2. 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.
3. ▪ Separation of components in a mixture in an electric field depends
on velocity.
▪ v=Eq/f
V = velocity of molecule
E = electric field
Q = net charge on a molecule
F = friction coefficient
4. 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
5. 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.
6. 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).
7. ▪ 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.
9. 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
10. 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.
11. ▪ 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.
13. 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)
15. 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
16. 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
17. 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
18. 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.
19. 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
20. 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
21. 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
22. 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.
25. 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).
26. ▪ 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.
27. 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).
28. 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.
29. 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
30. – 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
32. 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
33. ▪ 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
34. ▪ Normal pattern in infants
< 6mnths
▪ Z9- Hb a
▪ Z7- Hb F (large peak)
▪ Z3: Hb A2
35. 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
41. Uses
▪ Intravascular hemolysis
▪ Nephrotic syndrome (↓albumin, α1 acid glycoprotein,
transferrin and haptoglobin)
▪ Nutritional problems: dec in albumin levels
42. 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
45. 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.
47. 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.
Editor's Notes
In CE separation occurs because analytes have different electrophoretic mobilities. In the simplest approximation, electrophoretic
mobility can be because of analyte charge and size. Large, singly charged analytes will travel slower than small,
singly charge analytes, and small, doubly charged ions will travel faster than larger, doubly charged analytes,
etc. In other forms of CE separation is more complicated.The electrical potential also effects this process.
Electro-osmosis: When the voltage is applied to the circuit, one electrode become net positive and the other net negative. The
(wall’s) immobile silanol anions pair with mobile buffer cations, forming a double layer along the wall (wall-->
buffer cations-->buffer anions-->bulk buffer solution). The remaining buffer cations are attracted to the negative
electrode, dragging the bulk buffer solution with them. This is electroosmotic flow. For an uncoated capillary,
the EOF is toward the negative electrode.
Detection in CE requires techniques for small samples.
In CE, analytes with different migration times also spend different times in the detector. Comparing peak areas requires a correction to obtain the corrected peak area
Chiral separation : is a process for the separation of racemic compounds into their enantiomers
Ag-Ab, Protein – DNA, DNA- dye, enzyme-substrate, enzyme-inhibitor, protein-inhibitor.......
Separation based on interaction between Stationary Phase & Mobile Phase. Stationary Phase is Analytical Cartridge; Mobile Phase is Buffer.
Compounds are separated to target analytes according to physical properties:- size, shape, charge, hydrophobicity & affinity for other molecules.
Neonatal diagnosis for Hb S and Hb D-Punjab. Analysis of RBC lysates obtained from a dried blood spot obtained during the neonatal screening programme done in our laboratory. Analysis by two methods is required for the diagnosis. (A) Isoelectric focusing: Hb S and Hb D‑Punjab have close isoelectric points and the resolution between the two bands is poor. The absence of HbA and thickening of HbS band help to diagnose. (B) Cation‑exchange HPLC shows two abnormal peaks: one is eluted in S window and the other in the D window. (C) Capillary electrophoresis: (above): newborn heterozygous for Hb D-Punjab and (below): newborn heterozygous for Hb S. The two Hbs have different migrations.
C migrate cathodaly to E, D and lepore migrate anodaly to s
Principle of IEC: Individual Hb molecule travel through gel until their individual PI are equal to the corresponding PH of gel : PH at which net charge of soule is 0 is PI
Three major haptoglobin phenotypes are known to exist (Hp 1-1, Hp 2-1, and Hp 2-2). Hp 1-1 is biologically the most effective in binding free hemoglobin and suppressing inflammatory responses associated with free hemoglobin. Hp 2-2 is biologically the least active, and Hp 2-1 is moderately active. The possible association of allelic polymorphism of haptoglobin with various pathologic conditions such as coronary artery disease has been studied