Name: electrophoresis in gel and capillary formats
Uploaded: 2024-12-05T18:32:01.000Z
Duration: 1 min 20 s
Channel: JoVE

Electrophoresis uses the differential migration rate of charged species in an applied electric field. The greater the charge-to-size ratio, the faster the rate of migration through the gel.
Slab electrophoresis is performed on a thin, flat layer of porous semisolid gel containing an aqueous buffer solution.
The samples are placed as spots or bands in the sample well, and an electric field is applied across the slab. Post-separation, the distinct components are visualized using UV light or other staining techniques.
It is widely employed in DNA sequencing and analysis of mixtures.
Capillary electrophoresis, or CE, is more suitable for quantitative analysis.
CE employs a conducting buffer within a capillary tube. The sample is injected into one end of the tube.
Migrating sample components are influenced by electrophoretic mobility, which is the solute&#39;s response to the electric field.
They are also affected by electroosmotic flow arising from the movement of buffer solution through the capillary due to the electric field.
The migrating components eluting from the capillary at different times are recorded in an electropherogram.

electrophoresis: overview

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There are two main formats for electrophoretic separations: slab electrophoresis and capillary electrophoresis. Slab Electrophoresis uses a gel matrix, such as agarose or polyacrylamide, which acts as a sieve, allowing molecules to move through the pores. Capillary electrophoresis employs a narrow capillary tube filled with a buffer solution, providing high-resolution separation with rapid analysis time and minimal sample requirements. Although slab electrophoresis is widely used in biochemistry and biology to separate high-molecular-mass species, capillary electrophoresis is a more recent development with several advantages.
Capillary electrophoresis is a separation technique used to analyze complex mixtures of analytes based on their differential migration rates in an applied electric field. In this method, a sample is injected into a narrow capillary tube filled with a buffered solution, and an electric field is applied to facilitate separation. The migration rate of the analyte depends on its charge and size. The smaller, more highly charged ions migrate faster than the larger, less charged ones. In capillary electrophoresis, the migration of sample components is influenced by two types of mobility: electrophoretic mobility and electroosmotic mobility. Electrophoretic mobility is the solute&#39;s response to the applied electric field, with cations moving toward the negatively charged cathode, anions moving toward the positively charged anode, and neutral species remaining stationary. Electroosmotic mobility occurs when the buffer solution moves through the capillary in response to the electric field, typically toward the cathode.
Under normal conditions, capillary electrophoresis separates cations first, followed by neutral species, and finally, the anions. This separation is based on their respective electrophoretic mobilities and electroosmotic flow velocities. Notably, all separated species are eluted from the same end of the capillary, allowing their detection using quantitative detectors. The detector signals produce an electropherogram resembling a chromatogram but feature narrower peaks.
Capillary electrophoresis is a powerful tool for qualitative and quantitative analysis of complex mixtures. It provides information similar to that obtained from other separation techniques, such as gas chromatography (GC) or high-performance liquid chromatography (HPLC).

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric ...

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Analytical Chemistry

Unit 1

Principles of Chromatography

KEY TERMS AND CONCEPTS

electrophoresis in gel and capillary formats

Electrophoresis separates charged molecules by making them move in an electric field. It is widely used in biochemistry, molecular biology, and analytical chemistry. The technique can separate complex mixtures of amino acids, nucleotides, carbohydrates, and proteins with strong resolution.

Two main formats are used for electrophoretic separations: slab electrophoresis and capillary electrophoresis. Slab electrophoresis uses a gel matrix, such as agarose or polyacrylamide, as a sieve. The pores in the gel slow molecules by size as they move through the matrix.

Capillary electrophoresis uses a narrow capillary tube filled with buffer solution. It offers high-resolution separation, fast analysis, and very small sample needs. Compared with slab methods, it is a newer approach with several practical advantages.

In capillary electrophoresis, a sample is injected into the capillary and an electric field is applied. The migration rate depends on charge and size. Smaller, more highly charged ions usually move faster than larger, less charged ones.

Two kinds of mobility control the movement of sample components in the capillary. Electrophoretic mobility is the solute response to the electric field, so cations move toward the negatively charged cathode, anions move toward the positively charged anode, and neutral species stay in place. Electroosmotic mobility is the movement of the buffer through the capillary in response to the field, usually toward the cathode.

Under normal conditions, capillary electrophoresis separates cations first, then neutral species, and finally anions. This happens because electrophoretic mobility and electroosmotic flow work together. The separated species leave the same end of the capillary, which makes detection easier with quantitative detectors.

The detector signal produces an electropherogram, which looks like a chromatogram but has narrower peaks. Capillary electrophoresis is useful for both qualitative and quantitative analysis of complex mixtures. It gives information similar to gas chromatography, or GC, and high-performance liquid chromatography, or HPLC.

Electrophoresis in Gel and Capillary Formats

electrophoresis-gel-and-capillary-formats

chromatographic methods: terminology

Chromatography Terms and Peak Analysis

Chromatography is an analytical technique used to separate the parts of a mixture and identify substances in it. It is used in chemistry, biology, environmental science, and pharmaceuticals. The method depends on two phases: the stationary phase, which stays fixed, and the mobile phase, which moves across it and carries the solutes. As the mobile phase travels, each mixture component interacts with the phases in a different way. That difference causes the components to separate. Chromatography...

chromatographic methods: classification

Chromatography Types and Separation Modes

Chromatographic techniques are grouped by the phases they use, how those phases interact, and the way they separate sample components. The mobile phase is usually a liquid or a gas. The stationary phase is usually a solid or a liquid layer on a solid surface. Many chromatography names start with the type of chromatography, then the mobile phase, and then the stationary phase. For example, gas-solid chromatography uses a gas mobile phase and a solid stationary phase. In liquid-liquid...

analyte adsorption and distribution

Chromatography: Polarity and Adsorption

Chromatography separates solutes by how they move between the mobile phase and the stationary phase. In many systems, this transfer happens through sorption, which usually means adsorption. The separation depends strongly on polarity, or the overall dipole moment of a molecule. During chromatography, the solute and the solvent compete for adsorption on the stationary phase. Highly polar compounds and polar solvents tend to adsorb strongly on polar surfaces. This affects how the mixture...

diffusion on chromatography columns

Band Broadening from Column Diffusion

Column diffusion causes band broadening in chromatography columns. When an analyte is added as a narrow band at the top of the column, the solutes begin to separate and spread out. As this happens, the band develops a Gaussian profile, which means the concentration is highest near the center and lower at the edges. Longitudinal diffusion is one reason this spreading occurs. In this process, solute molecules in the mobile phase move from the more concentrated center of the band into the more...

chromatographic resolution

Peak Separation in Chromatography

Peak separation in chromatography depends on how far apart two neighboring peaks appear in a chromatogram. As a solute moves through a chromatographic column, it spreads into a band with a Gaussian shape. The longer the solute stays in the column, the broader that band becomes. This broadening can cause peaks to overlap and reduce separation efficiency. Chromatographic resolution is the measure used to judge that separation. It is the ratio of the difference in retention time or retention...

column efficiency: plate theory

Theoretical Plates in Chromatography Columns

Theoretical plates help explain how chromatography columns achieve good separation. Column efficiency is measured by band broadening, and that efficiency depends on the number of theoretical plates, or N. In plate theory, a column is treated as a continuous series of imaginary plates where solute equilibration happens between the stationary and mobile phases. A higher number of theoretical plates means better column efficiency and stronger separation ability. Plate height affects both band...

column efficiency: rate theory

Chromatography Band Broadening in Columns

Chromatography band broadening in columns explains why separated solute zones spread out as they move through a column. The rate theory of chromatography gives a quantitative way to describe the shape and width of elution bands. These bands follow a Gaussian profile, which means they form a smooth bell-shaped curve as the molecules travel. As a solute moves through the column, it makes many random transitions between the stationary phase and the mobile phase. Its residence time in each phase...

optimizing chromatographic separations

Improving Chromatography Resolution

Chromatography resolution improves when bands stay narrow and the two solutes separate clearly in the column. Good separation often depends on band broadening, plate height, capacity factor, and separation factor. These ideas help explain why some mixtures come apart quickly while others take more time. Band broadening is the spreading of solute bands as they move through the column. When bands spread out, resolution drops. Plate height, or H, is the length needed for one theoretical plate. A...

silica gel column chromatography: overview

silica-gel-column-chromatography-overview

Silica Gel Column Chromatography and Polarity

Silica gel column chromatography separates compounds by polarity using a column packed with silica gel. The silica gel acts as the stationary phase, and the solvent is the mobile phase. As the mixture moves through the column, the compounds separate into distinct bands. Polar compounds tend to bind more strongly to the silica gel. Because of this stronger interaction, they move more slowly through the column. Nonpolar compounds bind more weakly to the silica gel. They usually travel faster...

thin-layer chromatography (tlc): overview

TLC Separation by Polarity

Thin-layer chromatography (TLC) separates compounds by polarity. It uses a polar silica gel plate as the stationary phase. Silica gel is a form of silicon dioxide, and its surface has hydroxyl (OH) groups that can form hydrogen bonds with polar compounds. This affects how strongly a compound sticks to the plate. To start a TLC analysis, a mixture is placed on the starting line with a thin capillary. The bottom of the plate is then set in the mobile phase, which is usually an organic solvent...

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Gas Chromatography: Separating Volatile Mixtures

Gas chromatography separates and analyzes volatile compounds in a sample. It is used to identify and measure the parts of complex mixtures. This makes GC useful in environmental analysis, pharmaceuticals, and petrochemicals. GC is also called vapor-phase chromatography (VPC) or gas-liquid partition chromatography (GLPC). In the process, the sample is vaporized first. It is then mixed with an inert carrier gas, which acts as the mobile phase and carries the sample through a column. The column...

gas chromatography: types of columns and stationary phases

gas-chromatography-types-of-columns-and-stationary-phases

Gas Chromatography Columns and Polarity

Gas chromatography uses stationary phases to separate and analyze parts of a sample. In GC, the stationary phase can be liquid or solid. Liquid stationary phases are non-volatile, thermally stable, and chemically inert liquids coated on the column. Solid stationary phases are adsorbent particles, such as silica gel or molecular sieves. A sample component must interact well enough with the stationary phase to stay in the column long enough to separate. This idea follows “like dissolves like,”...

gas chromatography: sample injection systems

gas-chromatography-sample-injection-systems

Gas Chromatography: Split vs Splitless Injection

Gas chromatography uses sample injection methods to control how much material enters the column. The sample is first introduced as a vapor plug into the carrier gas stream. This helps produce high efficiency and good resolution. A microsyringe places the sample solution into a heated sample port. The heat vaporizes the sample, and it then mixes with the carrier gas. This step prepares the sample for analysis. Some thermally sensitive samples can be injected directly into the column. In that...

gas chromatography: overview of detectors

Gas Chromatography Detector Types and Uses

Gas chromatography detectors turn chemical properties into signals that appear on a chromatogram. These signals help identify and measure the parts of a mixture. In GC, detectors are often grouped as destructive or non-destructive. A non-destructive detector analyzes a sample without changing or using it up. That means the sample can still be collected after detection for more testing. Thermal conductivity detectors and electron capture detectors are examples of this type. A destructive...

gas chromatography: types of detectors-i

Gas Chromatography Detectors: Choosing the Right One

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gas chromatography: types of detectors-ii

Specialized Detectors in Gas Chromatography

Gas chromatography uses specialized detectors to identify compounds that general-purpose detectors may miss. These detectors are chosen for specific analytical needs. They can target certain elements, functional groups, or trace analytes with greater precision. Thermionic detectors are selective for organic compounds that contain phosphorus and nitrogen. They are especially sensitive to phosphorus compounds compared with the flame ionization detector, or FID. That makes them useful for...

high-performance liquid chromatography: introduction

high-performance-liquid-chromatography-introduction

HPLC Separation, Columns, and Detection

High-performance liquid chromatography (HPLC) separates, identifies, and quantifies the parts of a complex mixture. It uses high pressure to push a liquid mobile phase through a tightly packed column. The result is a precise separation process that is useful for analyzing many samples. In HPLC, the mobile phase is the liquid solvent that carries the sample through the system. The solvent is chosen for its polarity, or how it interacts with other substances. The polarity index helps guide the...

high-performance liquid chromatography: elution process

high-performance-liquid-chromatography-elution-process

HPLC Solvent Flow and Elution Types

High-performance liquid chromatography (HPLC) uses solvent flow and elution to separate analytes in a column. Elution is the way compounds move through the column and separate because of how they interact with the mobile phase and the stationary phase. These interactions affect retention times, peak shape, and resolution in the chromatogram. Knowing how elution works helps students read chromatographic data and understand how mixtures are separated. HPLC relies on highly pure HPLC-grade...

high-performance liquid chromatography: instrumentation

high-performance-liquid-chromatography-instrumentation

HPLC Parts and How They Work

High-performance liquid chromatography, or HPLC, uses a set of connected parts to separate liquid samples under high pressure. The system begins with mobile phase reservoirs, which are glass bottles that hold the solvents. HPLC-grade solvents are used to keep the mobile phase as pure as possible. A degasser removes dissolved gases from the solvents before they move through the instrument. The solvent then reaches the pump, which sends it into the analytical column. Some systems use a...

high-performance liquid chromatography: types of detectors

high-performance-liquid-chromatography-types-of-detectors

HPLC Detector Choices for Analyzing Solutes

High-performance liquid chromatography (HPLC) uses detectors to analyze solutes as they leave the chromatographic column. The detector recognizes a property of the solute and turns it into an electrical signal. A computer then converts that signal into a graph called a chromatogram, which shows detector response over elution time. HPLC includes several detector types, and each one has strengths and limits based on the analyte and the sensitivity needed. No single HPLC detection method can...

ion-exchange chromatography

Separating Ions with Ion-Exchange Columns

Ion-exchange chromatography separates ions by how strongly they bind to a charged stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic groups. These groups may be positively charged or negatively charged, which creates cation exchangers and anion exchangers. The choice of resin depends on the analyte and the separation needed. A cation exchanger contains a polymeric anion with active cations, while an anion exchanger contains a polymeric cation...

size-exclusion chromatography

Size-Exclusion Chromatography in Protein Analysis

Size-exclusion chromatography separates molecules by size, and it is often used for large molecules such as polymers and biomolecules. It is also called molecular-exclusion chromatography or gel-permeation chromatography. In this method, the stationary phase is made of micron-sized particles or resin beads that contain pores. The two main stationary phases are silica particles and cross-linked polymer resin beads. Both are porous, but their pore sizes can differ a lot. Silica particles are...

supercritical fluid chromatography

SFC for Fast Chemical Separations

Supercritical fluid chromatography (SFC) is a separation method used for compounds that can be hard to analyze with gas chromatography (GC) or liquid chromatography (LC). It is useful for nonvolatile or thermally unstable samples that do not work well in GC and for compounds that do not have the right functional groups for HPLC analysis. SFC combines useful features of both GC and LC while keeping the analysis efficient. SFC uses a supercritical fluid as the mobile phase, most often carbon...

affinity chromatography

Affinity Chromatography for Biomolecule Separation

Affinity chromatography separates and purifies specific biomolecules from a complex mixture. It depends on a highly selective match between an analyte and a binding partner, such as an antibody and an antigen. The binding partner is fixed on the stationary phase, which forms the affinity column. The stationary phase is usually a solid support such as agarose or porous glass beads. The mobile phase helps the analyte bind to the ligand and later helps release it. When a sample passes through the...

Electrophoresis separates charged molecules by making them move in an electric field. It is widely used in biochemistry, molecular biology, and analytical chemistry. The technique can separate complex mixtures of amino acids, nucleotides, carbohydrates, and proteins with strong resolution. Two main formats are used for electrophoretic separations: slab electrophoresis and capillary electrophoresis. Slab electrophoresis uses a gel matrix, such as agarose or polyacrylamide, as a sieve. The pores...

capillary electrophoresis: instrumentation

capillary-electrophoresis-instrumentation

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...

capillary electrophoresis: applications

Capillary Electrophoresis: Separation Modes

Capillary electrophoresis uses several separation modes to analyze charged molecules, size-based samples, and even neutral compounds. Each mode works a little differently and is used for different kinds of lab problems. Together, they show why capillary electrophoresis is a flexible tool in analytical chemistry. Capillary zone electrophoresis (CZE) separates ionic components by their electrophoretic mobility. It can separate proteins, amino acids, and carbohydrates in a short time, so it is an...

key terms and concepts

Explore gel and capillary electrophoresis, including how charge, size, and buffer flow separate molecules and produce electropherogram peaks.

Electrophoresis in Gel and Capillary Formats | JoVE K12

analytical chemistry

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Electrophoresis in Gel and Capillary Formats

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