Capillary Electrophoresis

Lecture 9Introduction to Capillary Electrophoresis

Lecture 9- PHCM662-SS2016

Dr. Rasha Hanafi

Lecture 9- PHCM662-SS2016

By the end of this session, the student should be able to:

1.Define general theory of capillary electrophoresis (CE).

2.Compare between CE and chromatography.

3.Define electrophoretic/electroosmotic mobilities, apparentmobilities, migration velocity.

4.Investigate the pumping mechanism in CE.

5.Compare between electroosmotic and hydrodynamic flowprofiles.

6.Investigate the effects of electrophoresis and electroosmosis onCE.

LEARNING OUTCOMES

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Electrophoresis is aseparation method based on thedifferential rate of migration ofions by attraction or repulsion ina buffer soln. across which hasbeen applied a dc electric field.

at steady speed:

Accelerating force = frictionalforce

qE = fvep

vep = (q/f)E=μepE

μep = Electrophoretic mobility

(α charge and inversely αhydrodynamic radius of ion)

Lecture 9- PHCM662-SS2016

What is Electrophoresis?

q.E

f.vep

q = charge of an ion.

E = field strength (V cm -1).

f= friction coefficient.

vep = migration velocity of charged particle in thepotential field (cm sec -1).

ep = electrophoretic mobility (cm2 V-1 sec-1).

V = applied voltage (V) (E=V/L).

L = length of capillary (cm).

The ion in an electric field

•When an ion of charge q (coulombs) is placed in an electric field E (VoltageV/ length of capillary m), the force on the ion is qE (newtons).

•In solution, the retarding frictional force is fvep, where vep is the velocity ofthe ion and f is the friction coefficient.

•The ion quickly reaches a steady speed when the accelerating force equalsfrictional force.

•Electrophoretic mobility ep is the constant of proportionality between thespeed of the ion and the strength of the electric field.

•ep is proportional to the charge of the ion and inversely proportional tothe friction coefficient.

•For molecules of similar size, the magnitude of mobility increases withcharge. The greater the radius the lower the mobility, i.e., as the radius ofthe hydrodynamic volume of the ion increases, “f” increases.

Lecture 9- PHCM662-SS2016

Set up of capillary electrophoresis instrument

Lecture 9- PHCM662-SS2016

All ions move to the cathode.

All???even the anions???? How????

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The basic instrument is made of

1.fused silica capillary.

2.a controllable high voltagepower supply.

3.2 electrode assemblies.

4.2 buffer reservoirs.

5.detector (ex: UV).

6.data acquisition system.

The ends of the capillary are placedin the buffer. After filling thecapillary with buffer, the sample canbe introduced by dipping the end ofthe capillary in the sample solution.Pressure, suction or application ofan electric field drives the sample inthe capillary.

In CE nothing is retained so theanalogous term to “retention time”is migration time.

How small is the capillary tube used in CE?

•In typical CE instruments, the inner diameter of the capillary is25 to 150 μm, the length is usually 50 cm.

•It also could be small enough to insert into a single, largeliving cell to analyze its contents (sensitivity of enzymeanalysis can reach zeptomol, 10-21 mol).

•Smaller cells can be taken one at a time into the capillaries,burst open and analyzed.

Lecture 9- PHCM662-SS2016

The inside wall of the capillary is covered by silanol groups (SiOH) that aredeprotonated at pH > 3  SiO-.

SiO- attracts cations of the buffer solution which become tightly adsorbed andhence immobile (THE RIGID LAYER) .

The following layer is rich in cations but still mobile (THE DIFFUSE LAYER).

These 2 layers represent an “electrical double layer”.

Lecture 9- PHCM662-SS2016

What happens in the capillary of CE?

Predominance of cations in the diffuse part produces ELECTROOSMOTICFLOW (EOF) towards the cathode, exceeding the opposite flow towards theanode.

i.e. Net flow occurs as solvated cations drag along the solution. The netflow becomes quite large for high pH situations – a 50 mM pH 8 buffer flowsthrough a 50-cm capillary at 5 cm/min with 25 kV applied potential.

Lecture 9- PHCM662-SS2016

Electroosmosis, the PUMP of CE…

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EOF contributes significantly to high resolutionunlike pressure-driven flow in LC and relatedtechniques, explain.

Lecture 9- PHCM662-SS2016

Electroosmotic versus hydrodynamic Flow Profiles

Cathode

Anode

Electroosmotic flow profile

Hydrodynamic flow profile

High

Pressure

Low

Pressure

1.Driven by charge alongcapillary wall.

2.no pressure drop isencountered.

3.flow velocity is uniform acrossthe capillary.

1.Driven by pressure difference.

2.Frictional forces at thecolumn walls causedifferences in flow velocityacross the capillary.

•veo = μeo E

μeo = Electroosmotic mobility is

1.proportional to the surface charge density on silica.

2.inversely proportional to the square root of the ionic strength of buffer.

i.e. electroosmosis decreases at low pH and high ionic strength.

Apparent Mobility μapp = μep + μeo

Lecture 9- PHCM662-SS2016

Electroosmosis, the pump of CE…

Electrophoretic mobility

Total apparent mobility

- -

Electroosmotic mobility

CATHODE

ANODE

Combining the two effects for migration velocity of an ion (also appliesto neutrals, but with ep = 0:

At pH > 2, cations flow to cathode because of positive contributionsfrom both ep and eo.

At pH > 2, anions flow to cathode in spite of the negative contributionfrom ep, due to the significant positive contribution from eo (if EOF isstrong enough). At low pH, eo is weak and anions may never reach thedetector, where ep predominates.

At pH > 2, neutrals flow to the cathode because of eo only.

Lecture 9- PHCM662-SS2016

Electrophoresis and Electroosmosis

Net EOF becomes large at higher pH:

–A 50 mM pH 8 buffer flows through a 50-cm capillary at 5 cm/min with 25kV applied potential

EOF can be quenched (stopped) by protection of silanols or low pH.

Uniform EOF contributes to the high resolution of CE, by minimizing peakwidth.

Lecture 9- PHCM662-SS2016

Silanols fullyionized abovepH = 9

Electroosmotic Flow Profile

Unprecedented resolution in CE! N= 50,000-500,000 routinely!!

WHY?????

Van Deemter Equation:

relates the plate height H to the velocity of the carrier gas orliquid mobile phase.

Where A, B, C are constants,and a lower value of Hcorresponds to a higherseparation efficiency.

Lecture 9- PHCM662-SS2016

CE versus chromatography (HPLC)

In CE, a very narrow open-tubular capillary is used

–No A term (multipath) because there is NO stationary phase (fused silicais not a stationary phase, i.e. There is no retention).

–No C term (mass transfer) because there is NO stationary phase.

–Only the B term (longitudinal diffusion) remains:

Cross-section of a capillary:

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Lecture 9- PHCM662-SS2016

CE versus chromatography (HPLC)

The flow of ions in the capillary generates heat. “Joule heating” is aconsequence of the resistance of the solution to the flow of current.Typically, the centerline of the capillary channel is 0.02 to 0.3 K hotterthan the edge of the channel.

 If heat is not sufficiently dissipated from the system the resultingtemperature and density gradients can reduce separation efficiency(effect on peak shape, how?) : viscosity is lower in the warmer regiondisturbing the flat electroosmotic profile of the fluid.

Heat dissipation is key to CE operation, for smaller capillaries (d=50μm) heat is already dissipated due to the large surface area tovolume ratio, yet it is a serious problem if d>1 mm.

The ability to dissipate heat results in extremely fast separations withexcellent resolution are possible by application of high potentials(30kV)

Lecture 9- PHCM662-SS2016

Joule Heating

Detectors are placed at the cathode sinceunder common conditions, all species aredriven in this direction by EOF.

Detectors similar to those used in LC, typicallyUV absorption, fluorescence, and Massspectrometry.

Lecture 9- PHCM662-SS2016

Detection and Quantitative Analysis

For quantitative analysis, normalized peak areas are used: analytes with differentapparent mobilities pass through the detector at different rates. The higher theapparent mobility, the shorter the migration time, and the less time the analytespends in the detector which may erroneously decrease the detector’s response to it.Hence, normalization involves dividing each peak area by its migration time.

Remember? In chromatography, each analyte passes through the detector at thesame rate under the effect of pressure, so peak area is proportional to the quantity ofanalyte.

•For maximum precision, mobilities are measured relative to an internal standard.Absolute variations from run to run should not affect relative mobilities.

Proteins (enzymes at zeptomol level 10-21!), Peptides, Aminoacids.

Nucleic acids (RNA and DNA) also analyzed by slab gelelectrophoresis.

Inorganic ions.

Organic bases, acids.

Whole cells (if large cells, the capillary is introduced intothem!).

Lecture 9- PHCM662-SS2016

Types of Molecules that can be Separated by CE

1.CE is based on the principles of Electrophoresis andElectroosmosis.

2.The speed of movement or migration of solutes in CE isdetermined by their electrophoretic mobility which dependson ions‘ charge and size. Small highly charged solutes willmigrate more quickly then large less charged solutes. Also thepH and ionic strength of the buffer change theirelectroosmotic mobilities.

3.Bulk movement of solutes is caused by EOF.

4.The speed of EOF can be adjusted by changing the buffer pH.

5.The flow profile of EOF is flat, yielding high separationefficiencies.

6.http://bio-animations.blogspot.com/2008/04/capillary-electrophoresis.html

Lecture 9- PHCM662-SS2016

Conclusion

QUIZ 2

Quiz 2 will take place on 14-04-2016 at 4:00PM and will include lectures 6, 7 and 8 +tutorials 6,7.

No make up is offered for the quiz

Please respect locations of your groups.

Lecture 9- PHCM662-SS2016

Hall

Date / Time

Groups

Thursday ,

14-4-2016,

4:00-4:30

1-4

6-9

H12

10-13

H13

14-17

H14

18-20

Location and time of Quiz 2

Lecture 9- PHCM662-SS2016

1.Textbook: Principles of instrumental analysis, Skoog et al.,chapter 30.

2.Textbook: Quantitative Chemical Analysis, Harris, chapter 26.

3.Capillary Electrokinetic Separations, lecture in University ofVillanova.

4.Extra resources on intranet folder:

V:\Faculties\Pharmacy & Biotechnology\PharmaceuticalChemistry\Instrumental Analysis II_ PHCM662_SS2014\Extra resources

Lecture 9- PHCM662-SS2016

References