gammaru_caspe

8/9/2019 gammaru_caspe

1/31

1

CapillaryCapillary

ElectrophoresisElectrophoresis


2/31

2

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


3/31

3

PRINCIPLE AND INSTRUMENTATIONPRINCIPLE AND INSTRUMENTATIONOF CAPILLARY ELECTROPHORESISOF CAPILLARY ELECTROPHORESIS

PRESENTED BY:Caspe, Nerlizabel RoseGammaru, Anabel A.

Bs biology 3-1d


4/31

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


5/31

5

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


6/31

6

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.


7/31

7

Types of ElectrophoresisTypes of Electrophoresis

Capillary

Native Polyacrylimide GelElectrophoresis (PAGE)

Slab

Paper


8/31

8

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


9/31

9

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


10/31

10

Cont.


11/31

11


12/31

12

Capillary ElectrophoresisCapillary Electrophoresis

ApparatusApparatus


13/31

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).


14/31

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.


15/31

Electroosmosis

A vitally important feature of CE isthe bulk flow of liquid through thecapillary.

This is called the electroosmotic flowand is caused as follows


16/31

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.


17/31

17

Sterns model of the double-layer charge distribution at a

negatively charged capillary wall leading to the generation of azeta otential and EOF


18/31

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


19/31

Effects of pH


20/31

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 %.

20


21/31

21

e


22/31

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


23/31

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

23


24/31

EquipmentEquipment Cont.Cont.

Detector

UV/Visible absorption

Fluorescence

Radiometric (for radioactive substances)

Mass Spec.

24

Eff f l d


25/31

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.


26/31

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.


27/31

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

27
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


28/31

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

28


29/31

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.

29


30/31

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.

30


31/31

Caspe, Nerlizabel Rose C.Gammaru, Anabel A.

BS. Biology 3-1D

Thank you !!!

gammaru_caspe

Documents