Capillary Electrophoresis (CE) PHAR

Lecture Objectives By the end of the lecture, students should be able to: 1.Illustrate the CE instrumental set up 2.Discuss the method of separation 3.Discuss the migration order 2

What is CE? Separation technique based on differential migration of molecules based on charge when subjected to an electric field. Uses narrow width fused-silica capillaries Electric fields about V/cm Provides short analysis time High separation 3

Introduction Rapidly expanding Rival to HPLC in its general applicability Used to separate and quantify many types of samples – Peptides, proteins, nucleic acid, inorganic anions, pharmaceutical drug compounds, etc. 1 major difference when compared to other separating method Uses an electric fields which causes the separation and not a flow of solute through the column 4

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Instrumentation Capillaries Detectors Power supplier Data handling 6

Capillaries Most use fused silica capillaries to ensure occurrence of an electro-osmotic flow 50-75μm i.d. and 375 μm outer diameter Small diameter were found to dissipate heat very well For good separation, use smallest i.d. In-expensive Easy to use Will last for a longer time 7

Detectors Absorbance, small window near the detection end Molecules passing the window are detected by their absorbance of a beam light passing through this window Often referred to as on-column detection Signal obtained is proportional to the path length, which is the i.d. Fluorescence, molecules are excited by a beam of light and their fluorescence is detected at right angles Method is more sensitive but have a disadvantage 8

Power supplier Is required to provide an electric field Most can be operated with a constant voltage or current Increasing voltage, will increase the EOF and reduces the migration time Leads to shorter analysis time 9

Data handling Electropherogram is a plot of detector response versus the migration time Qualitative analysis can be done by measuring the migration time of the unknown analyte and comparing it to a standard Peak height and areas are proportional to concentration 10

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Sample separations The separation column is the crucial component Separation due to 2 major factors 1.Electrophoretic migration 2.Electro-osmotic flow (EOF) 12

Electrophoretic migration Movement of charged molecules towards an electrode of opposite polarity in the electric field Different velocities of different compounds migrate through the capillary at different rates – Size and charge Typically, the larger the compounds the slower it will migrate Charged ions are strongly dependent on pH Therefore, for good separation the pH of the buffer used should be between the pK values of the sample analytes Change in pH of the buffer may alter the mobility of the different analytes 13

Electro-osmotic Flow (EOF) the silanol groups on the wall bears a negative charge The pKa of the acidic silanol group ranges from and the amount of negative charge on the wall will increase as pH rises Cations in the running buffer are attracted to the negative charge on the wall When a potential is applied across the capillary, the cations in solution migrate towards the cathode As the cations migrate they will pull along the buffer solution with them So the net flow of the buffer from the positive to the negative end of the capillary in the presence of an electric field is called electro-osmotic flow 14

EOF (2) Rate of EOF is pH dependent since the negative charge on the silanol groups increases with pH EOF decreases with buffer strength since a larger concentration of anions in the buffer will reduce the positive potential at the capillary wall 15

EOF differs from pressure driven flow When pressure is applied at one end of a fluid filled tube, fluid will begin to move through the tube Because of this friction, the velocity of pressure driven flow is greatest at the center of the tube, and slowest at the walls. This is known as Parabolic or Laminar flow 16

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Migration in CE Existence of EOF means that all species, will move towards the cathode Cations, move at a rate determined by their ion mobility plus the EOF Neutral compounds move at the same rate as the EOF Anions, move at the rate of EOF minus their ion mobility 18

Migration (2) Cations migrates most quickly, with smaller cations reaching the cathode first Neutral species move at the same rate as EOF Anions migrate most slowly, with the smallest anions reaching the cathode last 19

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An over view of the system A capillary is filled with a conductive fluid of a certain pH Sample is introduced into the capillary by an injection method High voltage are applied across the capillary Due to this electric field the sample components move (migrate) through the capillary at different speeds Therefore, causing compounds to separate As compounds migrate through the capillary, they will be detected and recorded Electropherogram which is a plot of detector response versus the time of migration Each peak corresponds to each compound contained in the sample solution 21

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Joule heating When electric field is applied, system tends to heat up This referred to as joule heating This heat will cause a temperature gradient within the system which could lead to extra dispersion and also lead to further change in viscosity of the system Therefore, crucial to control or minimize this heat Most effective way is to use an external method – E.g. by blowing air outside of the capillary 23

Types of CE Capillary zone electrophoresis (CZE) Capillary gel electrophoresis (CGE) Micellar electrokinetic chromatography (MEKC) Capillary electro chromatography 24

Capillary Zone Electrophoresis (CZE) Most standard form Buffer is flushed through the capillary by pressure Sample is injected and high voltage is applied Highly dependent on the polarity of the EOF Each analyte will migrate through the capillary at its own speed Only the difference in mobility will cause the separation 25

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Capillary Gel Electrophoresis (CGE) Capillary contains a gel matrix Compounds with different size but the same mobility are separated with this technique Analytes are slowed down by the gel Frequently used for the separation of DNA and proteins 27

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Micellar Electrokinetic Chromatography (MEKC) The electrophoresis micelles are generated in the buffer These micelles have a non polar cavity and a polar (or charged) surface Depending on the affinity of the micelles, the analytes in the buffer solution will divide over the micelles and the buffer solution When the migration speed of the buffer differs from the speed of the micelles, separation is achieved due to the different affinity for the micelles 29

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Capillary Electro Chromatography Uses a capillary which is partly packed with silica based particles as a stationary phase When high voltage is applied, the buffer fluid will start to migrate due to the EOF Separating force in this technique will be the affinity of the sample for the stationary phase High voltage is used to pump the mobile phase through the packed bed in the capillary 31