CAPILLARY ELECTROPHORESIS

1. differential migration of charged sp. in applied DC
2. rate depends on CHARGED:SIZE RATIO
3. migration rate ν= μeE

   Nota:

   • μe = electrophoretic mobility  α = ionic charge 1/α = frictional factors (size & shape, viscosity) E = applied field strength

Nota:

• V: voltage L: length of slab/capillary

Nota:

• D: diffusion coefficient of solute

1. V = I x R

   Nota:

   • I = current R = resistance
   1. if use gel - produce heat. limit V applied for slab elect. ~500v
2. Capillary typically 10-100μm ID and 30-100cm long (large surface:volume ratio = better cooling)
   1. P = I^2/R

      Nota:

      • P = power dissipation = amt heat produced long capillary and small cross sectional area = high R
3. 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)

1. high V applied - buffer-filled-Si cap. - bulk liq twds CATHODE
2. pressure-induced
3. electroosmotic flow
4. EO Flow generally > electrophoretic migratn
5. buffer = mob. phase
6. EO flow = mob. phase pump
7. all analytes are swept through column regarding the charged (CATION - NEUTRAL - ANION)
8. electroosmotic flow velocity ν= μeoE

   Nota:

   • μeo= electroosmotic mobility
9. ion velocityν= (μe+ μeo)E
10. migration rate tm= l/ (μe+ μeo)E = IL(μe+ μeo)V

    Nota:

    • L= Length of capillary l = length to detector
    1. N = 16(tm/W)^2

1. Small vol- cap (4-5μL), injector/detector < 10nL (the result not concentrated)
2. Cap: fused silica, external coating of polyimide (flexibility & durability)
3. can reverse the polarity: anions elute first
4. Sample Injection
   1. 5 -50 nL
   2. tips that can sample directly from cell/cell substructure
   3. Can be automated
      1. electrokinetic

         Nota:

         • Voltage--&gt; sample flow with EO flow + ion movement Injects larger amts of more mobile ions
      2. pressure
5. Detectors (= HPLC)
   1. Optical
      1. Problem with low volumes = short pathlength = low absorbance or fluorescence
      2. modify cell to increase the pathlength
         1. Z-cell

            Nota:

            • - path length 10 x the cap. diameter - increase path length -&gt; decreases in peak efficiency = add spherical ball lenses btwn source-zcell-detector.  - Increase sensitivity
         2. Bubble cell

            Nota:

            • bubble formed near the end of the capillary
         3. Multireflection cell

            Nota:

            • a reflective coating of silver is deposited on the end of the capillary
   2. Adsorption & fluoro. (limits in conc terms, but equal or better in mass detection limits)
6. Indirect detection
   1. for compounds that don't have suitable chromophore/molar absorptivity - add ionic chromophore in the buffer, see any reduction in that signal
   2. Electrochemical
      1. conductivity & amperometry
   3. MS
      1. small volume of flow rate (1uL/min) allow to couple w MS
      2. common use of electrospray Ionisation (ESI)
      3. use volatile buffer
      4. CE/MS - use to detect proteins, DNA fragments n peptides. (large biomolecules)

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

      Nota:

      • amphiprotic - sp in a soln is capable to donate or accept a proton.
   2. buffer mixture that continuously varies in pH along its length