capillary electrophoresis by sachin kuhire

By Sachin Kuhire

Junior Research FellowNational Chemical Laboratory,

PuneDate :- 01/10/2012

IntroductionInstrumentationSample introduction DetectionWorking Advantages DrawbacksExamplesReferences

Contents

Ref. –www.directindustry.com

What is Capillary Electrophoresis?

Capillary electrophoresis is a separation method based on the differential rates of migration of charged species in an applied dc electric field

Electrophoresis was first developed by Swedish chemist Arne Tiselius in the 1930’s(serum proteins)

He was awarded the Nobel prize for his work (1948) The speed of movement or migration of solutes in CE

is determined by their charge and size ratios. Small highly charged solutes will migrate more quickly then large less charged solutes.

Types of Molecules that can be Separated

by Capillary Electrophoresis

• Proteins ,vitamins• Peptides • Amino acids • Nucleic acids (RNA and DNA)• Inorganic ions • Organic bases • Organic acids • Enzymes• etc.

.

• A buffer filled fused-silica capillary

10-100 µm in internal diameter & 40-100 cm long• Two electrode(platinum) • High voltage supply (5 to 30 kv)• Sample injector (by pressure or vacuum)• Detector• Buffer solution (like sodium dihydrogen

phosphate,NaH2 PO4)

Instrumentation

Generic diagram of a capillary electrophoresis system

Ref.science vol 142

Electrophoretic Mobility• It is the process in which sample ions move under the

influence of an applied voltage.• The ion undergoes a force that is equal to the product of

the Electrophoretic mobility and the electric field strength.• The flow of ions is toward the opposite charged electrode.

Where,

µEP = Electrophoratic Mobility

q = Charge on ions

η = Viscosity

r = Radius

E = Electric field strength.

& VEPF = µEP .E µEP =6ηπr

q

Electroosmotic FlowThe heart of capillary electrophoresis

is electroosmotic flow(EOF).This is the mobile phase ‘’pump’’ in

capillary electrophoresis.The rate of EOF is generally greater

than the electrophoretic migration velocities

This flow occurs when buffer pH greater than 3 and the SiOH groups lose a proton to become SiO- Increase the pH intensity of EOF also increases.

V total = VEO + VEP

V electrophoretic (EP)

V electroosmatic (EO)

Electroosmotic Flow

Cross sectional flow profile Due to electro osmotic flow

Cross sectional flow profile Due to Hydrodynamic flow

Pressure

· Electroosmotic flow does not contribute significantly to band broadening like pressure-driven flow in LC and related techniques

Where,μEOF =Electro osmotic mobility.C= Dielectric constantE= Electric field strength ζ= Zeta potential.η= ViscosityVEOF=Velocity

μEOF = C.ζ 4πη & VEOF= μEOF.E

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

● Bulk movement of solutes is caused by EOF● EOF is usually sufficient to sweep all +ve,

neutral, -ve species towards the same end.

The velocity of ions is sum of velocity of EOF and EPF ( VTotal=VEOF+VEPF

.

• If the analyst wants the EOF in opposite Direction then the capillary can be coated with a cationic Surfactant or added to the buffer.

• Ex. Trimethylchlorosilane.• Coated capillary also available in

market.• This flow is toward the positively

charged electrode.

Reversed EOF

Ref- camis.sr.unh.edu

• Hydrodynamic injection• By applying pressure• By applying vacuum.• By gravitation

• Electrokinetic injection• By using Electric supply

Sample injection.

.

• Detectors similar to those used in GC,HPLC• majority of instruments have UV detectors

available. • Alternative detector modes include commercially

available fluorescence, laser induced fluorescence, conductivity and indirect detection.

• The mass spectrometers is frequently used to give structural information on the resolved peaks.

• Sensitive detectors are needed for small concentrations in CE

Detectors

· Electropherogram is like a chromatogram · A plot of the time from injection on X-axis Vs The

detector signal on Y- axis.· The general layout of an electropherogram

Det

ecto

r Re

spon

se

Time

Anion

Neutral

Cation

The Electropherogram.

Capillary Zone electrophoresis (CZE)

Capillary gel electrophoresis (CGE)

Capillary electrochromatography (CEC)

Capillary isoelectric focusing (CIEF)

Capillary isotachophoresis (CITP)

Micellar electrokinetic capillary chromatography (MEKC)

Common Modes of CE in Analytical Chemistry

Capillary Gel Electrophoresis (CGE

Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary .CGE uses separation based on the difference in solute size as a particle migrate through the gel.Gels prevent the capillary walls from absorbing then solute

Capillary Isoelectric Focusing (CIEF)

Capillary Isoelectric Focusing (CIEF) is a technique commonly used to separate peptides and proteinsThese molecule are called zwitterionic compounds.So, each molecule has a specific isoelectric point (pI).If pH = pI then molecule become a neutral.

Capillary Isotachophoresis (CITP)

• Capillary Isotachophoresis (CITP) is a focusing technique based on the migration of the sample components between leading and terminating electrolytes.

• Micellar Electrokinetic Capillary Chromatography

• MEKC is a separation technique that is based on solutes partitioning between micelles and the solvent

• Without micelles neutral molecule will migrate with the electroosmotic flow and no separation occurs.

• The aggregates have polar negatively charged surfaces and are attracted to the positively charged anode.

Some Examples

From P.Jandik, W. R. Jones, O. Weston, andP. R. Brown, LC-GC, 1991, 9, 634.

Separation of Metals ions

Detection:UV,214 nm.Peaks: 1 = rubidium (2 ppm),2 = potassium (5 ppm), 3 = calcium (2ppm), 4 = sodium (1 ppm), 5 =magnesium (1 ppm), 6 = lithium (1ppm),

13 = gadolinium (5 ppm),14 =terbium (5 ppm), 15 = dysprosium(5ppm) 16 =holmium (5 ppm) 17 =erbium (5 pprn),18 = thulium (5 ppm), 19 = ytterbium (5 ppm).

7 = lanthanum (5 ppm), 8 =cerium (5 ppm), 9 = praseodymium (5ppm),10 = neodymium (5 ppm) 11 =samarium (5 ppm), 12 = europium (5ppm)

Here UV detector used (254 nm) 1-thiosulphate2-bromide3-chloride4-sulfate5-nitrite6-nitrate7-molybdate8-azide9-tungate10-monoflorosulphate11-chlorate12-citrate13-fluoride14-formate15-phosphate16-phosphite17-chlorite18-galactarate19-carbonate20-acetate

21-ethanesulphonate22-propionate23-propanesulphonate24-butyrate25-Bu-sulphonate26-varalate27-benzoate28-l-glutamate29-pn-sulphonate30-d-gluconate

Ref. W.A.Jones andP.Jandik,J.Chromatogr.,1991,546,445

Electropherogram of some ions

From P.Jandik, W. R. Jones, O. Weston, andP. R. Brown, LC-GC, 1991, 9, 634.

Separation of Metals ions

Detection:UV,214 nm.Peaks: 1 = rubidium (2 ppm),2 = potassium (5 ppm), 3 = calcium (2ppm), 4 = sodium (1 ppm), 5 =magnesium (1 ppm), 6 = lithium (1ppm),

13 = gadolinium (5 ppm),14 =terbium (5 ppm), 15 = dysprosium(5ppm) 16 =holmium (5 ppm) 17 =erbium (5 pprn),18 = thulium (5 ppm), 19 = ytterbium (5 ppm).

7 = lanthanum (5 ppm), 8 =cerium (5 ppm), 9 = praseodymium (5ppm),10 = neodymium (5 ppm) 11 =samarium (5 ppm), 12 = europium (5ppm)

peakA= unknown impurity;B=labeled lysine; C= dilabeled lysine;D= leucine;E= serine; F= glycine; G and H =unknown impurities; I= dilabeled cystine; J= glutamic acid;K= aspartic acid; L= cysteic acid.

Electropherogram of

amino acids

Ref. Science,Vol-222, Pg.266

• Advantages• Offers new selectivity, an alternative to HPLC • Easy and predictable selectivity • High separation efficiency (millions of theoretical plates) • Small sample required (1-10 nl) • Fast separations (1 to 45 min) • Can be automated • Easily coupled to MS• Different “modes”

• Disadvantages• Can not do preparative scale separations• Low concentrations and large volumes difficult

Advantages and Disadvantages.

.

• “Principles of instrumental analysis”, 6th edition, by holler skoog & crouch, page 1003-1013.

• Manuel J. Gordon,Xiaohua,Stephen L.,science Vol.242 page 224-228.

• James W Jorgenson science Vol.222, page 266-272

References:

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