Capitulo III Electroforesis capilar video 16

Introduction to Capillary Electrophoresis

In this section, the principles governing immigration processes in capillary electrophoresis are discussed, along with different names for capillary electrophoresis.

Principles of Capillary Electrophoresis

• Capillary electrophoresis is known by various names such as high-performance capillary electrophoresis (HTC), free solution capillary electrophoresis (FSC), and capillary zone electrophoresis (CC TAE).

• Various compounds can be separated using capillary electrophoresis, including amino acids, peptides, proteins, nucleotides, small organic molecules, and pharmaceutical industry components.

Components and Setup of Capillary Electrophoresis

This section delves into the components and setup of a basic micro-capillary force apparatus used in capillary electrophoresis.

Components of Micro-Capillary Force Apparatus

• A basic micro-capillary force apparatus consists of a capillary connected to two buffer reservoirs serving as the anode and cathode.

• The detection equipment is attached to the capillary along with a data acquisition system. The dimensions typically range from 25 to 100 microns.

Electrophoresis Capillary Techniques

The discussion delves into the principles and instrumentation of capillary electrophoresis, highlighting factors affecting efficiency and separation in this technique.

Principles of Capillary Electrophoresis

• High voltages lead to more theoretical plates, enhancing separation for analytes with high mobility.

• Factors influencing capillary electrophoresis efficiency include dispersion, analyte-wall interactions, temperature effects on diffusion and resolution.

Instrumentation Components

• Key components: capillary for separation, sample introduction system, power source, and cooling system.

• Equipment includes a schematic overview comprising the mentioned components.

Capillary Structure and Function

• Capillaries resemble gas chromatography columns but lack a stationary phase. They are thin-walled with diameters below 100 microns.

• Capillaries are critical for separation due to their inert nature; efficient cooling is crucial to maintain temperature stability.

Capillary Electrophoresis Instrumentation

This segment explores sample introduction methods, detection techniques like fluorescence and mass spectrometry in capillary electrophoresis.

Sample Introduction Methods

• Various sample introduction systems exist: hydrodynamic injection by gravity or pressure, electrokinetic injection for precise control.

Detection Techniques

• Fluorescence detection involves laser-induced fluorescence for sensitive analysis. Other detectors include conductometric or amperometric sensors.

• Mass spectrometry integration via electro-spray interfaces enhances sensitivity and allows gaseous ion detection.

Detector Characteristics

• Different detectors offer varying limits of detection; electrochemical and mass spectrometry detectors are commonly used in capillary electrophoresis.

Zone Electrophoresis in Capillaries

Zone electrophoresis is discussed as a simple mode where analytes migrate based on charge differences within the capillary setup.

Operating Principle

Graphical Representation and Applications of Capillary Electrophoresis

The section discusses the graphical representation of migration order in capillary electrophoresis and its application for compounds with differences in electric point. It also touches on the separation technique using capillaries, particularly for molecules like proteins.

Graphical Representation and Migration Order

• Capillary electrophoresis is used for compounds with differences in electric point.

• Proteins and other molecules are separated using capillaries.

Capillary Electrophoresis in Gel and Its Applications

This part delves into capillary electrophoresis in gel as a variant that has been crucial in biochemistry for several decades, primarily focusing on separating proteins, peptides, and nucleic acids.

Capillary Electrophoresis in Gel

• Capillary electrophoresis in gel separates proteins, peptides, and nucleic acids.

• The method generates an effect allowing separation by size while reducing dispersion.

Comparison Between Chemical and Physical Gels

Here, the discussion centers on the differences between chemical gels with linked chains that are challenging to remove from capillaries versus physical gels made of linear polymers easily reloadable for new separations.

Chemical vs. Physical Gels

• Chemical gels have linked chains making them hard to remove from capillaries.

• Physical gels consist of linear polymers entangled together, facilitating easy reloading.

Micellar Electrokinetic Capillary Electrophoresis

This segment explores micellar electrokinetic capillary electrophoresis as a method to separate neutral molecules by including surfactants like SDS to induce partitioning based on interactions with charged micelles.

Micellar Electrokinetic Capillary Electrophoresis

• Micellar electrokinetic capillary electrophoresis uses surfactants like SDS for neutral molecule separation.

• Neutral solutes move along with charged micelles due to strong interactions.