capillary electrophoresis gammaru_caspe

8/9/2019 Capillary Electrophoresis Gammaru_caspe

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Capillary Capillary ElectrophoresisElectrophoresis


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1. Introduction2. Electrophoresis Overview3. Importance Of separation technique4. Why capillary Electrophoresis5. What is CE6. Types of CE

7. CE The Basics of the instrumentation8. Theory of Capillary Electrophoresis9. Electroosmotic Flow10.Electroosmotic Mobility11.Flow in CE12.The Electrophoregram13.Equipment of CE14.Methods For Improving Efficiency of CE15.Application

16.Summary and conclusion17.

CONTENTCONTENT

SS


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PRINCIPLE AND INSTRUMENTATIONPRINCIPLE AND INSTRUMENTATIONOF CAPILLARY ELECTROPHORESISOF CAPILLARY ELECTROPHORESIS

:PRESENTED BY,Caspe Nerlizabel Rose

, .Gammaru Anabel A-Bs bilogy 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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ElectrophoresisAnElectrophoresisAnOverviewOverview

Definition: The differential movementfor migration of ions by attractionor repulsion in an electric field.

Separation of components of amixture using 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- overviewcontcont ..

Can determine the size, shape, andcharge of a molecule

Different forms of electrophoresisare used for each of these factorsindependently or in combination.


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Types of Electrophoresis Types of Electrophoresis Capillary Native Polyacrylimide Gel

Electrophoresis (PAGE) Slab Paper


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BasicsBasicscont.cont.

A photocathode is thenused to measure theabsorbencies of themolecules as they passthrough the solution

The absorbencies areanalyzed by acomputer and they are

represented graphically


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Capillary Electrophoresis TheCapillary Electrophoresis TheBasics Of InstrumentationBasics Of Instrumentation

Electrophoresis in a buffer filled, narrow-bore capillaries

Each capillary is about 25-100 m in

internal 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


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Cont.


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Capillary ElectrophoresisCapillary ElectrophoresisApparatusApparatus


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Electrophoresis a separation technique based on

the differential transportation of charged species in an electric fieldthrough a conductive medium.

Primary candidates for CE separationare ions.

The basic instrumental set-up, consistsof a high voltage power supply (0 to30 kV), a fused silica (SiO2) capillary,two buffer reservoirs, two electrodes,and an on-column detector.


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Capillary electrophoresis(CE)

is a family of related techniques that employ

narrow-bore (20-200 mm i.d.) capillaries toperform high efficiency separations of bothlarge and small molecules .

separations are facilitated by the use of high voltages, which may generateelectroosmotic and electrophoretic flowof buffer solutions and ionic species,respectively, within the capillary .

The properties of the separation and theensuing electropherogram havecharacteristics resembling a cross

between traditional polyacrylamide gelelectro horesis (PAGE) and modern hi h


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Electrophoresisterminology

under ideal conditions, nothing isretained, so the analogous termbecomes migration time.

The migration time ( tm) is thetime it takes a solute to movefrom the beginning of the capillaryto the detector window.

Other fundamental terms are definedbelow. These include theelectrophoretic mobility, mep(cm2/Vs), the electrophoreticvelocity, vep (cm/s), and the


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h e r e l a t i on sh ip s .between these factors are shown in Equation 1

Equation 1 is only useful for determining the.apparent mobility To calculate the actual

,mobility the phenomenon of electroosmotic flow.must be accounted for To perform reproducible

,electrophoresis the electroosmotic flow must be.carefully controlled


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Electroosmosis This phenomenon is a consequence

of the surface charge on the wall of the capillary.

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 a

,voltage is applied across the capillary cations in the diffuse portion of the double layer migrate in the direction of the, .cathode carrying water with them

The result is a net flow of buffer solution in the direction of.the negative electrode


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-tern s model of the double layer charge distribut ion at a

egatively charged capi llary wall leading to the generation f a zeta potential and EOF


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Electroosmotic MobilityElectroosmotic Mobility

Zeta Potential The change

in potentialacross adoublelayer

Proportionalto thecharge onthe capillarywalls and tothe


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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

parabolic flow profile generated by an externalpump, as used for HPLC.

EOF has a flat profile because its driving force (ie.,charge on the capillary wall) is uniformly

distributed along the capillary, which means thatno pressure drops are encountered and the flowvelocity is uniform across the

capillary.

In HPLC, in which frictional forces at the column wallscause a pressure drop across the column, yieldinga parabolic or laminar flow profile.

The flat profile of EOF is important because itminimizes zone broadening, leading to highseparation efficiencies that allow separations onthe basis of mobility differences as small as 0.05


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Electropherogram

The data output from CE ispresented in the form of an electropherogram,which is analogous to achromatogram.

An electropherogram is a

plot of migration time vs.detector response. The detector response is

usually concentrationdependent, such as UV-visible absorbance orfluorescence.

The appearance of a typicalelectropherogram isshown in Figure for theseparation of athreecomponentmixture of cationic, neutral and


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EquipmentEquipment Capillary tube

Varied lengthbutnormally25-50 cm

Small boreandthickness of

the silicaplay a role

Using asmallerinternaldiamet


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EquipmentEquipment Cont.Cont. Detector

UV/Visible absorption Fluorescence Radiometric (for radioactive

substances) Mass Spec.


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Effects of voltage andtemperature

Both the electroosmotic andelectrophoretic velocities are directlyproportional to the field strength, so theuse of the highest voltages possible will

result in the shortest times for theseparation. Short separation times will give the

highest efficiencies since diffusion is the

most important feature contributing tobandbroadening.

The limiting factor here is Joule heating.Experimentally, the optimal voltage isdetermined by performing runs at


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The electrophoreticmobility and theelectroosmoticflow expressions bothcontain a viscosity term in thedenominator.

Viscosity is a function of temperature;therefore, precise temperature controlis important. As the temperatureincreases, the viscosity decreases;thus, the electrophoretic mobilityincreases as well.

Some buffers such as Tris are known tobe pH-sensitive with temperature. Forcomplex separations such as peptide

maps, even small pH shifts can alter


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HINTS FOR IMPROVING EFFICIENCY OF 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
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/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/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 of carbohydrates

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 of electrophoresis.

2. The speed of movement or migration of solutes in CE is determined by their

3. size and charge. Small, highly chargedsolutes will migrate more quickly thanlarge, less charged 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 an

electropherogram.


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Conclusion It is the most efficient separation

technique available for the analysisof both large and small molecules.

DNA Profiling, protein identification,inorganic metals and ions can be

detected easily by this method.


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Modes of capillaryelectrophoresis

Capillary zone electrophoresis Isoelectric focusing Capillary gel electrophoresis Isotachophoresis Micellar electrokinetic capillary

chromatography


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Capillary zoneelectrophoresis

Simplest form of CE. Capillary zone electrophoresis (CZE),

(free solution capillary

electrophoresis) The separation mechanism is based

on differences in the charge-to-

mass ratio Fundamental to CZE are

homogeneity of the buffersolution and constant field


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The net charge is usually pH

dependent. Separations of both large and small

molecules can be accomplished byCZE.

Even small molecules, where thecharge-to-mass ratio differencesmay not be great, may still beseparable.


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Effect of pH. At a high pH where EOF is substantial, the

order of migration will be cations, neutrals,and anions. None of the neutral molecules willbe separated since the net charge is zero.

The anions will still migrate toward the cathodebecause the EOF is greater than theelectrophoretic migration.

At lower pH where the EOF is greatly reduced,both cations and anions can still bemeasured, although not in a single run. Tomeasure anions, the anode must be beyondthe detector window. Likewise, to measurecations, the cathode must reside beyond thedetector window.

The proper electricalconfiguration is achievedby simply reversing the polarity of the


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Buffers A wide variety of buffers can beemployed in CZE. buffer is most effective within one or

two pH units of its pI. Zwitterionic buffers such as bicine,

tricine, CAPS, MES, and Tris are alsocommon, particularly for protein andpeptide separations. The advantageof the zwitterionic buffer is its lowconductivity when the buffer isemployed around the pI.

The advantage therein is the lowcurrent draw andthus reduced oule


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Isoelectric focusing The fundamental premise of isoelectric

focusing (IEF) is that a molecule willmigrate so long as it is charged.

IEF is run in a pH gradient where the pH islow at the anode and high at the cathode .

The pH gradient is generated with a seriesof zwitterionic chemicals known ascarrier

ampholytes. When a voltage is applied, the

ampholyte mixture separates in thecapillary.

Ampholytes that are positively charged will

migrate towards the cathode while thosene ativel char ed mi rate towards the


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Application In addition to performing high

resolution separations, IEF isuseful for determining the pI of aprotein.

IEF is particularly usefulforseparating immunoglobulins,

hemoglobin variants and post-translational modifications of recombinant proteins.


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Capillary gelelectrophoresis

Traditional gel electrophoresis isconducted in an anticonvectivemedium such as polyacrylamide oragarose.

The composition of the media canalso serve as a molecular sieve to

perform size separations


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physical gel Chemical gels

There are two fundamentalclasses of gels


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Isotachophoresis was the most widely used

instrumental capillaryelectrophoretic technique, althoughthe capillaries were quite wide(250-500 mm) by todaysstandards.


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Micellar electrokineticcapillary chromatography

Micelles are amphiphilic aggregatesof molecules known assurfactants.

They are long chain molecules(10-50 carbon units) and arecharacterized as possessing a long

hydrophobic tail and a hydrophilichead group.


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Caspe, Nerlizabel Rose C.Gammaru, Anabel A.

BS. Biology 3-1D

!!!Thank you

capillary electrophoresis gammaru_caspe

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