ELECTROPHORESIS

SLAB (THIN LAYER GEL) AND

CAPILLARY METHODS

A. General Introduction
• Electrophoresis: a separation method based on
differential rate of migration of charged species in an
applied dc electric field
• Rate of migration
– Depends on charge and size
– Separation based on differences in charge-to-size
ratios
• High efficiency and resolution

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 Historical notes
• Initially developed by Arne Tiselius in the 1930’s
– Separated serum proteins
• Slab gel electrophoresis: developed in the 1950’s
• Capillary electrophoresis:
– developed in the 1980’s
– Narrow bore tubes used
• Applications of electrophoresis:
– Separation of proteins and nucleic acids (single
nucleotide differentiation capability!)
– The human genome Project
• Human DNA: ~three billion nucleotides

B. Principle and Theory of

Electrophoresis
• Basic requirements
– Conducting medium (aqueous buffer/ electrolyte/
run buffer)
– Applied Electric Field
– Positively charged species move to the cathode (-)
– Negatively charged species move to the anode (+)
• Two Basic Techniques
1. Free solution: narrow capillary bore (Instrumental)
2. Non-conducting matrix (Agarose, Polyacrylamide
gel/PAGE)
a. Joule heating is minimal
b. Sieving effect of gel allows separation of species with
same charge to size ratio if they have different sizes.

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• Efficiency depends on:
– Electrophoretic mobility (µep)
– Electroosmotic flow of the bulk solution (EOF)
– Joule heating

B-1 Electrophoretic Mobility

• Ion placed in an electrical field (E) experiences
force Fef that is proportional to field strength and
the charge (q) on the ion (Fef=q.E)
• As the ion moves, a frictional force (ffr) opposes
the forward movement of the ion
• In a constant electrical field the velocity of particle
is constant and depends on the balance between
the two forces
• Electrophoretic mobility is the fundamental
parameter which determines the efficiency of
separation based on charge to size ratio (q/r)
• Change in pH effectively alters the charge on the
ions and their electrophoretic mobility.

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Electrophoretic Mobility
Fef = q × E

F fr = 6 × π × η × r × v ep

η : vis cos ity

r : radius
v ep : electropho retic − migration − velocity

Fef = F fr

q×E
v ep =
6 × π ×η × r
v ep q q
µ ep = = = const ×
E 6 × π ×η × r r
µ ep : electropho retic − mobility

B-2 Joule Heating

• Ohmic/Joule heating
– heating occurs as charged particles move within the conducting
buffer upon application of an electrical field.
• Temperature gradient are generated leading to convective
flows within the electrolyte
• Convective flow leads to band broadening
• Increase in temperature can also damage
macromolecules.
• Method for decreasing joule heating:
– Decrease applied voltage- leads to long analysis time
– Dissipate heat- use thin gel or small diameter
capillaries which have large surface-to-volume ratio.
Their electrical resistance is high, thus current flow is
reduced for a given voltage.

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 B-3 ElectoOsmotic Flow (EOF)
• EOF refers to migration of the bulk liquid toward the cathode
• Origin: formation of a double layer at the wall of the capillary.
– Glass, fused silica, agarose exibit surface charges
– Silanol groups are deprotonated at pH higher than 4.
• Potential difference is established
• Positive ions in solution migrate to the wall. A double layer is developed at the
wall of the capillary.
• Stern layer (SL): layer of positive charge that compensate for the negative
charge on the surface
• Diffuse layer (DL): layer adjacent to the Stern layer: layer of mobile cations

SL Incomplete neutralization

B-3 ElectroOsmotic Flow (EOF)

• Upon application of the electrical field,
cations within the double layer are ε ×ς × E
attracted towards the cathode and drag v EOF =
4 × π ×η
the bulk solvent with them.
• EOF is in opposite direction to analyte ε : dielectric − cons tan t
electrophoretic flow (generally) ς : zeta − potential ( SL − DL)
• EOF is practically equal across the v EOF
capillary, thus band broadening is µ EOF =
E
minimal
• EOF are not reproducible thus leading v EOF = µ EOF × E
to irreproducible separation efficiencies
• EOF in GE is minimal or practically
inexistent

HPLC flow
EOF

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Use of EOF in Capillary Electrophoresis

• If EOF is more important that

electrophoretic mobility, all analytes
++ _
are dragged by the bulk solvent ⊕ +
towards the negative electrode
(Mobile Phase??!!)
• Electropherogram -
--
• The order of elution is: fastest cation,
slower cations, neutrals (single zone),
slowest anion, faster anions ++

-
--

Control of EOF in Capillary Electrophoresis

• Work at low pH
• Dynamic coating of the channel walls
– With Polyethylene glycol (PEG) added to the buffer
– Polymer layer masks charges and suppress EOF

• Chemical modification
– With trimethylchlorosilane-TMCS bonds to the surface and reduces the
number of silanol groups- low EOF
– Sulfonic acid- high EOF
– Problem: long term stability
• Use additives to change the viscosity and zeta potential
– Hydroxy ethyl cellulose or polyvinyl alcohol increase viscosity of the run
buffer and thus reduce vEOF.
– Organic solvents: methanol (reduce) and acetonitrile (increase)
– Cationic surfactants such as dodecyl trimethyl ammonium bromide
(DoTAB) adsorb onto the capillary walls and thus change the surface
charge

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B-4 Separation Efficiency and Resolution

• Efficiency and resolution depend on both the

electrophoretic flow and the EOF
• Apparent mobility is given by:
µ app = µ ep + µ EOF
• If EOF dominates, all analytes move towards
the cathode (-)

Efficiency and Resolution

• If EOF is more important that
electrophoretic mobility, all analytes are
dragged by the bulk solvent towards the
negative electrode (Mobile Phase??!!)
• Electropherogram
• The order of elution is:
1. fastest cation
2. slower cations
3. neutrals (single zone),
4. slowest anion
5. faster anions

( )
v = µ ep + µ EOF E = µ app
V
L
L L2
t = =
v µ appV

t : migration − time
L : length − of − capillary

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Band Broadening
• Main contribution to band
broadening: longitudinal
diffusion
• Peak “dispersion” is 2 × D × L2
proportional to the diffusion σ = 2× D×t =
2
coefficient, D and the µ app × V
migration time of the
analyte
• In theory: efficiency superior L2 µ app × V
to LC. Calculated N at N= =
operating voltages are on σ2 2× D
the order of 106.
• In practice: joule heating,
sample injection and
adsorption of analyte to
matrix decrease efficiency

Resolution

• Resolution depends on
the difference in
electrophoretic mobility
– What is the equivalent in
LC?
1 1
• Resolution depends on Rs = ∆µep × V × ×
the applied voltage V, µapp 4× 2× D
the apparent
electrophoretic mobility
and the diffusion
coefficient D.
– What are the equivalents
of these terms in LC?
• Optimizing resolution:

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 C. GEL ELECTROPHORESIS
• Matrix: electrically non-conductive hydrogel containing buffer
– Agarose
– Polyacrylamide
• Advantages
– Porous gel acts as a sieve
– Gel limits diffusion of sample molecules
– EOF is suppressed
– Joule heating is suppressed
• Disadvantages
– Slow, labor intensive and not readily automated
• Techniques
– Native gel electrophoresis: analyte separated according to differences in
apparent mobility
– Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE):
analyte separated according to size
– Isoelectric Focussing (IEF)
– Two dimensional gel electrophoresis (2D-GE)

C-1 Instrumentation for Gel Electrophoresis

• Power Supply
– 200-500 V
– 400 µA -100 mA
• Electrophoresis Chamber with Buffer
Reservoir
• Electrodes in buffer reservoir
• Mini Gel 8 cm x 8 cm
• Larger gel 40 x 20 cm

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Gel Media
• Agarose
– 1g in 50 ml (2%)
– Dissolve, heat, cool, pour in casting stand
– Large pore size (e.g. 150 nm for 1%): no
sieving
– Charged surfaces: EOF present

Polyacrylamide Gel
• Polyacrylamide
– Copolymerization of
acrylamide and N,N’-
methylene-bisacrylamide
– Initiator: TEMED
(N,N,N′,N′-
Tetramethylethylenedia
mine) and Ammonium
Persulfate(NH4)2S2O8 )
– Sieving effect present: pore
size depends on total gel
concentration (%T: 5 -20%)
and degree of cross linking
– SDS for denaturinf and
coating proteins
– Mercaptoethanol for
reducing dissulfide bonds

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Sample Preparation
• Buffer:
– Tris (pH 8), Tris-glycine (pH 9.1)
– 50-100 mM
• Amount of sample: µg
• Sample volume: depends on size of well (
µL to mL)
• Denaturing agent: SDS
• β-mercaptomethanol: to reduce disulfide
bonds

Visualization and detection

• Staining
– Coomasie brillant blue
(alchoholic solution of dye)
– Silver nitrate
• Fluorescent reagents
– Ethidium Bromide for DNA

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 SDS PAGE

• Proteins are totally denatured and coated SDS

charge (negative): rod-like shaped
• Charge on protein depend on its size: constant
net charge per unit mass: same electrophoretic
mobility
• Separation based on size/ molecular weight
• Sample preparation: heating (90) in the
presence of SDS and β-mercaptoethanol
• Used to determine MW of proteins
• Molecular weight standards used

Isoelectric Focussing (IEF)

• Separation based on pI • Apply field:
• Agarose gel used – Negatively charged
• pH gradient from cathode to anode amphoyte will towards
• Several ampholytes with different pI the anode (lowest pI at
used (same concentration to maintain end).
conductivity constant) – Positively charged
• Gel immersed in medium pH mixture (1 ampholytes will move
– 2 % carrier ampholyte) towards the cathode
(highest pI at end).
• Most ampholytes are charged – Cathodic side becomes
• Anode compartment: low pH buffer in more acidic
(lower than the lowest pI) – Anodic side becomes
• High pH buffer in cathode more basic
compartment (higher the highest pI) – pH range determined
by choice of
ampholytes

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2D GEL
• First dimension separation in single lane:
IEF
• Second dimension separation: SDS-PAGE
• 1000 to 2000 proteins can be separated

Capillary Electrophoresis Instrumentation

• Capillary
– Fused silica
– Diameter: 20 to 100µm
• Length 10 -100 cm
• Typical voltages: 10-30 kV
• Current 300µA
• Temperature control to control EOF and Joule
heating
• Buffer (10 – 100 mM): pH and conductivity control
• Detection: UV, fluorescence, refractive index, etc.

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 CE: Performance Characteristics
• EOF present
• Convective flows due to joule heating
• Excellent for both large and small
biomolecules (amino acids, peptides)
• Short separation time
• Small sample: nL
• Solvent consumption low: few mL
• Neutral analytes can be separated using
Micellar ElectroKinetic Chromatography
(MEKC).

• Automatic pressure or
electrokinetic sample injection
- Built-in capillary rinsing
system
- Convenient access to the
vials and capillary
- Possibility of visual check of
the position of the vials and
capillary rinsing
- Liquid cooling system in
CAPEL103 and in CAPEL-105,
CAPEL-105M
- Built-in monochromator (190 -
380 nm) in CAPEL-105 and
CAPEL-105M

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 Capillary Zone Electrophoresis (CZE)
• Controlled electroosmotic flow used
• Separates anions as well as cations
• Separation of cations: no coating of walls
– Positively charged “mobile phase” moves towards
cathode
• Separation of anions: treat walls with an alkyl
ammonium salt: reverse osmotic flow
– Negatively charged “mobile phase” moves
towards anode
• Apparent mobility

µ app = µ ep + µ EOF

Separation of 30 anions including glutamate

, galactarate, (2 – 10 ppm)

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CZE of Anti-inflammatory drugs

• (1)naporexen
• (2)ibuprofen
• (3) tolmetin

CZE separation of protein mixture

1. Cytochrome c
12,400, pI = 10.7
2. Lysozyme, 14,100, pI
= 11.1
3. Trypsin, 24,000, pI =
10.1 pH = 2.7
4. Trypsinogen, 23,700,
pI=8.7
5. Trypsin inhibitor, 20,
100, pI= 4.5

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 Capillary Isoelectric Focussing (CIEF)
• Isoelectric focusing
– Capillary filled with ampholyte mixture
– Electric field applied: pH gradient develops within capillary
– Sample can be introduced with ampholyte mixture
• Capillary must be coated to suppress EOF
• Detection at a fixed point
– Mobilization of the focused bands is necessary for detection
• Mobilization Methods
– Hydrodynamic mobilization
• After focussing
• Capillary attached to a pressure or vaccum pump
– Electrophoretic (salt) mobilization
• After focussing
• Addition of salt to cathode buffer (NaCl): disrupts pH gradient
(proteins dragged towards the cathode)

CIEF of Proteins

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 Micellar Electrokinetic Chromatography
(EKC)/CE and LC
• Micelles included in run buffer
– SDS - (CMC: 8 mM) (25 – 150 mM used)
• Partitioning of analyte between core of micelles (hydrophobic) and
run buffer
• Both micelles and buffer are moved by applied electrical field
– Two mobile phases?!
• Neutral analytes are separated according to their hydrophobicity
• Elution in SDS
– Micelle is negatively charged: low apparent mobility due to
electrophoretic mobility towards the anode
– Hydrophilic substances elute with the buffer (t0)
– Very Hydrophobic substances which are completely solubilized by the
micelle elute with the micelles (tmc)
– Others elute between the two
• Applications:
– separation of amino acids, oligopeptides, nucleic acids, fatty acids,
steroids and pharmaceutical drugs

Capillary Gel Electrophoresis

• Gel
– anti-convective medium
– sieving medium
• Separation of analytes with similar mobilities
• Advantages
– Speed
– Efficiency
– Fully automated
– No casting of gels, no staining, no scanning!!! NO WORK!?
• Disadvantages
– No multisample capability
– No 2D capability
• Applications
– Separation of ssDNA, dsDNA, RNA
– Sizing of DNA fragments

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CGE separation of SDS denatured proteins in
polyethylene glycol column

(1) lactalbumin
(2) Soybean trypsin inhibitor

(4) Ovalbumin

(5) Bovine serum albumin

(3)Carbonic anhydrase

(6) Phosphorylase

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