Summary of analytical separation methods part 4. Will be taught in the 3rd bachelor, 1st semester, UGent, in chemistry. There may be minor errors, feel free to send a message because I still have my own summary with extra notes on it.
H4 Electrophoresis (electrodriven
separation techniques)
4.1 Introduction: from gel to capillary electrophoresis
another technique just like chromatography to separate molecules from each other
Electrophoresis (Tiselius,1837) = differential
movement of charged species (ion) by attraction of
repulsion in an electric field
the basic problem (in free solution):
⇒
limited by thermal diffusion & convection (
the basic problem excessive band broadening) ⇒ opl: performed in
anti-convective (stabilized) media (polyacrylamide,
agarose gels)
→ separation altered by addition of gel, as solutes
are now separated by q/m in combination with
sieving mech due to restriction of the gel
→ gel in slab or tube format for size dependent
separation (macromolecules, proteins; DNA analysis)
drawbacks
classic solution: eg DNA fragments - loaded (phosphate
backbone) ⇒ voltage applied ⇒
move to anode, longer chains → time-consuming technique
will face more resistance & will move slower ⇒ separation → impossible to automate
based on length
→ after analysis: colour zones to make visible (less
gel functions qualitative interpretation)
1. counteract peak broadening convection and → difficult to obtain homogeneously thick gel
diffusion processes → puur accuracy and precision (peak I varies to
2. allow additional size based separation of much)
molecules
alternative to slab gel: narrow-bore open tubes /
capillaries
, these capillaries limit convention & diffusion within → often as confirmatory for HPLC
internal dimensions, and are ± anti-convective
because low conductance (narrow); small heat
produce
→ 25-75 μmdia + filled with buffer
→ high electric resistance enable application of very
high electric field with minimal heat generation
4.2 Practical aspects of CE
Simple instrumentation
Easily automated
High efficiencies
Short analysis times
Small sample volumes required (±nL)( ⇒ no
syringe injection)
⇒ ends of narrow-bore capillary placed in buffer vials
Consumes limited quantities of chemicals and
reagents
⇒ high V connected to them (by electrodes) ⇒
optical detection will be performed on capillary itself
Numerous modes to vary selectivity
Extremely versatile technique
→ hydrodynamic (pressure) injection:
the injection side of the capillary and sampling solution is, for a controlled time, raised to a higher level
(siphoning). As alternative a vacuum can also be applied to the detector side (sucking) or pressure to the
inlet side. In all these cases the injection time determines to a large extent the reproducibility, and electronic
control and automation are recommended.
→ injection through electromigration
the electrode and capillary are brought into the sample solution and the voltage applied during a few
seconds. After that the electrode and capillary are transferred again to the elution buffer. The amount
injected is proportional to the applied voltage and injection time, but also to the mobility of the components
⇒ fastest eluting components are therefore enriched.
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