Capillary Electrophoresis Basics

• Capillary electrophoresis (CE) is a method using an electrical field to separate mixture components within a narrow tube.

Basic Principles

• In CE, ionic substances dissolve in a suitable solvent like water, and without an electric field, ions move randomly.
• When an electric field is applied, charged species move, causing a basic separation.
• Electrophoresis considers the mass-charge ratio (m/z) where the force on each singly charged particle is equal (Force = mass x acceleration).
• The technique exploits molecule differences or creates them, such as altering pH.
• Glycine and acetic acid have the same charge (-1) at pH 10.0, but glycine has a near-neutral charge at pH 7.0 while acetic acid remains at -1, affecting separation.
• Additional factors include the hydrodynamic radius of molecules, medium viscosity, and temperature.

Capillary Electrophoresis

• CE uses fused silica capillaries with inner diameters of 100 µm or less, typically 20-100 µm, with a circular cross-section.

Advantages

• Capillary is filled with a conductive buffer solution at a specific pH.
• Sample introduction is via pressure or electrokinetic injection, applying a high voltage (over 300 V/cm).
• Sample components migrate at varying speeds based on charge.
• Positive components move to the cathode, negative to the anode, and a detector observes fast components first.

Mobility

• Ion charge depends strongly on pH, influencing separation, by using a buffer at a certain pH.
• Acetic acid (pK value 4.756) is almost fully negative at pH 7, having high mobility, but at pH 3, lower mobility occurs.
• Altering buffer pH changes component mobilities for optimal separation, generally using a pH between the pK values of the sample components.

Endo Osmotic Flow (EOF)

• Bare fused silica capillaries with silanol groups (Si - O - H) are common.
• At higher pH, these groups become negatively charged (Si – O-), attracting positive charges in the buffer.
• Applying high voltage causes these positive charges to migrate towards the cathode, dragging the buffer and producing (Electro) Endo Osmotic Flow (EOF).
• Higher pH results in more negative charges and a stronger EOF.

Instrumentation

• Includes a cathode (negative electrode) and anode (positive electrode)
• Requires a power supply for the electric field.
• Catholyte (cathode buffer) and anolyte (anode buffer) solutions are needed, along with capillary (25-100mm).
• The instrument also utilizes a detector and output device.

Sample Injection

• CE can inject small sample volumes from picoliters to nanoliters, using hydrodynamic or electrokinetic methods.
• Hydrodynamic injection applies pressure or vacuum to move liquid into the capillary.
• Electrokinetic injection applies voltage while the capillary is in the catholyte and anolyte, moving ions into the capillary in 1-5 seconds.

Detection

• Separation detection occurs via UV or UV-Vis absorbance using the capillary as a detection cell, maintaining resolution.
• Capillaries are coated with polymers like polyimide or Teflon for flexibility.
• Fluorescence detection is used for naturally fluorescent samples or those chemically tagged, offering high sensitivity but limited to fluorescent compounds.

Modes of Capillary Electrophoresis

Capillary Zone Electrophoresis (CZE or FSCE)

• CZE, or free-solution CE, is the most standard CE form.
• Buffer is flushed through the capillary, sample is injected, and high voltage is applied.
• EOF direction depends on polarity, and sample components migrate at their own speed, separating based on mobility differences.

Capillary Gel Electrophoresis (CGE)

• CGE incorporates a gel matrix inside the capillary, separating components of varying sizes but similar mobility.
• Larger components move slower through the gel, and is frequently used with proteins and DNA.

Micellar Electrokinetic Chromatography (MECC or MEKC)

• Micelles with a nonpolar interior and polar surface generate in the buffer.
• Sample components partition between micelles and buffer.
• Separation occurs due to affinity differences, with buffer and micelle migration speed differences, used with HPLC and GC.

Non-Aqueous Capillary Electrophoresis (NACE)

• Separates water-insoluble components using organic solvents.
• Solvent viscosity and dielectric constants affect ion mobility and electroosmotic flow.

Iso Electric Focusing (IEF)

• Applying a pH gradient and voltage causes components to migrate to their isoelectric point (pI).
• Components move to different positions and pressure moves the pH gradient through the capillary for detection.

Capillary Electro Chromatography (CEC)

• Capillary is packed with silica particles with a stationary phase.
• Buffer fluid migrates due to EOF, and samples separate based on stationary phase affinity, similar to HPLC.

Electro Chromatography (EKC)

• Uses varying interactions between enantiomers and cyclodextrins to separate chiral compounds, used for natural products, toxicology, food analysis, forensics, fingerprinting, etc.

Capillary IsoTechoPhoresis (ITP)

• Employs two buffer types: a leading buffer with high mobility and a terminating buffer with low mobility.
• Sample component mobility must fall between these buffers- all components migrate at the same velocity.

Applications

• Determines ions like NH4+, Na+, K+, Mg2+, and Ca2+ simultaneously in saliva.
• Develops methods for DNA amplification and detection using PCR in forensic science.
• Types STR from biological samples to profile highly polymorphic genetic markers to differentiate individuals.
• Detects specific mRNA fragments and analyzes ink in forensic applications.

Polyacrylamide Gel Electrophoresis (PAGE)

• Matrices use agarose and polyacrylamide, which differ in preparation and separation capabilities

Agarose gel electrophoresis (AGE)

• AGE's advantages include a nontoxic gel medium, quick and easy gel casting, separating large DNA molecules, and sample recovery by melting the gel.
• Disadvantages include the high cost of agarose and poor separation of low molecular weight samples.

Polyacrylamide gel electrophoresis (PAGE)

• PAGE involves chemically cross-linked gels via acrylamide polymerization with a cross-linking agent (N,N'-methylenebisacrylamide).
• Reaction uses ammonium persulfate as the initiator and TEMED as the catalyst.
• Advantages include greater resolving power, accommodation of larger DNA quantities, high DNA purity, and controllable pore size using monomer concentrations; polyacrylamide is a neurotoxin when unpolymerized.
• Advantages include stable chemically cross-linked gels, sharp bands, and good separation of low molecular weight fragments.
• Disadvantages include toxic monomers and tedious gel preparation and leakage.

Gel concentration

• Agarose concentration is determined by fragment size, is referred to as a percentage of agarose to buffer volume (w/v), and is normally 0.2%-3%.
• Lower concentrations are better for large DNA fragments, and high concentrations, for small ones.
• Polyacrylamide gels separate proteins using chemical polymerization of acrylamide and N,N'methylenebisacrylamide.
• Pore size is controlled by adjusting acrylamide concentration and the ratio of acrylamide to bisacrylamide.

Applications of DNA PAGE

• For denaturing gels (8-20% concentrations), there is oligonucleotide purification and separation of single-stranded DNA.
• There is isolation of radiolabeled DNA probes, S1 nuclease assay, DNA footprinting, and RNase protection assays.
• For non-denaturing gels (3-20% concentrations) it is often used for separation of di-nucleotide repeats and of DNA ranging 20 bp - 2000 bp in length.

SDS-PAGE

• Proteins denature by boiling in sodium dodecyl sulfate (SDS) and 2-mercaptoethanol.
• Heat and detergent break noncovalent bonds, and 2-mercaptoethanol disrupts covalent bonds between cysteine residues.
• SDS, an amphipathic molecule, has a hydrophobic 12-carbon chain and hydrophilic sulfate group which permeates the protein interior, and binds to hydrophobic groups.
• Denatured proteins bind about 1.4 g SDS/g protein, with ~one SDS molecule for every two amino acids.
• The Laemmli SDS-PAGE system has stacking and running gels with varying pore sizes, ionic strengths, and pH levels.
• Electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH ~8.3) contains glycine, which impacts the separation.

Gel function

• With a applied voltage, chloride ions move to the positive pole with the negatively charged protein-SDS complexes.
• Glycine migrates at the the the rear, and this sets up a steep voltage gradient that "stacks up" into a region at the interface between the gels until glycine reaches the boundary.
• Dramatic changes occur once the ions ions enter the running gel, and the pH of the running gel is closer to the pka of the glycine amino groups, a significant fraction of the glycine molecules assume a negative change, and migrate at at the same rate.
• Because of this, pores, and frictional resistance occurs, because of the "sieving properties" which dictates different rates of protein migration in the gel is inversely proportional to the logarithm of its MW.
• Proteins are stained with dyes the bind non-covalently and with very little specificity to proteins.
• Proteins also "fixed", making them insoluble and unable to diffuse.
• Coomassie Blue staining intensity is considered quantitative, with the intensity of a stained band directly proportional to the amount of protein present.