change in chargemass ratio Capillary Electrophoresis Total mobility or velocity is Apparent mobility Electrophoertic electroosmotic 26 we MW 1 i os i Some practical issues 1 Injection ILI Pressure hydrodynamic injection Electrokinetic EOF caused by Q difference in charge Separation 3 Detection Detector Capillary Electrophoresis 39 7 rz4 777907E s zsvwW A B A c BA FIHDI ESEEI IEE atl 25039 2DD39 150 mut 50 Capillary Electrophoresis I 39 I quot l l 39 Iquot 3 E E I I I J E 3 3 I m a E I I 3 a I II I 3 a l I I m I 5 I 5 I H 39 I i I I D I39 quot I 7 II 39 I I I I39 39 T III 39 I T I39 II I i 1l 1 39 IJI Lc ml qu J u LIL 1Lf 2 l1 2 5 3 III 3 51 4 4 5 Tlme ulna Injactlun a Capillary Electrophoresis Capillary Electrophoresis 5mm Capillary Electrophoresis Capi11ary Zone Electrophoresis Mice11ar Electrokinetic Chromatograpy Capi11ary Electrochromatography Isoelectric Focusing Capillary Gel Electrophoresis CEOptical e g Absorbance LIF CEElectrochemical Detection CEMass Spec CENMR CEBiosensors Capillary Electrophoresis 10 ls O X nm 20 Practical Aspects of CE What diameter capillary What buffer What voltage What length capillary v w J 3 2 392 9 x z 1 k J 40 so I20 dum FIGURE 8 Separaiion ef ciency as a function of column inner diameicr From Lukacs K D and Jorgenson L W 1 High Res71m Chromatogr Clxmmalogr CrmmuuL 8 407 1985 With permission FIGURE 9 Separation ef ciency as a function of column length Fmin Lukacs K D and lorgcnson J W I High RemML Chromamgn Chromamgr Commun 8 407 I985 With permission Dispersion Mechanisms of theoreticaI pIate Length of separation channel N 3 2 OTotal 2 2 N 2 2 2 aTotal Z 0 j N aThermal UHydrOdynamic UDiffusion j 2 aDl39 usion 2 D t Real Protein Separation Interactions with wall mixture electrolytes Peak broadening asymmetry rreproducibe migration times Low mass recovery irreversible adsorption Additives to reduce adsorption Additive Types Theory Examples Neutral Polymers Ionic Salts Zvvitterions Amine Modifiers Surfactants Other IonPairing Agents Denaturing Agents Adsorb at the interface to increase the local viscosity in the double layer and masking silanol groups on the surface Silanol groups on the wall are considered as cation exchange site which can be suppressed by increasingthe ionic strength of solution aminogroup hydrogenbond to non derivatized silanol groups and reduces adsorption Surfactant can adsorb on wall OR bind to protein selectively Also improves dispersion Ionpair formation of protein can reduce net positive charge on protein and reduce Coulombic interaction with wall promote deaggregation and disruption of hydrophobic noncovalent interactions Methyl celluloses Polyvinyl alcohol Dextran Polyethylene oxide Polyethylene glycol Cs larger metal ion the better Trimethylammonium Propylsulfonate AccuPure Z1 methyl Trimethylglycine betaine 14 diaminobutane putrescine Morpholine Hexamethonium Bromide Polyethyleneimine PEI Fluorosurfactant fluorad F01 34 Oetyl trimethylammonium bromide Brij 35 Tween 2080 Triton X 100 sodium salt of Phytic acid alkylsulfonates tetraalkylammonium salts Urea high concentration Formic ac39 Trifluoroacetic Acid Good review article Corradini D J Chrom B 699 1997221256 Effect of Additives on EOF o At acidic pH ionization of silanol is suppressec and zetapotential approaches zero Ionic Strength Adsorption of CounterIons Can reduce zeta potential or even change it to positive and reverse the direction of EOF Viscosity and Dielectric Constant a iquot 7T E vs C I 2 l 5 l quot v 41 i j r 21l 1 I l o z a 2 4 s 39TKlT39 72 1214 Figure 1 Eltact of pH on the etactroosmottc mobility In iused silica capillaries Ionic strength at 001 The sulld line represents data obtalned on golng from alkaline to acidic conditions while the dashed line represents data with Increasing pH Error bars Indicate the standard deviatlon n 418 W J L Lambert D Middleton Analy Chem 62 1990 15851587 Joule Heating Volumetric heating rate 2 Q 2 0E aao1altT Iggt Increase in T RUNAWAY CONDITIQ liase M 73T 5 btFadl392331 ONARCIT39G quot quotquot quot quotty J Draws more current Strategies to deal with Joule heating Convection cooling thermostating 360 mums Reduce bufferion concentration Organiclow conductivity g 340 electrolyte buffer PET Keeping channel crosssectional 320 c area small 300 I 6393 Different materials a 05 1 Thermal Conductivity k WEm K Increase surface to volume rat1o Lim et at J cmom A in press Other effects Isotachophoresis Discontinuous electrolyte zones before and after analyte injection to setup a voltage gradient gt component zones move a the same velocity one after another Isoelectric Focusing using a pH gradient to con ne moving protein into a zone Where it becomes neutrally charged mmuclnr clrcH mm T and mmnlc mixmrc An in jcmml in human er Sow murmur pl nfilc alum me cap hty m vctnm he mluly 1 starts a mum m scpnmum E mums and n Mandy71m I lnrm ivl swarm of hn separmian by isoelmnr fncusing smimlc pmm39n I J migrmts almp me linmr H mm m nrmcd m m capillary umi n madlux llu pmmnn 0 Where m msullmg chums it c 7Imrllnscmrnm 1 mm new Remove Salt if Possible Salt in analyte can change axial A electric eld distribution g ltbuffer ionic strength will 4 cause sample stacking a preconcentration g 1 sharpens peak g EL gtbufferwi11 cause peak 1 c broadening Salt can be removed by E PAL P ultra ltration dialysis 4 D centrifugation g i 1m mm FIGURE 5 Ellen of mm m the sampl am an pm height and peak shape of browtwc peptides my 30 mM sodium chlunde 10s injcuion m so am indium chloride inf quotwanquot 0 100 mM sodium chlorwr 107 ammoquot n mo quotm sodium chloride 203 ille CHul39l hum Particular issues with chipbased CE Voltage balancing Turn Effect Y Figure 1 Schematic repmentalion of 2 turn and straight channel segment The turn radius and channel width are assumed 11 hi ccunslanl A sample band lravels along the straight channel seamen al a fixed speed U Grif ths et al Anal Chem 74 2002 2690 2697