CAPILLARY ELECTROPHORESIS

CAPILLARY ELECTROPHORESIS

1. ELECTROPHORESIS: HOW DOES IT WORK?

   • differential migration of charged sp. in applied DC
   • rate depends on CHARGED:SIZE RATIO
   • migration rate ν= μeE
     • μe = electrophoretic mobility  α = ionic charge 1/α = frictional factors (size & shape, viscosity) E = applied field strength

2. electric field strength related to magnitude and length over which

3. migration rate : ν= μe x V/L

   • V: voltage L: length of slab/capillary

4. Plate #: N= μeV/2D

   • D: diffusion coefficient of solute

5. RESISTANCE (joule heating)

   • V = I x R
     • I = current R = resistance
     • if use gel - produce heat. limit V applied for slab elect. ~500v
   • Capillary typically 10-100μm ID and 30-100cm long (large surface:volume ratio = better cooling)
     • P = I^2/R
       • P = power dissipation = amt heat produced long capillary and small cross sectional area = high R
   • voltages we can use in cap.ele. typical applied voltage 10-25kV = E of 100-400 Vcm-1 typical N = 100,000 –200,000(HPLC 5,000 –20,000)

6. ELECTROOSMOTIC FLOW

   • high V applied - buffer-filled-Si cap. - bulk liq twds CATHODE
   • pressure-induced
   • electroosmotic flow
   • EO Flow generally > electrophoretic migratn
   • buffer = mob. phase
   • EO flow = mob. phase pump
   • all analytes are swept through column regarding the charged (CATION - NEUTRAL - ANION)
   • electroosmotic flow velocity ν= μeoE
     • μeo= electroosmotic mobility
   • ion velocityν= (μe+ μeo)E
   • migration rate tm= l/ (μe+ μeo)E = IL(μe+ μeo)V
     • L= Length of capillary l = length to detector
     • N = 16(tm/W)^2

7. CE HARDWARE

   • Small vol- cap (4-5μL), injector/detector < 10nL (the result not concentrated)
   • Cap: fused silica, external coating of polyimide (flexibility & durability)
   • can reverse the polarity: anions elute first
   • Sample Injection
     • 5 -50 nL
     • tips that can sample directly from cell/cell substructure
     • Can be automated
       • electrokinetic
         • Voltage--&gt; sample flow with EO flow + ion movement Injects larger amts of more mobile ions
       • pressure
   • Detectors (= HPLC)
     • Optical
       • Problem with low volumes = short pathlength = low absorbance or fluorescence
       • modify cell to increase the pathlength
         • Z-cell
           • - path length 10 x the cap. diameter - increase path length -&gt; decreases in peak efficiency = add spherical ball lenses btwn source-zcell-detector.&nbsp; - Increase sensitivity
         • Bubble cell
           • bubble formed near the end of the capillary
         • Multireflection cell
           • a reflective coating of silver is deposited on the end of the capillary
     • Adsorption & fluoro. (limits in conc terms, but equal or better in mass detection limits)
   • Indirect detection
     • for compounds that don't have suitable chromophore/molar absorptivity - add ionic chromophore in the buffer, see any reduction in that signal
     • Electrochemical
       • conductivity & amperometry
     • MS
       • small volume of flow rate (1uL/min) allow to couple w MS
       • common use of electrospray Ionisation (ESI)
       • use volatile buffer
       • CE/MS - use to detect proteins, DNA fragments n peptides. (large biomolecules)

8. APPLICATIONS OF CE

   • Capillary Zone Electrophoresis
     • small ions
       • cation separations- wall untreated, analytes flow with EO flow twrds cathode
       • Anions separation- wall treated, reversed EO flow twrds anode
     • molecular sp.
       • ions or non-ion (derivatization) - herbicides, persticides, proteins, aa. etc
     • - buffer composition is constant - ionic components migrate into zones (complete resolved zone have buffer between zones)
   • Capillary Gel Electrophoresis
     • performed in porous gel polymer matrix w a buffer mixture that fills the pores of the gel
     • types of gel : polyacrylamide polymer, agarose, methyl cellulose etc.
   • Capillary Isoelectric Focusing
     • separate amphiprotic sp. (aa, proteins - have weak COOH and AMINE gp.)
       • amphiprotic - sp in a soln is capable to donate or accept a proton.
     • buffer mixture that continuously varies in pH along its length

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