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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.