Basic CE instrument: High voltage power, capillary, 2 buffer reservoirs, detector → simple, low cost, easily miniaturized. Inside the capillary: 1) Covered deprotonated (SiO - ) at pH>2(or adsorption ions from buffer) ; 2)Attracts cations inside capillary. 3) Stern double-layer or diffuse double layer (more tightly held fixed layer; not tightly mobile layer). 4) Stern plane: between fixed and mobile layer 5) Slipping plane: in mobile layer separates mobile fluid from fluid attached to surface. Electroosmosis: Under electric field, excess cations mobile layer pushed towards anode→ Net flow as solvated cations drag along solution (EOF) Zeta potential: ζ = 4πδe/ε - Zeta potential [potential difference at interface between fixed and mobile layers (relative to the bulk liquid)] proportional to EOF. - ζ =Zeta potential ε= dielectric constant , δ= thickness diffuse layer, e= charge per unit surface area Electroosmotic vs hydrodynamic: 1) EOF flow profile: no pressure drop, flow velocity uniform across capillary. no band broadening → sharp peaks and higher resolution (N) ,better separation efficiencies 2) Hydrodynamic flow profile: frictional forces at column wall → pressure drop across column. Electroosmotic mobility: μ EOF = ζε / 4πη Mobility depends buffer, independent electric field. μ EOF = cm 2 V -1 S -1 , ζ= cm 3 V -1 S -1 , η= cm Electroosmotic velocity: v EOF = ζεE/4πη Velocity depends electric field( E=V), buffer pH and concentration(higher, higher viscosity) organic solvent , surface of capillary. Higher E, sharper peaks, faster separation. Joule heating: resistance solution, reduced separation efficiency. Temp not controlled lower viscosity buffer heating dissipation: 1) P/L = χCr 2 V /L 2 =power/length, r 2 =capillary area, smaller area better) C= buffer concentration χ= molar conductance solution . 2) smaller capillaries, heat dissipated due large surface area volume ratio (capillary internal surface area = 2πrL) Control EOF by chemical modification of capillary inner surface: 1)Chemicals bonded capillary wall or dissolved in buffer (dynamic coating- temporary) → lower EOF due to shielding charge on capillary,increased viscosity. 2) For dynamic coating, surfactants and hydrophilic polymers, e.g (CTAB, cationic surfactant). Measurement EOF: (1) Injection neutral marker (nm): v EOF = I e / t nm I e = effective length(cm), Criteria neutral marker: uncharged at pH used, detectable, no interaction capillary wall, soluble in buffer used, (2)Gravimetric method: weighing buffer flushed out of capillary given period time → measure additional mass, accurate balance needed, prevent evaporation. Reverse EOF: anions analyzed, reverse direction EOF, reverse polarity voltage → anions migrate faster and separation time reduced. How to reverse EOF: 1) quanternary amines (e.g. CTAB, CTAC, TTAB, TTAC), 2) proteins (e.g. alpha-lactalbumin) 3) Negative SiO - capillary wall attract positively charged quarternary amines 4) Hydrophobic ends quarternary amines associate other quarternary ends 5) exposed positive charges attract negatively charged anions,migration anode Hysteresis: 1) starting acidic pH ,H + removed pH increase. Starting high pH, H + added pH decrease. 2) Competition between anionic enrichment due adsorption and anionic exclusion due to surface potential repulsion. To minimize effect of hysteresis of EOF: - New capillary prior first use: (1) flush MeOH, water, NaOH, (2) flush buffer. Conditioning between runs 3) try not expose capillary to pH extremes, (4) flush neutrals or basic buffer with NaOH (5) flush acidic buffers with phosphoric acid Effect EOF on resolution: Increasing EOF decrease time, higher separation efficiencies , too fast EOF , low resolution. Electrophoretic mobility: μ ep = q/(6πηr) q= charge solute, η =viscosity, r= solute radius greater (q/r), higher μep Electrophoretic velocity: v ep = μ ep E Total velocity analyte (Observed): v obs = v ep + v eof μ obs = μ ep + μ eof V ep = I e /t m – I e /t nm μ ep = (I e /t m – I e /t nm )(L/V) t m: time migrate from injection end to detector , L=capillary length, E=V/L (Vcm -1 ) t m = I e L / [(μ ep + μ eof )V] Migration velocity: 1) cations flow cathode +ve μep 2) anions tend to anode -Ve μep but EOF>ep, flow cathode. Resolution: H = A + B/u + Cu. In CE, no A (multipath) as tube open, no C(mass transfer) since no stationary phase, B(longitudinal diffusion) remains, good resolution, high efficiency. N = L/H H = B/v = 2D/v, B,D=m 2 s -1 v = μE = μV/L N = L / [2D/( μV/L)] = μV/2D Efficiency: number of theoretical plates (N). N = 16 (t m /w) 2 N = 5.54 (t m /w 1/2 ) 2 Peak variance: σ 2 = 2Dt σ 2 = 2DI e L / [(μ ep + μ eof )V] N = L 2 / σ 2 N = (μ ep + μ eof )V / (2D) Selectivity: α = (t 2 – t nm ) / (t 1 – t nm ). Resolution: R = ∆t/w = (t 2 – t 1 ) / [(w 1 + w 2 ) / 2] = 0.25 N 1/2 (∆v/v ave ) = 0.25[(μ ep +μ eof )v/2D] 1/2 [(μ - μ 2 )/(μ ave +μ eof )] = 0.177 (μ 1 – μ 2 ) {V/[(μ Ave + μ EOF )D]} 1/2 sharper peak and longer migration time, higher efficiency(provided solute zone not diffuse more) Types molecules separated by CE: proteins, peptides, amino acids, nucleic acids, inorganic ions, organic bases, organic acids, whole cells. Applications of CE: simultaneous and fast detection toxic metals and bacteria (advantages: current methods require several days, false positives common, techniques like ELISA, PCR, hybridisation are specific to certain microorganisms, readily miniaturized). Advantages of CE: new selectivity an alternative to HPLC, easy and predictable selectivity, high separation efficiency (10 5 to 10 6 theoretical plates), small sample sizes (1-10μl), fast separations (1- 45 min), can be readily automated, quantitation (linear), easily coupled to MS, different modes. Disadvantages: cannot preparative scale separations – small capillary, low concentrations , large volumes difficult, sticky compounds, species difficult dissolve in aqueous buffer, reproducibility problems – handling very small amounts. Capillary zone: 1) Separation charged analytes based differences electrophoretic mobility 2) Positive peaks: UV absorbing ions displaced detection zone 3) Negative peaks: UV absorbing ions concentrated detection zone 4) Advantages: faster detection than ion chromatography, able to measure both ions. Capillary gel electrophoresis: 1) Separation differences in solute size as migrate through pores gel-filled capillary → ‘molecular sieving’ 2) Gels anti-convective minimize peak broadening due diffusion, prevent solute adsorption capillary and eliminate EOF (EOF not favorable ,may destroy gel) 3) Gels temperature stability and suitable pore sizes 4) Extremely high efficiencies 5) Pore size polyacrylamide gel determined total gel (acrylamide) concentration (%T = [bis+acryl]/V) and concentration cross linking agent bisacrylamide (%C = [bis/(bis+acryl)]) 6)Increase %T decreases pore size → suitable separating smaller proteins and DNA , 5% C smallest pore sizes for all %T 7) Polymer solutions forming macromolecules can used as size sieving media in CE,replaceable gels (dilute polymer solutions, if not viscous and band broadening) 8) Slab gel vs CGE:CGE faster (mins vs hours), peaks more quantitative than bands in slab gels 9) Log (MW) vs migration time gives linear plot, normalized (divided by reference tm, neutral marker or first peak) 10) SDS denatures proteins and confers negative charges to polypeptide in proportion to length 11)To overcome limit DNA size: pulsed electric field and voltage gradient (increase voltage separation larger DNA) Micellar electrokinetic chromatography: 1) V x = V eo + [k * /(1+k * )]/V mc k * phase capacity ratio = moles in micelle / moles in buffer 2) Capacity factor: k’ = (t m – t o ) / [t 0 (1-t m /t mc )] t o= time complete insoluble micelles 3) Resolution: R = 0.25 N 1/2 [(s-1)/s] [k 2 ’/(k 2 ’+1)] [(1- (t 0 /t mc ))/(1+(t 0 /t mc )k ’)] s separation factor = k2’/k1’ 4) separation partitioning between (organic) micellar phase and (aqueous) solution phase 5) Separation both neutral and charged species 6) Micelles form in solution when surfactant is added in concentration above critical micelle concentration (CMC), aggregates surfactant molecules lifetimes less ~10μs 7) Most commonly used SDS (CMC=0.008M) anionic surfactant 8) Anionic micelles move slower than neutral and positive charged ions. 8) very hydrophobic molecules spend all time inside micelles and migrate slower than neutral. Capillary electrochromatography: 1) Separation based distribution equilibria, utilizes a packed or coated capillary 2)Packing materials may enhance EOF due to charges on surfaces 3) Enhanced Veo since particle has electrical double layer 4) interaction with stationary phase(retains strongly, migrates slower), analytes charged or neutral. Capillary isoelectric focusing (CIEF): 1) Separation amphoteric species based differences isoelectric points (for proteins seperation) 2) solution forms pH gradient inside capillary (mixture ampholytes), anodic end in acidic solution (anolyte) cathodic end in basic solution (catholyte) 3) In electric field, charged proteins migrate until reside in region pH electrically neutral and stop migrating, zones focused until steady state. 4) After focusing, zones mobilized from capillary hydrodynamically or adding salt to anolyte (chemical mobilization) 5) Detection without mobilization: by scanning but not preferable since removal polymer coating makes gel fragile. 6) Cathodic mobilization: proteins move towards cathode, Cl - added catholyte, higher pH shift. 7) Anodic mobilization: proteins move towards anode, Na + added to anolyte, lower pH shift. Capillary isotachophoresis: 1) Performed in discontinuous buffer (different buffers 2 ends) 2) leading electrolyte (low electric field, slower) , termination electrolyte( high electric field, faster) 3) graph: series of steps, each step an analyte zone → zone length proportional amount sample 4) On-column preconcentration: inject large plug, concentrate small plug, higher sensitivity. - Injection step: sample injected into column filled leading electrolyte - Focusing step: terminating electrolyte placed into reservoir, voltage applied - Separation step: reservoir replaced leading electrolyte and separation voltage applied - Qualitative: RSH, ratio of the step height of the analyte to that of the terminator 5) iso(same) + tacho(velocity)+ phoresis Inclusion complexes with cyclodextrins : 1) Cyclic oligosaccharide molecules built of D- (+)- glucopyranose units bonded via α- (1,4) linkages 2) Slightly soluble in water but cavities are non-polar which form inclusion complexes 3) Capacity for highly hydrophobic solute, k’ = n mc /n CD = KV mc / V CD where n = total amounts solute, K =distribution coefficient between micelles and CD V=volume mc=micelles 4) Ratio solute incorporated micelle depends on hydrophobicity but inclusion complex formation depends matching solute molecular size with cavity diameter CD 5) α,β,γ consists of 5,7,8 glycopyranose units and diameters of 0.47-0.52, 0.60-0.64, 0.75-0.83 nm 6) analyte higher affinity CD faster, follow EOF and analyte higher affinity micelles slower attract anode Chiral separation : 1) Advantage: high resolution, low cost, simple & versatile 2) Disadvantages: Microscale, low UV sensitivity 3) capillary electrophoresis: CD derivatives (CDen, THCMH) , carbohydrates (HS-Cys), ionic liquids (organic salts with mp<100 0 C, soluble in both polar and non-polar)leucinol and pyrrolidinol derived 4) Capillary electrochromatography: silica monoliths, polymer monoliths, particle-fixed Effect of adsorption: H ad =2C (1-C) [ μ ep + μ eo ]Et ad 1) H ad is contribution adsorption to plate height, C fractional concentration free solute, t ad mean residence time adsorbed solutes 2) Results peak distortion , irreproducibility (large molecules such as proteins are vulnerable) Effect of mobility difference 1) Fronting: solute anion mobility higher than buffer 2) Tailing: solute anion mobility lower than buffer 3) match buffer and analyte mobility for symmetrical peak Sample introduction : 1) On-column injection 2) Injection volumes: 1- 50nL (should be small to minimize zone spreading (loss efficiency, resolution except on-column pre- concentration dilute sample Hydrodynamic/hydrostatic injection: 1) performed by gravity, pressure or vacuum suction, 2) advantage no sample bias Gravity flow injection (siphoning): 1) Sample vial, capillary raised distance, H, above destination vial, sample solution siphon into capillary. 2) Volume sample injected depends on H, length time vial is raised, sample solution viscosity and capillary dimensions 3 ) Volume injected: q = ρ g π r 4 ∆ ht i /(8 η L)=B ∆ ht i q = volume (cm 3 ), p= density (gcm -3 ), B= cm 2 s -1 t i= injection time 4) Amount injected: w = B ∆ ht i C 5) disadvantage= limited by height Pressure or vacuum injection: 1)Amount injected: π r 4 ∆ PCt I / (8 η L) = B’ ∆ Pt i C 2 )Correction capillary travel time between reservoirs: w tot = w i + 2w T = ∆ hC(t i +2t T )B , w i =amount injected during actual injection, w T = amount injected during travel time either before or after injection t T = travel time, t i= actual injection time. 3) disadvantage = viscous sample cannot Pressure injection: 1) Sample vial pressurized, forcing sample into capillary. 2) Volume sample injected depends on magnitude, duration pressure applied, sample solution viscosity and capillary dimensions Vacuum injection: 1) vacuum applied to destination vial, pulling sample into capillary. Volume sample injected depends on magnitude, duration vacuum applied, sample viscosity, capillary dimensions 2) vacuum limited compare pressure Electrokinetic / electromigration injection: 1) Applying voltage while capillary inserted into sample vial 2) Capillary and anode placed sample vial, voltage applied causes sample migrate into capillary due to μeof and μep 3) Amount sample injected depends on electrophoretic mobility , EOF flow rate, voltage, capillary dimensions ,solute concentrations 4) Length of sample zone: ℓ = (v ep + v eo ) t i = ( μ eof + μ ep ) V i t i / L , ℓ = length zone , t i= injection time 5) Amount of sample injected: w = π r 2 ℓ C = π r 2 C ( μ eof + μ ep ) V i t i / L 6) For best resolution and peak shape, concentration injected sample less than concentration buffer, i.e. dilute samples 7) Amount sample injected depends migration velocity (sample bias due to effect applied voltage) Sample bias: 1) Due differences in mobility – faster migrating ions injected more 2) Due difference in conductivities between sample solution and buffer 3) For same sample, different species: w (1) / w (2) = b C (1) / C (2) b = ( μ (1) + μ eo ) / ( μ (2) + μ eo ) b = t m,2 / t m,1 t m,i = z / v tot,I = z / [( μ i + μ eo ) E] , z= distance injector to detector, v tot,I = total velocity 4) For 2 different samples: w (i;s1) / w (i;s2) = v tot(i;s1) C (i;s1) / v tot(i;s2) C (i;s2) v tot = total volume Field-Amplified sample injection: 1) v ep increases at high field and analytes reaches boundary between sample zone and background buffer (stacking → enhance efficiency due preconcentrate samples) 2) plug sample in buffer lower ionic strength injected column filled with buffer higher ionic strength, ions migrate rapidly to boundary between sample and buffers under voltage, resulting stacking sample plug 3) lower ionic strength= lower conductivity= higher field 4) Conventional amount injected = ( μ eof + μ ep )AC i Et, A= area , plug length = ( μ eof + μ ep )Et 5) FASI amount injected = γ ( μ eof + μ ep ) AC i Et, plug length = ( μ eof + μ ep / γ )Et , γ = ratio concentrations background buffer to sample buffer 6) larger γ, more sample injected and plug length shorter when FASI used. 7) samples prepared in low conductivity solution (water) 8) Injecting plug water before sample introduction to enhance field strength 9) To pre- concentrate negative ions: FASI polarity switching Advantages stacking: 1) higher sensitivity 2) sharper peaks, better resolution. Impact of injection size (100nL vs 5nL): LOD improved by factor of 13, some injection mediated band broadening can occur Stacking of cations in a low pH buffer: 1) When positively charged solutes migrate out of injection zone and encounter BGE, field strength drops and vep slows down 2) Solutes at middle to rear of injection still exposed high field strength and continue move forward full speed 3) Ions in injection band continue narrow until all migrated into BGE Stacking of anions in a high-pH buffer: Negatively charged anions migrate towards anode and cross boundary between injection solution and BGE at rear of injection zone Anti-stacking: 1) When sample with high ionic strength relative BGE injected, electric field over injection zone declines 2)When positive ion electrophoreses into BGE, exposed to high field strength over BGE 3) cation accelerates away from those cations still remaining in injection zone → anti-stacking 4) band broadening occurs pH mediated stacking : 1) Upon application voltage, negatively charged peptides(in high pH) migrate toward anode 2) peptides at rear band enter acidic buffer (low pH),