theory of electro phoresies and types and detection technique

1. ELectrophoresis

2. Introduction
• Electrophoresis is a separation method based on the differential
rate of migration of charged species in an applied DC electric
field.
• Electrophoresis on a macro scale has been applied to a variety
of difficult analytical separation problems: inorganic anions and
cations, amino acids, catecholamines, drugs, vitamins,
carbohydrates, peptides, proteins, nucleic acids, nucleotides,
polynucleotides, and numerous other species.

3. Introduction
• An electrophoretic separation is performed by injecting a small band of
the sample into an aqueous buffer solution contained in a narrow tube or
on a flat porous support medium such as paper or a semisolid gel.
• A high voltage is applied across the length of the buffer by means of a
pair of electrodes located at each end of the buffer. This field causes ions
of the sample to migrate toward one or the other of the electrodes
• The rate of migration of a given species depends on its charge and its
size. Separations are then based on differences in charge-to-size ratios for
the various analytes in a sample. The larger this ratio, the faster an ion
migrates in the electric field.

4. The Basis for Electrophoretic Separations
• The migration rate v of an ion (cm/s) in an electric fiel is equal to the product of the field
strength E (V cm '1 and the electrophoretic mobility M, (cm2 V-I S-1)
• The electrophoretic mobility is in turn proportional to the ionic charge on the analyte and
inversely proportional to frictional retarding factors. The electric field acts on only ions.
• If two species differ either in charge or in the frictional forces they experience while
moving through the buffer, they will be separated from each other. Neutral species are not
separated.
• The frictional retarding force on an analyte ion is determined by the size and shape of the
ion and the viscosity of the migration medium.
• The ions are separated on the basis of charge-to-size ratio.
• For ions of the same size, the greater the charge, the greater the driving force and the
faster the rate of migration. For ions of the same charge, the smaller the ion, the smaller
the frictional forces and the faster the rate of migration.

5. Types of Electrophoresis
• Slab Electrophoresis
• Capillary Electrophoresis

6. Slab Electrophoresis
• It is the classical method that has been used for many years to separate
complex, high-molecular-mass species of biological and biochemical
interest.
• Separations are carried out on a thin flat layer or slab of a porous
semisolid gel containing an aqueous buffer solution within its pores.
• Samples are introduced as spots or bands on the slab, and a dc electric
field is applied across the slab for a fixed period. When the separations
are complete, the field is discontinued and the separated species are
visualized by staining.

7. Capillary electrophoresis (CE)
• CE is performed in capillary tubes. It yields high-speed, high-resolution separations on
exceptionally small sample volumes (0.1 to10nL in contrast to slab electrophoresis, which
requires samples in the µL range).
• The separated species are eluted from one end of the capillary. so quantitative detectors,
similar to those found in HPLC can be used.
•

8. Migration Rates in CE
• The migration rate of an ion v depends on the electric field strength. The electric field in
turn is proportional to the magnitude of the applied voltage V and inversely proportional
to the length L over which it is applied. Thus
Where, Electrophoretic mobility=μe
• This relationship indicates that high applied voltages are desirable to achieve rapid ionic
migration and a fast separation.

9. Plate Heights in CE
• In chromatography, both longitudinal diffusion and mass-transfer resistance contribute to
band broadening. However, because only a single phase is used in electrophoresis, in
theory only longitudinal diffusion needs to be considered.
• In electrophoresis, we calculate the plate count N by
where D is the diffusion coefficient of the solute (cm2/S).
• Because resolution increases as the plate count increases, it is desirable to use high
applied voltages to achieve high-resolution separations. With gel slab electrophoresis,
joule heating limits the magnitude of the applied voltage to about 500 V.

10. • In CE, the capillary is quite long and has a small cross-sectional area, the solution
resistance through the capillary is exceptionally high. Because power dissipation is
inversely proportional to resistance, much higher voltages can be applied to
capillaries than to slabs for the same amount of heating. Electric fields of 100-400
V/cm are typically used. High-voltage power supplies of 10-25 kV are normal.
• Additionally, the high surface-to-volume ratio of the capillary provides efficient
cooling. As a result of these two factors, band broadening due to thermally driven
convective mixing does not occur to a significant extent in capillaries.
• CE normally yields plate counts in the range of 100,000 to 200,000, compared to
the 5,000 to 20,000 plates typical for HPLC.

11. Electroosmotic flow
• A unique feature of CE is electroosmotic flow. When a high voltage is applied
across a fused-silica capillary tube containing a buffer solution, electroosmotic
flow usually occurs, in which the bulk liquid migrates toward the cathode.
• For example. a 50 mM pH 8 buffer flows through a 50 cm capillary toward the
cathode at approximately 5 cm/min with an applied voltage of 25 kV4

12. • As shown in Figure, the cause of electroosmotic flow is the electric double layer that
develops at the silica-solution interface.
• At pH values higher than 3, the inside wall of a silica capillary is negatively charged
because of ionization of the surface silanol groups (Si-OH). Buffer cations congregate in
the electrical double layer adjacent to the negative surface of the silica capillary.
• The cations in the diffuse outer layer of the double layer are attracted toward the cathode,
or negative electrode, and because they are solvated, they drag the bulk solvent along with
them.

13. • The electroosmosis leads to bulk solution flow that has a flat profile across the tube
because flow originates at the walls of the tubing.
• This profile is in contrast to the laminar (parabolic) profile observed with the pressure-
driven flow encountered in HPLC. Because the profile is essentially flat, electroosmotic
flow does not contribute significantly to band broadening the way pressure-driven flow
does in liquid chromatography.
• The rate of electroosmotic flow is generally greater than the electrophoretic migration
velocities of the individual ions.
• Even though analytes migrate according to their charges within the capillary, the
electroosmotic flow rate is usually sufficient to sweep all positive, neutral, and even
negative species toward the same end of the capillary, so all can be detected as they pass
by a common point.
• The resulting electropherogram looks like a chromatogram but with narrower peaks.

14. • As a result of electroosmosis, order of elution in a typical electrophoretic separation is,
first, the fastest cation followed by successively slower cations, then al the neutrals in a
single zone, and finally the slowest anion followed by successively faster anions
• It is possible to reverse the direction of the normal electroosmotic flow by adding a
cationic surfactant to the buffer. The surfactant adsorbs on the capillary wall and makes
the wall positively charged. Now buffer anions congregate near the wall and are swept
toward the cathode, or positive electrode. This method is often used to speed up the
separation of anions.
• Electroosmosis is often desirable in certain types of CE, but in other types it is not.
Electroosmotic flow can be minimized by modifying the inside capillary walls with a
reagent like trimethylchlorosilane that bonds to the surface and reduces the number of
surface silanol groups.

15. Instrumentation
• A buffer-filled fused-silica capillary, typically 10 to 100 µm in internal diameter and 30 to
100 cm long, extends between two buffer reservoirs that also hold platinum electrodes.
• The outside walls of the fused-silica capillary are typically coated with polyimide for
durability, flexibility, and stability.
• The sample is introduced at one end and detection occurs at the other.
• A voltage of 5 to 30 kV DC is applied across the two electrodes to allow rapid separation
of ions.
• The volume of a normal capillary is 4 to 5 µL, injection and detection volumes must be
on the order of a few nanoliters or less.

16. Sample Introduction
• In electrokinetic injection, one end of the capillary and its electrode are removed from
their buffer compartment and placed in a small cup containing the sample. A voltage is
then applied for a measured time, causing the sample to enter the capillary by a
combination of ionic migration and electroosmotic flow. The capillary end and electrode
are then returned to the regular buffer solution for the duration of the separation. This
injection technique discriminates by injecting larger amounts of the more mobile ions
relative to the slower moving ions.
• In pressure injection, the sample-introduction end of the capillary is also placed in a
small cup containing the sample, but here a pressure difference drives the sample solution
into the capillary. The pressure difference can be produced by applying a vacuum at the
detector end, by pressurizing the sample, or by elevating the sample end.

17. Detection
Absorption Methods
• A small section of the protective polyimide coating is removed from the exterior of the
capillary by burning or etching. That section of the capillary then serves as the detector
cell.
• Several cell designs have been used for increasing the measurement path length to
improve the sensitivity of absorption methods.

18. • In the commercial detector shown in Figure (a), the end of the capillary is bent into Z
shape, which produces a path length as long as ten times the capillary diameter.
• Figure (b) shows a second way to increase the absorption path length. In this example, a
bubble is formed near the end of the capillary.
• A third method for increasing the path length of radiation by reflection is shown in Figure
(c). In this technique, a reflective coating of silver is deposited on the end of the capillary.
The source radiation then undergoes multiple reflections during its transit through the
capillary, which significantly increase the path length.

19. Indirect Detection
Indirect absorbance detection has been used for species of low molar absorptivity
that are difficult to detect without derivatization. Here, an ionic chromophore is
placed in the electrophoresis buffer. The detector then receives a constant signal due
to the presence of this substance. The analyte displaces some of these ions so that
the detector signal decreases during the passage of an analyte band through the
detector. The analyte is then determined from the decrease in absorbance.

20. • Electrochemical Detection-Two types of electrochemical detection have been
used with CE, conductivity and amperometry. The method for isolation involves
inserting a porous glass or graphite joint between the end of the capillary and a
second capillary containing the detector electrodes.
• Mass Spectrometric Detection-The most common sample-introduction and
ionization interface for this purpose is currently electrospray, although fast atom
bombardment, matrix-assisted laser desorption-ionization (MALDI) spectrometry
can be used. Because the liquid sample must be vaporized before entering the
mass spectrometry (MS) system, it is important that volatile buffers be used.