Capillary electrophoresis

Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other

The instrumentation needed to perform capillary electrophoresis is relatively simple. A basic

The separation of compounds by capillary electrophoresis is dependent on the differential migration of analytes in an applied electric field. The electrophoretic migration velocity (${\displaystyle u_{p}}$) of an analyte toward the electrode of opposite charge is:

${\displaystyle u_{p}=\mu _{p}E\,}$

The electrophoretic mobility can be determined experimentally from the migration time and the field strength:

${\displaystyle \mu _{p}=\left({\frac {L}{t_{r}}}\right)\left({\frac {L_{t}}{V}}\right)}$

where ${\displaystyle L}$ is the distance from the inlet to the detection point, ${\displaystyle t_{r}}$ is the time required for the analyte to reach the detection point (migration time), ${\displaystyle V}$ is the applied voltage (field strength), and ${\displaystyle L_{t}}$ is the total length of the capillary.^[4] Since only charged ions are affected by the electric field, neutral analytes are poorly separated by capillary electrophoresis.

The velocity of migration of an analyte in capillary electrophoresis will also depend upon the rate of

The velocity of the electroosmotic flow, ${\displaystyle u_{o}}$ can be written as:

${\displaystyle u_{o}=\mu _{o}E}$

where ${\displaystyle \mu _{o}}$ is the electroosmotic mobility, which is defined as:

${\displaystyle \mu _{o}={\frac {\epsilon \zeta }{\eta }}}$

where ${\displaystyle \zeta }$ is the zeta potential of the capillary wall, and ${\displaystyle \epsilon }$ is the relative permittivity of the buffer solution. Experimentally, the electroosmotic mobility can be determined by measuring the retention time of a neutral analyte.^[4] The velocity (${\displaystyle u}$) of an analyte in an electric field can then be defined as:

${\displaystyle u_{p}+u_{o}=(\mu _{p}+\mu _{o})E}$

Since the electroosmotic flow of the buffer solution is generally greater than that of the electrophoretic mobility of the analytes, all analytes are carried along with the buffer solution toward the cathode. Even small, triply charged anions can be redirected to the cathode by the relatively powerful EOF of the buffer solution. Negatively charged analytes are retained longer in the capillary due to their conflicting electrophoretic mobilities.

In certain situations where strong electroosmotic flow toward the cathode is undesirable, the inner surface of the capillary can be coated with polymers, surfactants, or small molecules to reduce electroosmosis to very low levels, restoring the normal direction of migration (anions toward the anode, cations toward the cathode). CE instrumentation typically includes power supplies with reversible polarity, allowing the same instrument to be used in "normal" mode (with EOF and detection near the cathodic end of the capillary) and "reverse" mode (with EOF suppressed or reversed, and detection near the anodic end of the capillary). One of the most common approaches to suppressing EOF, reported by Stellan Hjertén in 1985, is to create a covalently attached layer of linear polyacrylamide.^[8] The silica surface of the capillary is first modified with a silane reagent bearing a polymerizable vinyl group (e.g. 3-methacryloxypropyltrimethoxysilane), followed by introduction of acrylamide monomer and a free radical initiator. The acrylamide is polymerized in situ, forming long linear chains, some of which are covalently attached to the wall-bound silane reagent. Numerous other strategies for covalent modification of capillary surfaces exist. Dynamic or adsorbed coatings (which can include polymers or small molecules) are also common.^[9] For example, in capillary sequencing of DNA, the sieving polymer (typically polydimethylacrylamide) suppresses electroosmotic flow to very low levels.^[10] Besides modulating electroosmotic flow, capillary wall coatings can also serve the purpose of reducing interactions between "sticky" analytes (such as proteins) and the capillary wall. Such wall-analyte interactions, if severe, manifest as reduced peak efficiency, asymmetric (tailing) peaks, or even complete loss of analyte to the capillary wall.

Efficiency and resolution

The number of theoretical plates, or separation efficiency, in capillary electrophoresis is given by:

${\displaystyle N={\frac {\mu V}{2D_{m}}}}$

where ${\displaystyle N}$ is the number of theoretical plates, ${\displaystyle \mu }$ is the apparent mobility in the separation medium and ${\displaystyle D_{m}}$ is the

The efficiency of capillary electrophoresis separations is typically much higher than the efficiency of other separation techniques like

The resolution (${\displaystyle R_{s}}$) of capillary electrophoresis separations can be written as:

${\displaystyle R_{s}={\frac {1}{4}}\left({\frac {\triangle \mu _{p}{\sqrt {N}}}{\mu _{p}+\mu _{o}}}\right)}$

According to this equation,

Besides diffusion and Joule heating (discussed above), factors that may decrease the resolution in capillary electrophoresis from the theoretical limits in the above equation include, but are not limited to, the finite widths of the injection plug and detection window; interactions between the analyte and the capillary wall; instrumental non-idealities such as a slight difference in height of the fluid reservoirs leading to siphoning; irregularities in the electric field due to, e.g., imperfectly cut capillary ends; depletion of buffering capacity in the reservoirs; and electrodispersion (when an analyte has higher conductivity than the background electrolyte).[12] Identifying and minimizing the numerous sources of band broadening is key to successful method development in capillary electrophoresis, with the objective of approaching as close as possible to the ideal of diffusion-limited resolution.

Applications

Capillary electrophoresis may be used for the simultaneous determination of the ions NH₄^+,, Na⁺, K⁺, Mg²⁺ and Ca²⁺ in saliva.^[13]

One of the main applications of CE in forensic science is the development of methods for amplification and detection of DNA fragments using polymerase chain reaction (PCR), which has led to rapid and dramatic advances in forensic DNA analysis. DNA separations are carried out using thin CE 50-mm fused silica capillaries filled with a sieving buffer. These capillaries have excellent capabilities to dissipate heat, permitting much higher electric field strengths to be used than slab gel electrophoresis. Therefore separations in capillaries are rapid and efficient. Additionally, the capillaries can be easily refilled and changed for efficient and automated injections. Detection occurs via fluorescence through a window etched in the capillary. Both single-capillary and capillary-array instruments are available with array systems capable of running 16 or more samples simultaneously for increased throughput.^[14]

A major use of CE by forensic biologists is typing of

Another application of CE in forensics is ink analysis, where the analysis of inkjet printing inks is becoming more necessary due to increasingly frequent counterfeiting of documents printed by inkjet printers. The chemical composition of inks provides very important information in cases of fraudulent documents and counterfeit banknotes. Micellar electrophoretic capillary chromatography (MECC) has been developed and applied to the analysis of inks extracted from paper. Due to its high resolving power relative to inks containing several chemically similar substances, differences between inks from the same manufacturer can also be distinguished. This makes it suitable for evaluating the origin of documents based on the chemical composition of inks. It is worth noting that because of the possible compatibility of the same cartridge with different printer models, the differentiation of inks on the basis of their MECC electrophoretic profiles is a more reliable method for the determination of the ink cartridge of origin (its producer and cartridge number) rather than the printer model of origin.[16]

A specialized type of CE, affinity capillary electrophoresis (ACE), utilizes intermolecular binding interactions to understand protein-ligand interactions.^[17] Pharmaceutical companies use ACE for a multitude of reasons, with one of the main ones being the association/binding constants for drugs and ligands or drugs and certain vehicle systems like micelles. It is a widely used technique because of its simplicity, rapid results, and low analyte usage.^[18] The use of ACE can provide specific details in binding, separation, and detection of analytes and is proven to be highly practical for studies in life sciences. Aptamer-based affinity capillary electrophoresis is utilized for the analysis and modifications of specific affinity reagents. Modified aptamers ideally exhibit and high binding affinity, specificity, and nuclease resistance.^[19] Ren et al. incorporated modified nucleotides in aptamers to introduce new confrontational features and high affinity interactions from the hydrophobic and polar interactions between IL-1α and the aptamer.^[20] Huang et al. uses ACE to investigate protein-protein interactions using aptamers. A α-thrombin binding aptamer was labeled with 6-carboxyfluorescein for use as a selective fluorescent probe and was studied to elucidate information on binding sites for protein-protein and protein-DNA interactions.^[21]

Capillary electrophoresis (CE) has become an important, cost-effective approach to do DNA sequencing that provides high throughput and high accuracy sequencing information. Woolley and Mathies used a CE chip to sequence DNA fragments with 97% accuracy and a speed of 150 bases in 540 seconds.^[22] They used a 4-color labeling and detection format to collect fluorescent data. Fluorescence is used to view the concentrations of each part of the nucleic acid sequence, A, T, C and G, and these concentration peaks that are graphed from the detection are used to determine the sequence of the DNA.^[22]

References