Instant Notes: Analytical Chemistry

surface to form an electrical double layer with an associated potential (zeta

potential) (Fig. 3). Application of a voltage across the capillary initiates an EOF,

the velocity or mobility, mEOF, of which is typically about an order of magnitude

greater than those of individual solutes. Hence, the total migration rate of a

solute, mTot,is

mTot= msolute+ mEOF (3)

and all solutes, are carried towards the cathodic end of the capillary by the EOF

where they pass through the detector cell.

Important characteristics of the EOF are:

● The velocity increaseswith increasing pHof the running buffer as more

silanol groups become ionized.

● The velocity increaseswith the magnitude of the applied potential.

● The velocity decreasesalong with the zeta potentialwith increasing ionic

strength.

● The velocity decreaseswith increasing viscosityof the running buffer.

● It has a flat flow profileacross the capillary in contrast to the parabolic

profile of a pumped HPLCmobile phase (Fig. 3). This minimizes band

spreading and results in very high efficiencies (plate numbers) for the

separating solutes.

● There is no pressure dropalong the capillary, as occurs when the mobile

phase is pumped through an HPLC column, because the electrically-

generated driving force acts equally along the whole length.

● The velocity can be controlled, reduced to zero or reversedby additives such

as surfactants, organic solvents or quaternary amine salts incorporated into

the running buffer, or by modifying the inside wall of the capillary.

D8 – Electrophoresis and electrochromatography 179

Table 2(a). Typical running buffers for electrophoresis

Buffer Useful pH range

Phosphate 1.1–3.1

Ethanoate 3.8–5.8

Phosphate 6.2–8.2

Borate 8.1–10.1

Zwitterionic buffers

MES 5.2–7.2

(2-(4-morpholino)ethanesulfonic acid)

TRIS 7.3–9.3

((2,3-dibromopropyl) phosphate)

Table 2(b). Typical buffer additives for electrophoresis

Additive Function

Inorganic salts Change protein conformations

Organic solvents Modify EOF, increase solute solubilities

Urea Solubilize proteins, denature oligonucleotides

Surfactants Form micelles, cationic ones reverse charge on capillary wall

Cyclodextrins Provide chiral selectivity