about capillary electrophoresis by pavani

1. By
G.Pavani
Y17mphpa426
Chalapathi Institute Of Pharmaceutical Sciences
Lam,Guntur.

2.  DEFINITION
 PRINCIPLE
 PROCEDURE
 INSTRUMENTATION
 SAMPLE INTRODUCTION TECHNIQUES
 CE-MS HYPHENATION TECHNIQUE
 APPLICATIONS.

3. Electro-Electricity
Phoresis-Separation
 Literally,the term electrophoresis means migration of ions with
electricity.
“It involves separation of Components of a sample by the
differential rate of migration of ions by attractions (or) repulsion
in an applied dc electric field”.
 The technique was first developed by Arne Tiselius in the 1930’s
for the study of serum proteins.
 He was awarded the 1948 Nobel Prize in Chemistry.
 It is been Principal method for the separation of Proteins
(Enzymes
Harmones
Antibiotics
Nucleic Acids)

4. “Electrophoresis Performed in a thin tube subjected to Capillary”.
Principle:Electrophoretic Mobility
Electro Osmotic Flow(EOF)
Electrophoretic Mobility
 The rate of Migration (usually/s)per unit electric field strength (usually
V/cm)of a charged particle in Electrophoresis.
 Symbol-µ
 It has been found that, the migration Velocity (v)(cm-1) of a molecule in an
electric field is given by,
V=µeE
Where,E= Strength of the electric field(Vcm-1)
µe= Electrophoretic mobility(cm2 V-1 S-1)
 The value of E depends upon the,
 Charge of the Analyte ion
 The frictional retarding forces which includes,
- Size and shape of the ion
- Viscosity of the medium, in which migration
occurs.

5. Where in HPLC- No.of Plates – Efficiency of Separation .
Plate count= n(16rt)2
W
 In CE, N= µeV
2D
Where,N= No.of Plates
µe = Electrophoretic Mobility
V = Migration Velocity
D = Diffusion co-efficient of the solute(cm-1 s-1)
 Plate count - resolution
 High voltage is applied to achieve high resolution separations in
CE.
 In gel slab electrophoresis,joule heating limits the magnitude of
applied voltage is about 500 V.
Plate heights in CE:

6.  In Capillary electrophoresis-
 Due to , small cross sectional area
Longer length of the capillary Broad banding is not seen
inCE.
 Electric fields of 100-400V/Cm are typically used.
 Plate count range of
CE- 100,000 to 200,000(1 lakh to 2 lakhs)
HPLC-5,000 to 20,000
 Plate counts of 3000,000 (3 lakhs ) have been reported for CZE of
dansylated amino acids.
 Plate counts of 10,00000 (1 lakh) have been reported for CGE of
Polynuclotides.

7. Electro Osmotic flow(EOF):
 A unique feature in CE is EOF.
“ Bulk flow of liquid throuh theCapillary is Called as EOF.
i.e,
o Silica Capillary Walls Contain Surface Silonal (SI-OH)- groups &
these are ionized at pH values higher than 3 and the wall becomes
(-ve)ly Charged.
o Wall attracts Cation and double layer forms.
o Cations inthe diffuse layer are attracted towards Cathod and
migrate in that direction as they are in solution, the buffer fluid is
also dragged along with them.
o The direction of flow of EOF may be reversed by the treatement
with cetyl trimethyl ammonium bromide.

10. CE-STEPS
Electrophoresis is done in bufferfilled, narrow bore capillaries.
Each Capillary is about 25-100 µm in internal diameter.
Small cross sectional area, long length, leading to high resistance, low
currents.
.Vmax =20-100Kv.
N = 100,000-10,000,000 high resolution.
When a Voltage is applied to the solution, the molecules move through the
solution towards the electrode of opposite charge
.

11. Depending on the Charge , the molecules move through at
different speeds.
Thus Separation is Achieved.
A suitable detector is then used to detect the solute as it
comes out from the end of the Capillary.
The data Obtained are analyzed by a Computer and
represented Graphically.

14.  The instrumentation of CE is relatively Simple.
 A buffer filled fused –Silica Capillary,typically 10 to 100 µm in
internal diameter& 30 to 100 cm long, extended between two
buffer reserviors that also hold platinum electrodes.
 Like the Capillary tubes used in GC, the outside walls of the
fused- silica Capillary are typically coated with Polyimide for
durability, flexibility,&Stability.
 The Sample is Introdused at one end & detection occurs at
another end.
 A Voltage of 5 to 30 Kvdc is applied across the two electrodes.

15.  The Polarity of this high Voltage canbe as indicated in fig
can be reversed to allow rapid separation of anions.
 High- Voltage Electrophoresis Compartments are usually
safety interloked to protect the user.
 Although the instrumentation is conceptually simple,
significant experimental difficulties in sample introduction
& detectionarise due to the Very small volumes involved.
Because the volume’s must be on the order of a few nl
(Or)less.

16. By 2 methods,-Electro kinetic injection
Pressure injection
i. Electro kinetic injection:-
One end of the Capillary & its Electrode are removed from
their buffer Compartment &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 &EOF.

17. The Capillary end & electrode are then returned to the regular
buffer solution for duration of the separation.
This injection technique discriminates by injecting larger amounts
of the more mobile ions relative to the slower moving ions.

18. The sample introduction end of the capillary is also placedin
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
vaccume at the detector end, by pressurizing the sample, (or)
by elevating the sample end (hydrodynamic injection)
Pressure injection does not discriminates because of ion
mobility, but it can’t be used in gel-filleed capillaries.

19.  For both Electrokinetic injection & Pressure injection the
Volume injected is Controlled by the duration of the
injection.
 Injections of 5 to 50nl are common and volumes below
100pl have been reported.
 For a buffer with density & Viscosity near the values for
water, a height differential of 5 cm for injects about 6nl
with a 75 µm inside diameter capillary.

20.  Micro injection tips constructed from capillaries drawn to
very small diameters allow sampling from picoliter
environments such as single cells (or) substructures with
in single cells.
 This technique have been to study aminoacids &
neurotransmitters from single cells.
 Other novel injection techniques have been described in
the literature, commercial CE systems are available with
thermostatted multiplication for automated sampling.

21. TYPE OF DETECTOR REPRESENTATIVE DETECTION
LIMIT (attamoles detected)
Spectrometry
Absorption 1-1000
Flourescence 1-0.01
Thermal lens 10
Raman 1000
Chemoluminiscence 1-0.0001
Mass spectrometry 1-0.01
Electrochemical
Conductivity 100
Potentiometry 1
Amperometry 0.1

22. CE-MS HYPHENATED TECHNIQUE

23.  The very small volumetric flow rates of less than 1 µl/min
from electrophoresis capillaries make it feasible to
couple the effluent directly to the ionization sources of
a mass spectrometer .
 The most common sample introduction and ionization
interface for this purpouse is currently,
electronspray
fast atom bombardment,
matrix-assisted laser desorption
ionization (MALDI) spectrometry,
Inductively coupled plasma mass spectrometry (ICPMS) have
also been used.
Because the liquid sample must be vapourised before
entering the mass spectrometry(MS) system,
It is imp to that volatile buffers be used.
CE-MS systems have become quite imp in the life sciences
for determining large biomolecules

24.  Proteins
 DNA fragments
 Peptides
 FIG-a typically electron spray interfacecoupled to a
quadrapole mass spectrometer,
 Note that the capillary is positioned b/w the isolated high-
voltage region and the electronspray source.
 The high-voltage end of the capillary was at 30 to 50 kV
with respect to common.
 The low-voltage end was maintained at 3-5kv and charged
the droplets.
 Similar electronspray instruments are available
commercially coupled with either quadrapole or ion-trap
mass spectrometers.
 Ion trap mass spectrometers can allow CE-MS/MS(Or) MSn
operation.

26.  Spectrum-For vasotocin,
 A polypeptide having mass of 1050.
 Note the presence of doubly and triply charged species.
 Wiith higher molecular mass species, ions are often
observed with charges of +12 or more.
 Ions with such charge make it possible to detect high
molecular mass analytes with a quadrapole instrument
with a relatively modest mass range.
 Typical detection limits for CE-MS are of a few tens of
molecules with molecular massess of 1000,000 (1 lakh) or
more.

27.  The vast applications of electrophoresis include,
 Vaccine analysis
 Protein and DNA analysis
 Used in forensic investigations
 Determination of impurities
 Chiral analysis
 Analysis of Carbohydrates and other
macromolecules
 Analysis of inorganic anions /metal ions.

28.  Vaccine Analysis is one of the many important applications
of electrophoresis.
 There are Several Vaccines that have been Purified,
Processed and analyzed through Electrophoresis ,such
asthe,
 Influenza vaccine,Hepatitis vaccine,Poli vaccine.

29.  Electrophoresis has advanced our understanding
on the structure and function of proteins.
 These molecules are needed by our body cells
and may be analyzed, for instance, by getting
blood and urine samples.
 Then through electrophoresis, the amount of
Proteins in your blood or in your urine is
measured and compared to establish normal
values ---lower or higher than the normal levels
usually indicates a disease.

30.  Atropine Sulphate i.v. Solution.
 Codeine Phosphate Syrup.
 Ketamine HCLi.v. Solution.
Have Successfully been assayed by this
Process.