Capillary Electrophoresis

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By: wendelfba (40 month(s) ago)

Very nice presentation, congratulations.

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Capillary Electrophoresis : 

Capillary Electrophoresis

Electrophoresis—An Overview : 

Electrophoresis—An Overview Definition: The differential movement for migration of ions by attraction or repulsion in an electric field. Separation of components of a mixture using an electric field v=Eq/f v = velocity of molecule E = electric field q = net charge of molecule f = friction coefficient

Electrophoresis- overview cont. : 

Electrophoresis- overview cont. Can determine the size, shape, and charge of a molecule Different forms of electrophoresis are used for each of these factors independently or in combination.

Types of Electrophoresis : 

Types of Electrophoresis Capillary Native Polyacrylimide Gel Electrophoresis (PAGE) SDS-PAGE Slab Paper

Capillary Electrophoresis Apparatus : 

Capillary Electrophoresis Apparatus

Capillary Electrophoresis – The Basics : 

Capillary Electrophoresis – The Basics Electrophoresis in a buffer filled, narrow-bore capillaries Each capillary is about 25-100 μm in internal diameter 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 Separation is achieved

Basics cont. : 

Basics cont. A photocathode is then used to measure the absorbencies of the molecules as they pass through the solution The absorbencies are analyzed by a computer and they are represented graphically

Electrophoretic Mobility : 

Electrophoretic Mobility The movement of ions solely due to the electric field, potential difference Cations migrate toward cathode Anions migrate toward anode Neutral molecules do not favor either

Electrophoretic Mobility : 

Electrophoretic Mobility v=Eq/f vep = μepE μ = q/(6πηr) η is buffer viscosity r is solute radius Properties that effect mobility Voltage applied Size and charge of the solute Viscosity of the buffer

Electroosmotic Flow : 

Electroosmotic Flow As the buffer sweeps toward the anode due to the electric field, osmotic flow dictates the direction and magnitude of solute ion flow within the buffer All ions are then swept toward the anode. Negative ions will lead the neutral ions toward the anode Positive ions will trail the neutral ions as the cathode pulls them

Electroosmotic Mobility : 

Electroosmotic Mobility veof = μeofE μeof = ɛζ / (4πη) ɛ = buffer dielectric constant ζ = zeta potential Zeta Potential The change in potential across a double layer Proportional to the charge on the capillary walls and to the thickness of the double layer. Both pH and ion strength affect the mobility

Total Mobility : 

Total Mobility vtot = vep + veof Migration times vtot = l/t l = distance between injection and detection t = migration time to travel distance l t = lL/((μep + μeof)V L = length of capillary V = voltage

Slide 14: 

Ooooo! It MOVES

Equipment : 

Equipment Capillary tube Varied length but normally 25-50 cm Small bore and thickness of the silica play a role Using a smaller internal diameter and thicker walls help prevent Joule Heating, heating due to voltage

Equipment : 

Equipment Because ions are in the bulk solution are about to travel through the capillary without interference from the capillary itself, there is no dramatic drop in potential within the capillary No meniscus is made

Equipment : 

Equipment Detector UV/Visible absorption Fluorescence Radiometric (for radioactive substances) Mass Spec. Yowzah!!!

Applications : 

Applications Analysis of carbohydrates Analysis of inorganic anions/metal ions DNA profiling Protein identification Advantages Fast Small Sample Relatively inexpensive Automated Disadvantages Cannot identify neutral species Joule Heating Cannot discern shape