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CapillaryCapillary
ElectrophoresisElectrophoresis
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1. Introduction2. Electrophoresis Overview3. Importance Of separation technique
4. Why capillary Electrophoresis5. What is CE6. Types of CE7. CE The Basics of the instrumentation8. Theory of Capillary Electrophoresis9. Electroosmotic Flow
10. Electroosmotic Mobility11. Flow in CE12. The Electrophoregram13. Equipment of CE14. Methods For Improving Efficiency of CE15. Application16. Summary and conclusion
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PRINCIPLE AND INSTRUMENTATIONPRINCIPLE AND INSTRUMENTATIONOF CAPILLARY ELECTROPHORESISOF CAPILLARY ELECTROPHORESIS
PRESENTED BY:Caspe, Nerlizabel RoseGammaru, Anabel A.
Bs biology 3-1d
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Definition of terms
CE- capillary electrophoresis E- electric field strength
EOF- electroosmotic
EPF-Electrophoretic flow
Ld- length of the capillary to the detector
Lt- total capillary length
Uep- electrophoretic mobility
pI- isolectric point
V-volt
V- voltage
VeO- electroosmotic flow velocity
Vep- electrophoretic velocity
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ElectrophoresisAnElectrophoresisAn
OverviewOverview Definition: The differential movement
for migration of ions by attraction orrepulsion in an electric field.
Separation of components of a mixtureusing an electric field
v=Eq/f v = velocity of molecule
E = electric field q = net charge of molecule
f = friction coefficient
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Electrophoresis- overviewElectrophoresis- overview
contcont.. Can determine the size, shape, and
charge of a molecule
Different forms of electrophoresisare used for each of these factorsindependently or in combination.
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Types of ElectrophoresisTypes of Electrophoresis
Capillary
Native Polyacrylimide GelElectrophoresis (PAGE)
Slab
Paper
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BasicsBasics
cont.cont. A photocathode is then
used to measure theabsorbencies of themolecules as they pass
through the solution
The absorbencies areanalyzed by a computerand they are represented
graphically
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Capillary Electrophoresis TheCapillary Electrophoresis The
Basics Of InstrumentationBasics Of Instrumentation Electrophoresis in a buffer filled, narrow-
bore capillaries
Each capillary is about 25-100 m ininternal diameter
When a voltage is applied to the solution,the molecules move through the solutiontowards the electrode of opposite charge
Depending on the charge, the moleculesmove through at different speeds Separation is achieved
C
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Cont.
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Capillary ElectrophoresisCapillary Electrophoresis
ApparatusApparatus
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Electrophoresisterminology
The migration time (tm) is thetime it takes a solute to movefrom the beginning of the capillary to
the detector window. Other fundamental terms are defined
below. These include theelectrophoretic mobility, mep(cm2/Vs), the electrophoreticvelocity, vep (cm/s), and the electricfield strength, E (V/cm).
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The relationships between these factors are shown in Equation 1.
Equation 1 is only useful for determining theapparent mobility. To calculate the actualmobility, the phenomenon of electroosmotic flow
must be accounted for. To perform reproducibleelectrophoresis, the electroosmotic flow must becarefully controlled.
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Electroosmosis
A vitally important feature of CE isthe bulk flow of liquid through thecapillary.
This is called the electroosmotic flowand is caused as follows
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The negatively-charged wall attracts positively-charged ions
from the buffer, creating an electrical double layer. When avoltage is applied across the capillary, cations in the diffuseportion of the double layer migrate in the direction of thecathode, carrying water with them.The result is a net flow of buffer solution in the direction of thenegative electrode.
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Sterns model of the double-layer charge distribution at a
negatively charged capillary wall leading to the generation of azeta otential and EOF
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Electroosmotic MobilityElectroosmotic Mobility
Zeta PotentialThe change in
potential across adouble layer
Proportional to thecharge on the
capillary walls andto the thickness ofthe double layer. Both pH and ion
strength affectthe mobility 18
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Effects of pH
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Flow Profile in CE A further key feature of EOF is that it has flat flow
profile, which is shown in Figure alongside the parabolicflow profile generated by an external pump, as used forHPLC.
EOF has a flat profile because its driving force (ie.,charge on the capillary wall) is uniformly distributedalong the capillary, which means that no pressure dropsare encountered and the flow velocity is uniform acrossthecapillary.
In HPLC, in which frictional forces at the column walls
cause a pressure drop across the column, yielding aparabolic or laminar flow profile.
The flat profile of EOF is important because itminimizes zone broadening, leading to high separationefficiencies that allow separations on the basis ofmobility differences as small as 0.05 %.
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e
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eElectropherogram
The data output from CE is
presented in the form of anelectropherogram, which isanalogous to achromatogram.
An electropherogram is a
plot of migration time vs.detector response. The detector response is
usually concentrationdependent, such as UV-visibleabsorbance or fluorescence.
The appearance of a typicalelectropherogram is shownin Figure for the separationof a threecomponentmixture of cationic, neutral
and anionic solutes. 22
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EquipmentEquipment
Capillary tube Varied length but
normally 25-50 cm
Small bore and
thickness of the silicaplay a role
Using a smallerinternal diameter andthicker walls help
prevent Joule Heating,heating due to voltage
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EquipmentEquipment Cont.Cont.
Detector
UV/Visible absorption
Fluorescence
Radiometric (for radioactive substances)
Mass Spec.
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Eff f l d
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Effects of voltage andtemperature
Both the electroosmotic and electrophoretic velocitiesare directly proportional to the field strength, so the useof the highest voltages possible will result in theshortest times for the separation.
Short separation times will give the highest efficiencies
since diffusion is the most important featurecontributing to bandbroadening.
The limiting factor here is Joule heating. Experimentally,the optimal voltage is determined by performing runs atincreasing voltages until deterioration in resolution is
noted.
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The electrophoretic mobility and the electroosmoticflow expressions both contain a viscosity term in
the denominator. Viscosity is a function of temperature; therefore,
precise temperature control is important. As thetemperature increases, the viscosity decreases;thus, the electrophoretic mobility increases as well.
Some buffers such as Tris are known to be pH-sensitive with temperature. For complex separationssuch as peptide maps, even small pH shifts can alterthe selectivity.
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HINTS FOR IMPROVING EFFICIENCYOF CE
Buffers Additives for CZE Additives for HPCE Hints Ionic Strength in HPCE PKa Values of Common Buffers
Proteins, Choosing a Proper Buffer
Capillaries Conditioning Dimensions, Changing How to Properly Cut A Capillary (Animated)
Storage
Data Analysis Migrating Peak Correction
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http://www.microsolvtech.com/cehint15.asphttp://www.microsolvtech.com/cehint2.asphttp://www.microsolvtech.com/cehint3.asphttp://www.microsolvtech.com/cehint14.asphttp://www.microsolvtech.com/cehint6.asphttp://www.microsolvtech.com/cehint16.asphttp://www.microsolvtech.com/cehint5.asphttp://www.microsolvtech.com/cehint13.asphttp://www.microsolvtech.com/cutcap.asphttp://www.microsolvtech.com/cehint4.asphttp://www.microsolvtech.com/cehint1.asphttp://www.microsolvtech.com/cehint1.asphttp://www.microsolvtech.com/cehint4.asphttp://www.microsolvtech.com/cutcap.asphttp://www.microsolvtech.com/cehint13.asphttp://www.microsolvtech.com/cehint5.asphttp://www.microsolvtech.com/cehint16.asphttp://www.microsolvtech.com/cehint6.asphttp://www.microsolvtech.com/cehint14.asphttp://www.microsolvtech.com/cehint3.asphttp://www.microsolvtech.com/cehint2.asphttp://www.microsolvtech.com/cehint15.asp -
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ApplicationsApplications
Analysis ofcarbohydrates
Analysis of inorganic
anions/metal ions DNA profiling
Protein identification
Advantages Fast
Small Sample
Relatively inexpensive
Automated
Disadvantages Cannot identify
neutral species
Joule Heating
Cannot discern shape
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Summary
1. CE is based on the principles ofelectrophoresis.
2. The speed of movement or migration ofsolutes in CE is determined by their
3. size and charge. Small, highly charged soluteswill migrate more quickly than large, lesscharged solutes.
4. Bulk movement of solutes is caused by EOF.
5. The speed of EOF can be adjusted by changing
the buffer pH used.6. The flow profile of EOF is flat, yielding high
separation efficiencies.
7. The data output from CE is called anelectropherogram.
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Conclusion
It is the most efficient separationtechnique available for the analysisof both large and small molecules.
DNA Profiling, protein identification,
inorganic metals and ions can bedetected easily by this method.
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Caspe, Nerlizabel Rose C.Gammaru, Anabel A.
BS. Biology 3-1D
Thank you !!!