Introduction to CE

What is Electrophoresis?

Movement of charged molecules under the influence of an applied electric field

cathode anode

Electrophoresis is a bi-directional process that involves charged

particles.

System Overview and Terminology

Electrolyte Buffer

Electrolyte Buffer

Reservoir Reservoir

HVPS

Electrode Electrode

Capillary Inlet

Capillary Outlet

Detector

Data Acquisition

Basic Principles of Capillary Electrophoresis

Basis of separation is the differential migration of molecules in a applied electric field

Electrophoresis, not Chromatography!

A Complementary Technique to LC

User controls the environment within the capillary!

Basic Principles of CE The environment within the capillary

The Environment

Analyte (Sample)

Buffer (Medium)

Electric Field

Capillary Wall

Basic Principles of CE Influential Properties of Analyte

The Environment

Analyte (Sample)

Buffer (Medium)

Electric Field

Capillary Wall

Size and Shape

Net Charge

Mass

Basic Principles of CE Influential Properties of Buffer

The Environment

Analyte (Sample)

Buffer (Medium)

Electric Field

Capillary Wall

Ionic Strength

Temperature

pH

Viscosity

Dielectric Constant

Basic Principles of CE Influential Properties of Electric Field

The Environment

Analyte (Sample)

Buffer (Medium)

Electric Field

Capillary Wall

Dependent on Applied Voltage

Dependent on Length

Driving Force of Electrophoresis

Basic Principles of CE Influential Properties of Capillary Wall

The Environment

Analyte (Sample)

Buffer (Medium)

Electric Field

Capillary Wall

Surface Charge

Coating

Electroosmotic Flow

CE Fundamental Equations

CE Fundamental Equations Terminology

Capillary Inlet

Reservoir

Capillary Outlet

Reservoir

Detector

Lt

Total Capillary Length

Ld

Length to Detector

The Electric Field Strength

E = Electric Field

V = Applied Voltage (Volts)

Lt = Total capillary length (cm) tL

VE

Electrophoretic Velocity

m

dep

t

Lv

vep = Velocity

tm = Migration Time (seconds)

Ld = Capillary length to the

detector (cm)

Mobility = velocity/unit field strength

Electrophoretic Mobility

E = Electric Field

vep = electrophoretic

velocity

E

vep

ep

Calculating Mobility

m

dep

t

Lv

tL

VE

Electrophoretic Velocity

Electric Field

Calculating Mobility

t

mdepep

LV

tLEv/Vs)(cm

/

//2

ep =electrophoretic mobility

ep = electrophoretic velocity

E = Electric Field

V = Applied Voltage

Lt = Total capillary length (cm)

Ld =Length to the detector (cm)

tm = migration time (seconds)

Mobility = velocity/unit field strength

Electrophoretic Mobility

st

epr

q

6

q = charge

= viscosity of the solution

rst = Stokes Radius of the ion

(hydrodynamic size)

Mobility is also dependent on

Charge/Mass Ratio

Electrophoretic Mobility –

Fundamental parameter governing Capillary Electrophoresis!

– Intrinsic property of an analyte

– Depends on the analyte’s charge/mass ratio

– Analyte charge related to Buffer pH!

UV detection at 214 nm.

Peak identification of the ACTH (Adrenocorticotropic hormone) fragments.

Reproduced with permission from Van de Goor et al., J. Chromatogr. 545, 379 (1991).

Effect of Buffer pH on Mobility

Effect of the Electric Field

McLaughlin, G.M., Nolan, J,A.,Lindahl,

et al. J. Liq. Chromatography,

15,961,1992

60 mM borate, 60 mM SDS, 15%

methanol pH 8.92

75 m X 50cm(effective length) 25

C

5 Vitamins

1.Niacinamide

2.Cyanocobalamine (B12)

3.Pyridoxine(B4)

4.Niacin

5.Thiamine(B1)

The Capillary Wall

The Environment

Analyte (Sample)

Buffer (Medium)

Electric Field

Capillary Wall

Surface Charge

Coating

Electroosmotic Flow

Basic Principles of CE Influential Properties of Capillary Wall

The Capillary Characteristics of the Capillary Surface

– Glass • bare fused silica

– Always coated externally • Polyimide

– Internal surface may carry a surface charge

– Surface charge may be manipulated • pH of Buffer

• dynamic internal coating

• permanent internal coating.

– Responsible for Electroosmotic Flow (EOF)

The Capillary Wall Ionization of the Silanols

The silanol groups are significantly ionized above pH 4.

The capillary wall will have a negative charge.

Amount of charge is controlled by the buffer pH!

Electroosmotic Flow

Mobility and Electroosmotic Flow

Migration time is the measurement of the apparent mobility.

EOF affects the Apparent Mobility of the analyte:

app = ep + eof

Thus: ep = app - eof

Electroosmotic Flow The Effect of pH

At constant ionic strength buffers (I=0.06) the µos can be described with a pka ~ 6.2

Lukacs & Jorgenson, J. High Resolut. Chromatogr. Chromatogr. Commun. 8, 407 (85).

µo

sm x

10

4(c

m2/v-s

ec)

µo

sm x

10

4(c

m2/v-s

ec)

Electroosmotic Flow Flow Profile

Electroosmosis generates an (almost) flat flow profile.

Result for CE separations

sharper peaks

better resolution

better detectability

In contrast, in HPLC the flow profile is parabolic in shape.

Modification of Capillary Surface

• Covalently bonded phase

– Neutral Capillaries

• Reduce Electroosmotic flow

• Reduce sample interaction with the capillary surface

• 2 Types

– Acrylamide – eCap Neutral capillary (cIEF applications)

– PVA – N-CHO capillary (Carbohydrates)

• Dynamic Coating

– Amine Capillaries

• Reverse Electroosmotic Flow

• Operate at a higher pH

• Reduce sample interaction with capillary surface

Basic Principles of CE

• Summary of Main Points

– Electrophoresis not HPLC

– Both analyte and buffer components move in the electric field

– Silica walls, when ionized have a negative charge.

– (+) ions of the buffer form a layer on the (– ) charged silica walls

– (+) ion layer moves toward the (– ) electrode to produce EOF

– Electrophoretic Mobility ( ep) of analyte = app - eof

– Higher applied voltage yields better peak efficiency

– Joule heating must be controlled