Capillary electrophoresis

Capillary electrophoresis

1. Capillary electrophoresis Presented by : k . jayalakshmi D/o k. kristaiah 1st yr M.pharm, pharmaceutical analysis 17-06-2020 1

2. Contents Introduction Principle Mechanism CE-steps & detectors Methods and modes Advantages & Disadvantage's Applications References 17-06-2020 2

3. Introduction Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. Electrophoresis of positively charged particles is sometimes called cataphoresis, while electrophoresis of negatively charged particles is sometimes called anaphoresis . Principles .   Electrophoresis  is a general term that describes the migration and separation of charged particles (ions) under the influence of an electric field . An  electrophoretic  system consists of two electrodes of opposite charge (anode, cathode), connected by a conducting medium called an electrolyte. 17-06-2020 3

4. TYPES  OF  ELECTROPHORESIS   Zone   Electrophoresis   Paper   Electrophoresis   Gel   Electrophoresis   Thin Layer  Electrophoresis   Cellulose acetate  Electrophoresis   Moving Boundary  Electrophoresis   Capillary   Electrophoresis   Isotachophoresis Isoelectric Focussing Immuno   Electrophoresis 17-06-2020 4

5. What is Capillary Electrophoresis? Electrophoresis : The differential movement or migration of ions by attraction or repulsion in an electric field Capillary electrophoresis is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. 17-06-2020 5

6. 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. 17-06-2020 6

7. 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). 17-06-2020 7

8. Types of Molecules that can be Separated by Capillary Electrophoresis Proteins Peptides Amino acids Nucleic acids (RNA and DNA) – also analyzed by slab gel electrophoresis Inorganic ions Organic bases Organic acids Whole cells 17-06-2020 8

9. Mechanism Capillary electrophoresis works on 2 mechanisms They are Electro osmatic flow Movement/direction of the bulk solution Electrophoretic mobility Separation of charged species Electro osmatic flow It is the movement of the separation buffer through the silica capillary is a results of the existence of a zeta potential at the solvent /silica interface. 17-06-2020 9

10. • Net flow becomes is large at higher pH: • Key factors that affect electroosmotic mobility: dielectric constant and viscosity of buffer (controls double-layer compression) • EOF can be quenched by protection of silanols or low pH • Electro osmotic mobility : Where : v = electroosomotic mobility εo = dielectric constant of a vacuum ε = dielectric constant of the buffer ζ = Zeta potential η = viscosity E = electric field 17-06-2020 10

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13. Capillary  Zone  Electrophoresis  (CZE), also known as free-solution CE, is the most standard form of CE.  Buffer  is flushed through the  capillary  by pressure, sample is injected and high voltage is applied. Dependable on the polarity the EOF is towards the inlet or the outlet. 17-06-2020 13

14. The Basis of Electrophoretic Separations Migration Velocity : Where: v = migration velocity of charged particle in the potential field (cm sec -1 ) µ ep = electrophoretic mobility (cm2 V-1 sec-1) E = field strength (V cm -1) V = applied voltage (V) L = length of capillary (cm) Electrophoretic mobility : Where : q = charge on ion η = viscosity r = ion radius 17-06-2020 14

15. CE-STEPS Electrophoresis is done in buffer filled , narrow bore capillaries Each Capillary is about 25-100 µm in internal diameter. Small cross sectional area, long length, leading to high resistance, low currents. Vmax =20-100Kv. N = 100,000-10,000,000 high resolution . When a Voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge Depending on the Charge , the molecules move through at different speeds. Thus Separation is Achieved. A suitable detector is then used to detect the solute as it comes out from the end of the Capillary. The data Obtained are analyzed by a Computer and represented Graphically. 17-06-2020 15

16. Detectors used in capillary electrophoresis 17-06-2020 16

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18. Common Modes of CE in Analytical Chemistry Capillary Zone electrophoresis ( CZE) Capillary gel electrophoresis ( CGE) Capillary isoelectric focusing (CIEF ) Capillary isotachophoresis (CITP ) Micellar electrokinetic capillary chromatography (MEKC) 17-06-2020 18

19. Capillary Zone Electrophoresis (CZE ) Capillary Zone Electrophoresis (CZE), also known as free-solution CE (FSCE), is the simplest form of CE (what we’ve been talking about ). The separation mechanism is based on differences in the charge and ionic radius of the analytes. Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. 17-06-2020 19

20. Capillary Gel Electrophoresis (CGE ) Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary using polymers in solution to create a molecular sieve also known as replaceable physical gel . This allows analytes having similar charge-to-mass ratios to also be resolved by size. This technique is commonly employed in Gel molecular weight analysis of proteins and in applications of DNA sequencing and genotyping. 17-06-2020 20

21. Capillary Isoelectric Focusing (CIEF ) Capillary Isoelectric Focusing (CIEF) allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. At the solute’s isoelectric point (pI), migration stops and the sample is focused into a tight zone. In CIEF, once a solute has focused at its pI, the zone is mobilized past the detector by either pressure or chemical means. This technique is commonly employed in protein characterization as a mechanism to determine a protein's isoelectric point. 17-06-2020 21

22. Capillary Isotachophoresis (CITP ) Capillary Isotachophoresis (CITP) is a focusing technique based on the migration of the sample components between leading and terminating electrolytes. (isotach = same speed ) Solutes having mobilities intermediate to those of the leading and terminating electrolytes stack into sharp, focused zones. Although it is used as a mode of separation, transient ITP has been used primarily as a sample concentration technique. 17-06-2020 22

23. Micellar Electrokinetic Capillary Chromatography Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a mode of electrokinetic chromatography in which surfactants are added to the buffer solution at concentrations that form micelles. The separation principle of MEKC is based on a differential partition between the micelle and the solvent (a pseudo-stationary phase ). This principle can be employed with charged or neutral solutes and may involve stationary or mobile micelles . MEKC has great utility in separating mixtures that contain both ionic and neutral species, and has become valuable in the separation of very hydrophobic pharmaceuticals from their very polar metabolites. Micellar 17-06-2020 23

24. Micellar Electrokinetic Capillary Chromatography 17-06-2020 24

25. The MEKC surfactants are surface active agents such as soap or synthetic detergents with polar and non-polar regions . At low concentration, the surfactants are evenly distributed At high concentration the surfactants form micelles. The most hydrophobic molecules will stay in the hydrophobic region on the surfactant micelle. Less hydrophobic molecules will partition less strongly into the micelle. Small polar molecules in the electrolyte move faster than molecules associated with the surfatant micelles. 17-06-2020 25

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27. Advantages and Disadvantages of CE 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 17-06-2020 27

28. Disadvantages Disadvantages Cannot do preparative scale separations “Sticky” compounds Species that are difficult to dissolve Reproducibility problems Aged , improperly stored blood samples – degradation products Abnormal Hb – use other means of identification 17-06-2020 28

29. Applications • Applications (within analytical chemistry) are broad: – For example, CE has been heavily studied within the pharmaceutical industry as an alternative to LC in various situations • detecting bacterial/microbial contamination quickly using CE – Current methods require several days. Direct inoculation (USP) requires a sample to be placed in a bacterial growth medium for several days, during which it is checked under a microscope for growth or by turbidity measurements False positives are common (simply by exposure to air) Techniques like ELISA, PCR, hybridization are specific to certain microorganisms 17-06-2020 29

30. References 1.Watson G.David,pharmaceutical analysis,2nd edi.2005,Churchill Livingstone,Pno.333-353. 2.Frank A. Settle,Handbook of Instrumental Techniques for Analytical chemistry,1st edi.,2004,Pearson education,Pno.165 . 3.http :// www.ceandcec.com/presentation.htm 4.http ://www.hbc.ukans.edu/CBAR/Electrochrom.htm 17-06-2020 30

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