Capillary Electrophoresis

FRSC Orgo and Bio

What occurs in Electrophoresis?

In electrophoresis, charged analytes move in an electric field. Differences in charge, size, and shape cause them to move at different velocities, resulting in particle separation.

What movement occurs in Electrophoretic flow?

Cations move toward the negatively charged cathode. Anions move toward the positive anode. Neutral species remain stationary.

What is electroosmotic flow (EOF)?

Electroosmotic flow (EOF) is the buffer movement that moves most solutes through the capillary to the cathode in response to an electric field.

What is the equation for Electrophoretic/Electroosmotic Flow velocity?

Electrophoretic (Vep) or Electroosmotic (Veof) velocity is, v = μE, where mobility (μ) and electric field strength (E) determines velocity.

What is the order of electrophoresis movements between a cation, neutral, and anion molecule?

Cation (Vtot=Vep+Veof), Neutral (Vtot=Veof), Anion (Vtot-Vep=Veof).

What is the relationship between Capillary Electrophoresis and Van Deemter?

In CE, two Van Deemter terms are zero (Eddy’s diffusion, A, and mass transfer, C) because separation is carried out in a single phase of carrier liquid (buffer). The only source of band broadening is longitudinal diffusion (B).

What is the equation for analyte mobility? Which analytes have the greatest mobility in CE?

μep = q/(6πƞr) where q is the analytes charge, ƞ is buffer viscosity, and r is the analytes radius. A small analyte with a high charge has the greatest mobility (fast).

What is the approximate pH of the buffer used in CE analysis of DNA? What happens to phosphate groups on DNA at pH 8?

pH 8. Phosphate groups deprotonate, giving DNA a negative charge.

What is used as the sieving mechanism in DNA capillary electrophoresis? What is its function?

A viscous polymer is used as the sieving mechanism, creating a resistance that separates DNA fragments according to size.

How are DNA fragments separated in CE?

DNA fragments are separated according to size base on migration time in the polymer filled silica capillary. Smaller fragments travel faster and larger fragments travel slower.

How is DNA detected in capillary electrophoresis (CE)?

DNA fragments are detected using Laser Induced Fluorescence (LIF). The laser excites fluorophores (fluorescent dyes) attached to DNA fragments, causing them to emit light that is measured by the detector. This signal intensity is proportional to the amount of DNA present.

What is the purpose of capillary electrophoresis (CE) in forensic DNA analysis?

The purpose of CE in DNA is to separate amplified STR fragments according to size.

What components make up the CE system?

Capillary, polymer, buffer vials, electrodes, laser, and camera.

What are the advantages and disadvantages of capillary electrophoresis?

Advantages: Automated, small sample, rapid separation, minimal contamination. Disadvantages: High instrumentation and maintenance costs.

What is the function of the camera in CE?

The camera functions as a CCD detector that detects fluorescence from dye labeled DNA fragments. The location of the signal identifies the dye color and the signal intensity indicates the amount of DNA.

What is the function of ILSS?

ILSS (Internal Lane Size Standard) is a standard containing DNA fragments of a known size and is used to correlate migration time to fragment size.

What is the function of Allelic Ladder?

Allelic Ladder is a standard containing known allele sizes and is used to correlate allele size to fragment size to make an allele call. It uses the same primers as the unknown sample.

How is an allele call made?

An allele call is made by using ILSS, which converts migration time into fragment size, and an allelic ladder, which converts fragment size into an allele size.