Capillary Electrophoresis Overview

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Questions and Answers

What is the primary advantage of capillary electrochromatography (CEC) over HPLC?

It combines the principles of electrophoresis and chromatography. (correct)

It exclusively separates charged solutes.

It operates at a lower temperature.

It requires less sample preparation.

In capillary isoelectric focusing (CIEF), what is the role of ampholytes?

To create a constant pH environment.

To establish a pH gradient for molecule migration. (correct)

To facilitate enzyme reactions.

To enhance the conductivity of the buffer.

What is capillary isotachophoresis (CITP) primarily used for?

Separating tiny ions and DNA. (correct)

Analyzing the composition of proteins.

Separating large biological macromolecules.

Estimating molecular weights of carbohydrates.

What causes the electric field used in capillary electrophoresis?

A high-voltage, variable-current power supply. Signup and view all the answers

Which types of substances can CEC effectively separate?

Amino acids, proteins, peptides, and carbohydrates. Signup and view all the answers

What is the first stage of capillary isoelectric focusing (CIEF)?

Loading. Signup and view all the answers

Which statement is NOT true regarding the use of capillary electrophoresis?

It is exclusively used in material science. Signup and view all the answers

What aspect of CEC helps in the separation of analytes?

Electrophoretic mobility or partition ratios. Signup and view all the answers

What is the primary advantage of applying pressure during hydrostatic injection?

It enhances the injection efficiency. Signup and view all the answers

Which injection method is less accurate than HPLC due to a lack of injection loops for small volumes?

Hydrostatic injection Signup and view all the answers

How does the flow rate in a tube relate to the capillary length L according to Poiseuille’s law?

The flow rate decreases with longer capillary length. Signup and view all the answers

What effect does doubling the radius of the capillary have on its volume?

Increases volume by a factor of 16. Signup and view all the answers

What is a key disadvantage of the electro-migration injection method?

It causes non-representative sample composition. Signup and view all the answers

Which detection technique is commonly used in capillary electrophoresis to analyze UV-visible absorbance?

Diode array detection Signup and view all the answers

What is the role of an internal standard in high-performance capillary electrophoresis (HPCE)?

To improve reliability of quantification. Signup and view all the answers

What must be removed from the capillary to utilize the diode array detector for UV detection?

The polyimide covering Signup and view all the answers

What are factors that influence band dispersion in capillary electrophoresis?

Length of the injection plug and size of the detector cell Signup and view all the answers

Which capillary electrophoresis type utilizes a gel as a molecular sieve for separation?

Capillary Gel Electrophoresis (CGE) Signup and view all the answers

What is one of the key mechanisms used to achieve separation of two bands in capillary electrophoresis?

Altering the electrophoretic mobility of the analytes Signup and view all the answers

In capillary zone electrophoresis (CZE), what primarily determines the velocity of the band?

Electrophoretic mobility and the electroosmotic flow of the analyte Signup and view all the answers

What type of analytes can be effectively analyzed using capillary zone electrophoresis?

Substances with molecular weights between 2,000 and 100,000 Signup and view all the answers

In Micellar Electrokinetic Capillary Chromatography (MEKC), what is added to the electrolytic solution?

A surfactant above critical micellar concentration Signup and view all the answers

How is average electrophoretic mobility calculated for two analytes?

μaep = ½ (μepb + μepa) Signup and view all the answers

Why do smaller molecules migrate more rapidly in capillary gel electrophoresis?

They navigate the gel network more easily. Signup and view all the answers

Which scientific discipline covers the study of the composition and properties of substances?

Analytical Chemistry Signup and view all the answers

What is one of the primary uses of capillary electrophoresis?

Analyzing biological molecules Signup and view all the answers

Which of the following topics is not categorized under Basic Chemistry?

Biochemistry Signup and view all the answers

How many topics does the 'Inorganic Chemistry' category include?

182 Signup and view all the answers

Which category has the least number of topics listed?

Book Reviews Signup and view all the answers

What role do charge, radius, and solvent viscosity play in capillary electrophoresis?

They affect the electrophoretic mobility of molecules. Signup and view all the answers

Which of the following is an advantage of capillary electrophoresis over high-performance liquid chromatography (HPLC)?

Increased separation efficiency. Signup and view all the answers

What is one key application of capillary electrophoresis?

Separation of large biological molecules. Signup and view all the answers

How does the narrow diameter of capillaries enhance the effectiveness of capillary electrophoresis?

By facilitating effective heat transfer. Signup and view all the answers

What does the high selectivity of capillary electrophoresis allow researchers to do?

Detect compounds in very low concentrations. Signup and view all the answers

What is an important aspect of capillary electrophoresis related to operational time?

Setup to separation can take up to one hour. Signup and view all the answers

Which of the following compounds can be analyzed using capillary electrophoresis?

Ions, tiny molecules, proteins, and DNA/RNA. Signup and view all the answers

What is the significance of the mass spectrometry technique in relation to capillary electrophoresis?

It enhances the sensitivity of detection for tiny compounds. Signup and view all the answers

Study Notes

Band Dispersion

Band dispersion can be influenced by various factors including the length of the injection plug, size of the detector cell, buffer reservoir unevenness, conductivity mismatch between the sample and buffer, sample adsorption to the capillary wall, and heat dispersion.

Separation of Bands

The separation of two bands in capillary electrophoresis can be achieved through optimizing the efficiency of each analyte and adjusting their electrophoretic and electroosmotic mobilities.

Capillary Electrophoresis Types

Capillary zone electrophoresis (CZE) employs a buffer solution within the capillary, allowing separation based on the electrophoretic mobility and electroosmotic flow of the analyte. This method is well-suited for analyzing compounds with molecular weights between 2,000 and 100,000.
Capillary gel electrophoresis (CGE) utilizes a gel-filled capillary functioning as a molecular sieve, separating molecules based on their size. Smaller molecules migrate faster than larger ones due to their ability to traverse the gel network. This technique excels in separating proteins, DNA fragments, and biological macromolecules with similar charge-to-mass ratios.
Micellar electrokinetic capillary chromatography (MEKC) introduces a surfactant above its critical micellar concentration to create a hybrid separation system combining electrophoresis and chromatography. This method facilitates the separation of both neutral and charged solutes by exploiting their partition coefficients between the micelle-based pseudo-stationary phase and the aqueous buffer.
Capillary electrochromatography (CEC) combines capillary electrophoresis and chromatography, specifically high-performance liquid chromatography (HPLC). Separation is achieved by electrophoretic mobility or partition ratios, while electroosmotic force drives the mobile phase through the chromatographic bed. CEC surpasses HPLC and capillary electrophoresis in resolving enantiomers of amino acids, proteins, peptides, and carbohydrates. It finds widespread applications in pharmaceutical and polymer analysis industries for distinguishing acidic and basic drugs.
Capillary isoelectric focusing (CIEF) utilizes a pH gradient generated by ampholytes in a separation buffer to enable the migration of charged molecules. This method is predominantly used for enantiomeric separations and the analysis of amino acids, proteins, peptides, and carbohydrates. It involves three stages: loading, focusing, and mobilization.
Capillary isotachophoresis (CITP) separates ions electrophoretically in a flowing buffer exhibiting a continuous and gradually decreasing conductivity gradient. Its primary application lies in the separation of small ions, such as DNA and other biological macromolecules.

Capillary Electrophoresis Instrumentation

Power supply: A high-voltage, variable-current power supply is the cornerstone of capillary electrophoresis equipment. It provides the electrical field necessary for sample separation.

Capillary Electrophoresis Procedures

Hydrostatic injection: A capillary is dipped into the sample solution, creating a vacuum at the opposite end. Applying pressure of approximately 50 mbars enhances this procedure.
Electro-migration injection: Similar to gel electrophoresis, this method involves partially immersing the capillary in a sample, creating a potential difference of suitable polarity relative to the other end. This technique can result in non-representative sample composition due to its discriminatory action on compounds.

Capillary Electrophoresis Detection Techniques

Direct UV/Vis detection: Typically, a diode array detector measures UV-visible absorbance. The polyimide coating is removed to create a small window allowing UV light detection. The detector cell is integrated into the capillary, minimizing space requirements.
Fluorescence detection: This method utilizes laser-induced fluorescence to detect labeled analytes, offering high sensitivity for fluorescent species.
Mass spectrometry detection: This method combines capillary electrophoresis with mass spectrometry, allowing the identification and quantification of separated compounds.

Capillary Electrophoresis Advantages

High efficiency: The small capillary diameter allows for effective heat transfer, rivaling the efficiency of liquid chromatography.
High resolution: CE enables the separation of ions and molecules based on size, charge, and hydrophobicity, resulting in highly accurate and comprehensive results.
High selectivity: The excellent detection sensitivity of CE allows for the analysis and detection of compounds in trace concentrations.
Versatility: CE can analyze various compounds, including ions, small molecules, proteins, and DNA/RNA.
Less time-consuming: Once the analyst becomes familiar with the software, the separation results are rapid, saving time and making the entire process (setup to separation) complete within one hour.

Capillary Electrophoresis Applications

Capillary zone electrophoresis finds application in the separation and analysis of large biological molecules, including DNA, mRNA, proteins, peptides, and biopolymers.

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Description

Explore the concepts of band dispersion and separation in capillary electrophoresis. This quiz covers different types of electrophoresis, including capillary zone electrophoresis and capillary gel electrophoresis. Test your knowledge of the factors affecting separation and the principles behind each method.