Capillary electrophoresis: principles and applications

Dr. Indira shastry Kasturba medical college Manipal university, Manipal

What is electrophoresis ?

▪ Separation of solutes based on different rates of migration though an electric field through background electrolyte [running buffer].

▪ Anions (-) move toward the anode (+) & vice versa. Charge and size influence the movement of charged particles, in opposite ways.

▪ Separation of components in a mixture in an electric field depends on velocity.

▪ v=Eq/fV = velocity of moleculeE = electric fieldQ = net charge on a molecule F = friction coefficient

Types of electrophoresis

▪ Gel electrophoresis▪ High resolution electrophoresis▪ Capillary electrophoresis▪ Isoelectric focussing▪ Immunochemical ▪ Pulsed field ▪ 2-D electrophoresis

▪ Cellulose acetate electrophoresis at alkaline pH 8.5

▪ Citrate agar or acid agarose gel electrophoresis at PH 6.0

▪ Isoelectric focusing (IEF)▪ Automated High Performance

Liquid Chromatography (HPLC)▪ Globin chain electrophoresis ▪ Capillary electrophoresis

Capillary electrophoresis: principle

▪ Capillary tube is placed between two buffer reservoir, and an electric field is applied, separation depends on electrophoretic mobility & electro-osmosis .

▪ Defined volume of analysate is introduced in to the capillary by replacing one buffer reservoir with sample vial.

▪ Electrophoretic separation is measured by detector.

Capillary electrophoresis

▪ Using narrow bore tubes, CE removes the Joule heating effect, which decreases band broadening, giving faster separations than gel.

▪ CE uses tubes 20-100mm diameter and 20-100 cm in length.▪ CE is used with/without gel. Longitudinal diffusion is the main

source of band-broadening.▪ Higher electric fields result in high efficiency and narrow

peaks (analyte migrates faster).

▪ All analytes travel the same distance, but the migration time (tm) for that distance is measured.

▪ Relate time to identity.

▪ Relate peak area or height to amount.

Electro-osmotic flow

Movement of Analyte

Analyte ν = µ E ν = velocity µ =

electrophoretic mobility E = Electric field

Electrophoretic mobility µ = q/[6πηr] q = charge η =

solution viscosity r = radius

Electro-osmotic flow νEOF = [ε/4πη]ζE ε = dielectric Constant

ζ = Zeta potential Flow of migration• ν = [(μEO + μe)V]/L• V = potential L =

length of capillary

Capillary electrophoresis

▪ The tube in CE is typically silica, which may be coated or uncoated.

▪ Uncoated silica lead to electro-osmosis when run at neutral or basic pH due to de-protonation of silanol groups.

▪ In “normal polarity mode,” a sample with many types of ions can be injected (at the + end), and they then travel in the same direction toward the negative electrode through a detector.

▪ Observed mobility will be the sum of inherent electro-osmosis plus electrophoretic mobility.

▪ These affect time, efficiency, and separation.

▪ If an analyte has a migration rate faster than electro-osmosis, it may flow in the opposite direction of the electro-osmotic flow. This is known as the “reverse polarity mode.”

▪ Changing the degree of de-protonation (altering the pH) of the silica will alter electro-osmotic flow. Analysis is done by injecting at the negative electrode.

▪ Using a neutral coating in the tube reduces electro-osmosis, while a positive coating will reverse direction of flow toward the positive end.

Various separation modes for CE

▪ Capillary zone electrophoresis (CZE)▪ Non-aqueous capillary electrophoresis (NACE)▪ Capillary gel electrophoresis (CGE)▪ Capillary electro-kinetic chromatography (CEKC) / Capillary

electro-chromatography (CEC)▪ Micellar electro-kinetic chromatography (MEKC)▪ Micro-emulsion electro-kinetic chromatography (MEEKC)

▪ Capillary isoelectric focusing (CIEF)▪ Capillary iso-tachophoresis (CITP)▪ Pressurized capillary electro-chromatography (pCEC)▪ Affinity capillary electrophoresis (ACE)▪ Imuno-affinity capillary electrophoresis (IACE)▪ Nano-capillary electrophoresis (NCE)▪ Microchip-based capillary electrophoresis (Microchip-

based CE)▪ Micro-fluidic capillary electrophoresis (MFCE)

Capillary zone electrophoresis

▪ Technique intermediary btw classical zone electrophoresis & liquid chromatography

▪ Charged molecules separated by their electrophoretic mobility in an alkaline buffer (pH 9.4)

• High voltage protein separation in silica capillary tubes • Direct Hb detection at absorbance wavelength of 415nm at

cathodic end• Cathode to anode Hb A

Capillary Gel Electrophoresis

▪ Used for size & shape separation.

▪ Separation based on differences in solute size.

▪ Detection is by UV absorbance of chromohore.

▪ DNA sequencing▪ Protein analysis

Capillary Iso-electric Focusing

▪ Depends on PH buffer gradient▪ The capillary is coated inside with an ampholyte , when

the field is applied, will create a pH gradient.▪ Molecules migrate under influence of electric feild ▪ Uses: – Separation of proteins – Peptides – Amino acids – Drugs

▪ Not useful for chiral compounds

Affinity capillary electrophoresis (ACE)

▪ uses a biologically active compound in the running buffer. ▪ Adv : Measure specific interaction of anylate with ligand

(receptor, antibodies ,etc)▪ ACE can separate chiral analytes.

Immunoaffinity capillary electrophoresis (IACE)

▪ Combine immunoassay and CE ▪ Three step procedure: – Bio-selective absorption– Subsequent recovery of compounds from immobilised affinity

ligand – Separation of enriched compounds

▪ Rapidly emerging : analysis of low- abundance biomarkers▪ Uses : – DNA analysis – Pharmacological – Forensic

Advantages of Capillary electrophoresis

▪ Simple ▪ Automated▪ High efficiency of separation ▪ Short analysis time ▪ Low sample volume ▪ Ease of operation ▪ Ability to separate both charged and non-charged molecules ▪ Different mechanisms for selectivity ▪ Low cost ▪ Use aqueous rather organic solvents hence environment

friendly

Disadvantages of CE

▪ Aged , improperly stored blood samples – degradation products

▪ Abnormal Hb – use other means of identification

▪ Migration of Hb variant close to HbA – underestimation of Hb A & variant + overestimation of HbA2

▪ Sensitivity & resolution limits

Capillary Electrophoresis V/S High Performance Liquid Chromatography (HPLC) 

▪ Advantages: – Automated, utilise less staff

time and permit processing of large batches.

– Very small sample sufficient for analysis : 5μl.

– Quantification of normal and variant Hb available in every sample.

▪ Disadvantages – Hb A is separated in to its

component fractions of A0 and A1 ( subdivides in to several peaks)

– Various abnormal and normal Hb can have same retention time

– HbE and Hb Lepore co-elute with A2.

– Retention time of glycosylated and other derivatives of Hbs can be same as HbA0 and A2.

Separates HbS, A, A2, F, S, C, D and G

A) Iso-electric focusing, B) Cation exchange HPLC, C) Capillary Electrophoresis

Capillary electrophoresis V/S cellulose acetate electrophoresis

▪ Labor-intensive.

▪ Inaccurate in quantification of low-concentration variants (HbA2) and in detection of fast variants (HbH, Hb Barts).

▪ The precision and accuracy for Hb A2 using scanning of electrophoretic gels is poor (in comparison to HPLC).

▪ Smaples showing single band either in S or C position should be analysed further by acid agarose / citrate agar gel electrophoresis, HPLC or IEF.

▪ Exclude heterozygote – SD, SG, CE, or CO.

CE V/S citrate agar electrophoresis

▪ Used to differentiate Hemoglobin variants that migrate together on the cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).

CE V/S Iso electric focusing

▪ Hb F is separated in to F1( acetylated F) and F11▪ Hb A can produce 5 bands – A0, A1,A (αmet), A

(βmet), and A (αβmet) : interpretation more difficult. ▪ Identification of variants are still provisional.

Applications

▪ Hemoglobin electrophoresis : abnormal Hb detection and characterization

▪ Immuno-typing : monoclonality ▪ Protein electrophoresis [capillary protein (E) 6]▪ High resolution (HR) : multifraction human serum proteins▪ Carbohydrate deficient transferrin : chronic alcohol abuse ▪ Molecular diagnosis : – DNA sequencing :▪ Analysis of DNA fragment length / restriction patterns/

microsatellites ▪ Analysis of single strand polymorphism

– Diagnosis of neoplastic disorders ▪ Loss of heterozygosity ▪ Microsatellite instability ▪ Monoclonality assay ▪ Analysis of tumor related mutations ▪ Single nucleotide polymorphisms

– Diagnosis of hereditary disease and prenatal testing – Diagnosis of infectious disease

▪ Pharmaceutical and biopharmaceutical applications ▪ Forensic applications

Abnormal hemoglobin detection by using CZE

Hb move from Cathode to anode

Dans cheque zone: potential variants located in each zone

Zones Hb variant Zones Hb variant

1 Hb δ A2 9 Hb A

2 Hb c 10 Hb M-Iwate, Hope

3 Hb A2 11 Denatured Hb A, vassa, provience

4 Hb E 12 Hb bart

5 Hb S 13 Hb N-baltimore, norfolk

6 Hb D-punjab, Hb G-Norfolk,

14 Hb N-seattle

7 Hb F 15 Hb H , I (I-Texas)

8 Hb Lansing, atlanta , hinsdale

▪ Normal adult pattern :

▪ Z9- Hb A▪ Z3- HbA2▪ Normal range : ▪ Hb A : 97%▪ Hb A2: 2.5-

3.5%▪ Hb F: <1%

Dans cheque zone

▪ Normal pattern in infants < 6mnths

▪ Z9- Hb a▪ Z7- Hb F (large peak)▪ Z3: Hb A2

Capillary Serum protein (E) 6 ▪ Proteins are clearly separated in to

5/6 fractions: – Albumin – Globulins – α1, α2, β1+/ β2 and γ

▪ uses: – Inflammatory response – Immune reaction – Quantification of proteins

▪ Adv: minimal TG /LP/bile interference with unsurpassed assay clarity (α1). Additional wash programme.

▪ Dis adv: monoclonality may not be detected

In hemolytic anemia

Capillary serum protein electrophoresis : high resolution (HR)

▪ Designed for multifraction human serum proteins : – Albumin– α1 acid glycoprotein (oromucoid)– α1 antitrypsin – Haptoglobulin – Transferrin – C3 complement – CRP – Gamma globulins

Can be quantified

α 1 Antitrypsin deficiency

Uses ▪ Intravascular hemolysis▪ Nephrotic syndrome (↓albumin, α1 acid

glycoprotein, transferrin and haptoglobin) ▪ Nutritional problems: dec in albumin levels

Capillary immunotyping

▪ Serum sample is mixed with individual specific antisera Ag-Ab complex is rapidly formed in liquid medium

▪ Treated samples are electrophoresed interpretation is accomplished by comparing reference pattern

▪ Used for immunoglobulin quantification and detect monoclonality

▪ Adv : – No sample incubation is required – Alternative to immunofixation – Allows easy identification of monoclonal peaks

▪ Immunofixation in a pt with IgG monoclonal gammapathy with κ restriction.

Carbohydrate deficient transferrin (CDT)

▪ The major form of the iron-transport glycoprotein transferrin.

▪ Contains 2 N-linked di-sialylated oligosaccharide chains (glycans) and is named di-sialotransferrin.

▪ Regular high alcohol consumption (mean of at least 50–80 g/day) alters the glycosylation profile of transferrin.

▪ Lack one (disialotransferrin) or both (asialotransferrin). ▪ CDT concentration normalizes with a half-life of 1.5–2

weeks.

References

▪ Sebia capillary 2 maual ▪ Overview of capillary electrophoresis and its application in

pharmasutical feild ; J pharm educ res Vol 2 ; 2011▪ Dacie & Lewis practical haematology 10th ed. ▪ Capillary electrophoresis: Anja bosserhoff and claus

hellerbrand ; molecular diagnosis ▪ Carrier diagnosis and prevention of hemoglobinopathies

using capillary electrophoresis : P.C.Giordano▪ David F Keren protein electrophoresis in clinical diagnosis.▪ Henri Wajcman & Kamran Moradkhani : abnormal Hb

detection and characterisation.