Name: capillary electrophoresis: sample separation setup
Uploaded: 2024-12-05T18:32:01.000Z
Duration: 1 min 20 s
Channel: JoVE

Capillary electrophoresis instrumentation includes a high-voltage power supply connected to an anode and a cathode placed in buffer reservoirs, a sample vial, a fused silica capillary tube, and a detector.
The buffer-filled capillary tube is coated with polyimide for mechanical strength and connected to two buffer reservoirs containing electrodes.
Sample introduction involves detaching one end of the capillary and its electrode from the associated buffer reservoir and placing it in the sample vial.
Hydrodynamic injection uses pressure, while electrokinetic injection applies an electric field to introduce the sample into the capillary tube.
If the sample concentration is too low, it is injected into a solution of lower ionic strength than the buffering solution to enhance its detection through a stacking technique.
Applying a high voltage across the buffer system causes charged species to move toward the cathode via electroosmotic flow.
The separation of components occurs based on their electrophoretic mobility within the electric field.
Detectors based on absorption, fluorescence, conductivity and mass spectrometry are commonly used in CE.

capillary electrophoresis: instrumentation

Capillary Electrophoresis: Instrumentation

Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary for the separation by connecting to an anode (the positively charged electrode) and a cathode (the negatively charged electrode) located in buffer reservoirs at each end of the capillary tube. The system includes a sample vial, a fused silica capillary tube coated with polyimide for mechanical strength through which the sample components migrate during separation, and a detector to analyze the separated components.
The sample must be introduced into the capillary tube to initiate the process. This is achieved by detaching one end of the capillary and its electrode from the associated buffer reservoirs and placing them in the sample vial. Sample introduction can be accomplished through hydrodynamic injection, which involves applying pressure to the sample vial, or electrokinetic injection, which relies on an electric field to drive the sample into the capillary.
When current flows through the capillary containing a conductive buffer solution, it leads to Joule heating due to the narrow bore of the capillary column and the relative thickness of the capillary&#39;s walls. Joule heating refers to the heat generated as an electric current passes through a conductive medium&#8212;in this case, the buffer solution inside the capillary. It is directly related to the energy dissipation in the form of heat, as defined by the equation Q = I&#178;Rt, where Q is the heat energy (in joules), I is the current, R is the resistance, and t is time. In Capillary electrophoresis, this heating can change the viscosity of the buffer solution, causing solutes in the center of the capillary to migrate faster than those near the walls, resulting in band broadening and degraded separation. Capillaries with smaller inner diameters generate less Joule heating, while those with larger outer diameters are more effective at dissipating heat.
A stacking technique may be employed to enhance detection sensitivity in cases where the sample concentration is low. This method involves injecting the sample into a solution with a lower ionic strength than the buffering solution, causing the sample components to concentrate at the interface between the two solutions.
Once the sample has been introduced, a high voltage is applied across the buffer system, prompting charged species to migrate toward the cathode through electroosmotic flow. The separation of components occurs based on their electrophoretic mobility within the electric field.
Various detectors, such as absorption, fluorescence, conductivity, and mass spectrometry, are commonly employed in capillary electrophoresis to detect and analyze the separated components.

Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary ...

capillary-electrophoresis-instrumentation

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JoVE Core

Analytical Chemistry

Unit 1

Principles of Chromatography

KEY TERMS AND CONCEPTS

capillary electrophoresis: sample separation setup

Capillary electrophoresis uses a high-voltage setup, a capillary tube, a sample vial, and a detector to separate charged molecules. The electric field comes from a power supply connected to an anode and a cathode in buffer reservoirs at each end of the capillary. The capillary is made of fused silica and coated with polyimide for added strength.

To start the separation, the sample must be placed inside the capillary. One end of the capillary and its electrode are removed from the buffer reservoir and moved into the sample vial. The sample can then enter by hydrodynamic injection, which uses pressure, or by electrokinetic injection, which uses an electric field.

As current passes through the conductive buffer in the narrow capillary, Joule heating can occur. Joule heating is heat made by electric current, and it follows the relation Q = I^2Rt, where Q is heat energy, I is current, R is resistance, and t is time. This heat can change buffer viscosity and make solutes in the center of the capillary move faster than those near the walls. The result can be band broadening and a poorer separation.

Capillary size helps manage this heating. Capillaries with smaller inner diameters produce less Joule heating, while capillaries with larger outer diameters remove heat more effectively. When the sample amount is low, a stacking step can improve sensitivity. In stacking, the sample is injected into a solution with lower ionic strength than the buffer, so the sample components collect at the boundary between the two solutions.

After sample introduction, a high voltage is applied across the buffer system. Charged species then migrate toward the cathode through electroosmotic flow, and they separate according to electrophoretic mobility in the electric field. Detectors such as absorption, fluorescence, conductivity, and mass spectrometry are used to measure the separated components.

Capillary Electrophoresis: Sample Separation Setup

capillary-electrophoresis-sample-separation-setup

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Capillary electrophoresis uses a high-voltage setup, a capillary tube, a sample vial, and a detector to separate charged molecules. The electric field comes from a power supply connected to an anode and a cathode in buffer reservoirs at each end of the capillary. The capillary is made of fused silica and coated with polyimide for added strength. To start the separation, the sample must be placed inside the capillary. One end of the capillary and its electrode are removed from the buffer...

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key terms and concepts

Learn how capillary electrophoresis uses a capillary, high voltage, sample injection, Joule heating control, stacking, and detectors to separate charged molecules.

Capillary Electrophoresis: Sample Separation Setup | JoVE K12

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Capillary Electrophoresis: Sample Separation Setup

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