Techniques of Capillary Electrophoresis: Introduction, Procedure

Introduction:

• Capillary Electrophoresis (CE) is a type of electrophoresis where the separation of molecules takes place within a narrow-bore capillary. Let's break down its concepts, mechanisms, and applications.

Basics of Electrophoresis:

• Electrophoresis is a laboratory technique used to separate charged particles under an electric field based on their size, charge, and shape.

• Molecules move towards the electrode with the opposite charge.

• The speed at which they move depends on their size, charge, and the nature of the medium in which they're moving.

Procedure:

1. Capillary Preparation:

• The capillary tube is first conditioned by rinsing with a buffer solution to establish the desired pH and ionic strength.

2. Sample Introduction:

• The sample can be introduced into the capillary by methods like hydrodynamic or electrokinetic injection.

3. Electrophoresis:

• The filled capillary is placed between two buffer reservoirs, each containing an electrode.

• Once the power supply is turned on, charged molecules migrate through the capillary toward the opposite electrode.

4. Detection:

• As molecules pass through a detection window near the capillary's end, they can be identified and quantified.

• Common detection methods include UV-Vis absorbance and fluorescence.

5. Data Analysis:

• Modern CE equipment often comes with software that facilitates data acquisition, peak integration, and quantitative analysis.

Here's the diagram illustrating the "Capillary Electrophoresis (CE)" procedure

Modes of Capillary Electrophoresis:

Depending on the nature of the separation medium and the sample, various modes of CE have been developed:

1. Capillary Zone Electrophoresis (CZE):

• Here, the separation occurs in a buffer-filled capillary based purely on the charge-to-size ratio of the analytes.

2. Capillary Gel Electrophoresis (CGE):

• A gel is used as the separation medium within the capillary.

• Commonly used for DNA fragment separations.

3. Micellar Electrokinetic Chromatography (MEKC):

• In this mode, surfactants are added to the buffer above their critical micelle concentration (CMC), forming micelles.

• These micelles can separate neutral as well as charged analytes.

4. Capillary Isoelectric Focusing (cIEF):

• Separation is based on the isoelectric point (pI) of proteins.

5. Capillary Electrochromatography (CEC):

• Combines principles of both CE and HPLC, where the capillary is packed with chromatographic material.

Here is a colored diagram illustrating the modes of Capillary Electrophoresis:

Detection Methods:

• UV/Vis Absorption: Most commonly used, it requires analytes to be UV active.

• Fluorescence: Either native fluorescence of compounds or by using fluorescent tags.

• Mass Spectrometry (MS): CE can be coupled with MS for detailed molecular identification.

Here is a colored diagram illustrating the detection methods of Capillary Electrophoresis:

Capillary electrophoresis: Application, Advantages, Disadvantages & Limitations