Capillary electrophoresis principles and applications

Brief description of principle and applications

1. Capillary electrophoresis: principles and applications Dr. Indira shastry Kasturba medical college Manipal university, Manipal

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 20-100 m m 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 (t m ) for that distance is measured. Relate time to identity. Relate peak area or height to amount.

8. Electro-osmotic flow

9. Movement of Analyte Analyte ν = µ E ν = velocity µ = electrophoretic mobility E = Electric field Electrophoretic mobility µ = q/[6 πη r] q = charge η = solution viscosity r = radius Electro-osmotic flow ν EOF = [ ε /4 πη ] ζ E ε = dielectric Constant ζ = Zeta potential Flow of migration ν = [( μ EO + μ e )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.

12. 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)

13. 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)

14. 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

15. 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

16. 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

17. 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 .

18. 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

19. 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

20. 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

21. 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 : 5 μ l. 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.

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

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

24. 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).

25. 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.

26. 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 ).

27. 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 ( α met), A ( β met), and A ( αβ met) : interpretation more difficult. Identification of variants are still provisional.

28. 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

29. 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

30. Abnormal hemoglobin detection by using CZE Hb move from Cathode to anode

31. 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

32. 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

33. Normal pattern in infants < 6mnths Z9- Hb a Z7- Hb F (large peak) Z3: Hb A2

34. Capillary Serum protein (E) 6 Proteins are clearly separated in to 5/6 fractions: Albumin Globulins – α 1, α 2, β 1+/ β 2 and γ uses: Inflammatory response Immune reaction Quantification of proteins Adv: minimal TG /LP/bile interference with unsurpassed assay clarity ( α 1). Additional wash programme. Dis adv: monoclonality may not be detected

35. In hemolytic anemia

36. Capillary serum protein electrophoresis : high resolution (HR) Designed for multifraction human serum proteins : Albumin α 1 acid glycoprotein ( oromucoid ) α 1 antitrypsin Haptoglobulin Transferrin C3 complement CRP Gamma globulins Can be quantified

37. α 1 Antitrypsin deficiency

38. Uses Intravascular hemolysis Nephrotic syndrome ( ↓ albumin, α 1 acid glycoprotein, transferrin and haptoglobin ) Nutritional problems: dec in albumin levels

39. 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

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

41. 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.

42. 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 10 th 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.