CAPILLARY ELECTROPHORESIS

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undergraduate degree analytical chemistry Mind Map on CAPILLARY ELECTROPHORESIS, created by chaz_id89 on 28/10/2013.
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Mind Map by chaz_id89, updated more than 1 year ago
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Resource summary

CAPILLARY ELECTROPHORESIS
  1. ELECTROPHORESIS: HOW DOES IT WORK?
    1. differential migration of charged sp. in applied DC
      1. rate depends on CHARGED:SIZE RATIO
        1. migration rate ν= μeE

          Annotations:

          • 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
          1. migration rate : ν= μe x V/L

            Annotations:

            • V: voltage L: length of slab/capillary
            1. Plate #: N= μeV/2D

              Annotations:

              • D: diffusion coefficient of solute
              1. RESISTANCE (joule heating)
                1. V = I x R

                  Annotations:

                  • 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

                      Annotations:

                      • P = power dissipation = amt heat produced long capillary and small cross sectional area = high R
                    2. 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)
                    3. ELECTROOSMOTIC FLOW
                      1. high V applied - buffer-filled-Si cap. - bulk liq twds CATHODE
                        1. pressure-induced
                          1. electroosmotic flow
                            1. EO Flow generally > electrophoretic migratn
                              1. buffer = mob. phase
                                1. EO flow = mob. phase pump
                                  1. all analytes are swept through column regarding the charged (CATION - NEUTRAL - ANION)
                                    1. electroosmotic flow velocity ν= μeoE

                                      Annotations:

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

                                          Annotations:

                                          • L= Length of capillary l = length to detector
                                          1. N = 16(tm/W)^2
                                        2. CE HARDWARE
                                          1. Small vol- cap (4-5μL), injector/detector < 10nL (the result not concentrated)
                                            1. Cap: fused silica, external coating of polyimide (flexibility & durability)
                                              1. can reverse the polarity: anions elute first
                                                1. Sample Injection
                                                  1. 5 -50 nL
                                                    1. tips that can sample directly from cell/cell substructure
                                                      1. Can be automated
                                                        1. electrokinetic

                                                          Annotations:

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

                                                                  Annotations:

                                                                  • - 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
                                                                  1. Bubble cell

                                                                    Annotations:

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

                                                                      Annotations:

                                                                      • 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)
                                                                  3. 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
                                                                      1. Electrochemical
                                                                        1. conductivity & amperometry
                                                                        2. MS
                                                                          1. small volume of flow rate (1uL/min) allow to couple w MS
                                                                            1. common use of electrospray Ionisation (ESI)
                                                                              1. use volatile buffer
                                                                                1. CE/MS - use to detect proteins, DNA fragments n peptides. (large biomolecules)
                                                                            2. APPLICATIONS OF CE
                                                                              1. Capillary Zone Electrophoresis
                                                                                1. small ions
                                                                                  1. cation separations- wall untreated, analytes flow with EO flow twrds cathode
                                                                                    1. Anions separation- wall treated, reversed EO flow twrds anode
                                                                                    2. molecular sp.
                                                                                      1. ions or non-ion (derivatization) - herbicides, persticides, proteins, aa. etc
                                                                                      2. - buffer composition is constant - ionic components migrate into zones (complete resolved zone have buffer between zones)
                                                                                      3. Capillary Gel Electrophoresis
                                                                                        1. performed in porous gel polymer matrix w a buffer mixture that fills the pores of the gel
                                                                                          1. types of gel : polyacrylamide polymer, agarose, methyl cellulose etc.
                                                                                          2. Capillary Isoelectric Focusing
                                                                                            1. separate amphiprotic sp. (aa, proteins - have weak COOH and AMINE gp.)

                                                                                              Annotations:

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