ch. 30 capillary electrophoresis, capillary electrochromatography and field - flow fractionation...

Ch. 30

Capillary Electrophoresis, Capillary Electrochromatography

and Field - Flow Fractionation

Electrophoresis = ?

Capillary Electrophoresis = ?

電 泳

1948 Nobel Prize

Q / d : charge-to-size ratio

Swedish chemist Serum proteins

Electrophoresis: a separation method based on

the differential rate of migration of charged species in a

buffer solution across which has been applied a dc

electric field.

Slab electrophoresisBio macromolecules:

Proteins (enzyme, hormones, antibodies)

Nucleic acids (DNA, RNA)

L. Stryer, Biochemistry, 4th ed., W. H. Freeman, 1999, p. 47.

平板

F. Rouessac and A. Rouessac, Chemical Analysis, Wiley, 2000, p.112

毛細管電泳

Human Genome: sequence for human DNA

Systematic apparatus for capillary electrophoresis

15 ~ 30 kV

25 ~ 75 μm i.d.

~ 50 cm long

G. D. Christian, Analytical Chemistry, 6th ed., John Wiley, 2004, p. 632

vs. slab eletrophoresis:no joul heating effect

FIGURE 30-1 Schematic of a capillary electrophoresis system.

D. C. Harris, Quantitative Chemical Analysis, 6th ed., 2003, p. 655.

FIGURE 30-2 Charge distribution at a silica-capillary interface and resulting electroosmotic flow.

buffer solution

e.g. Na+Ac

-

Acts as a pump

帶動整體溶液電 滲 流

OH pH > 3 O-

Si Si

electricaldouble layer

Microfluid

微流體

D. C. Harris, Quantitative Chemical Analysis, 6th ed., 2003, p. 658.

+ > -

+ = -

FIGURE 30-4 Velocities in the presence of electroosmotic flow. The length of the arrow next to an ion indicates the magnitude of its velocity; the direction of the arrow indicates the direction of motion. The negative electrode is to the right and the positive electrode to the left of this section of solution.

In general,

νeo > νe

Separation is based on differences in electrophoretic mobility.

電滲流

電泳 ++ -

charge / size

migration velocity

淨移動速率

FIGURE 30-3 Flow profiles for liquids under (a) electroosmotic flow and (b) pressure-induced flow.

electroosmotic hydrodynamic

parabolicflat

pump

+ -

D. C. Harris, Quantitative Chemical Analysis, 6th ed., 2003, p. 658.

pump

ms

D. C. Harris, Quantitative Chemical Analysis, 7th ed., 2007.

ms

D. C. Harris, Quantitative Chemical Analysis, 6th ed., 2003, p. 656.

van Deemter equation:

D. C. Harris, Quantitative Chemical Analysis, 6th ed., 2003, p. 656.

Advantages of CZE:

• High separation efficiency (N > 105)

• Low reagent consumption

• Low sample consumption (< 10 nL)

Sample introduction in CZE:

1. Electrokinetic injection

2. Pressure injection

a. pressure

b. vacuum

c. height difference (gravity)

TABLE 30-1 Detectors for CE

(UV/Vis)