Capillary electrochromatography

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Mechanism of capillary electrochromatography CEC schematic diag.png
Mechanism of capillary electrochromatography

In chemical analysis, capillary electrochromatography (CEC) is a chromatographic technique in which the mobile phase is driven through the chromatographic bed by electro-osmosis. [1] [2] Capillary electrochromatography is a combination of two analytical techniques, high-performance liquid chromatography and capillary electrophoresis. Capillary electrophoresis aims to separate analytes on the basis of their mass-to-charge ratio by passing a high voltage across ends of a capillary tube, which is filled with the analyte. High-performance liquid chromatography separates analytes by passing them, under high pressure, through a column filled with stationary phase. The interactions between the analytes and the stationary phase and mobile phase lead to the separation of the analytes. In capillary electrochromatography capillaries, packed with HPLC stationary phase, are subjected to a high voltage. Separation is achieved by electrophoretic migration of solutes and differential partitioning.

Contents

Principle

Capillary electrochromatography (CEC) combines the principles used in HPLC and CE. The mobile phase is driven across the chromatographic bed using electroosmosis instead of pressure (as in HPLC). Electroosmosis is the motion of liquid induced by an applied potential across a porous material, capillary tube, membrane or any other fluid conduit. Electroosmotic flow is caused by the Coulomb force induced by an electric field on net mobile electric charge in a solution. Under alkaline conditions, the surface silanol groups of the fused silica will become ionised leading to a negatively charged surface. This surface will have a layer of positively charged ions in close proximity which are relatively immobilised. This layer of ions is called the Stern layer. The thickness of the double layer is given by the formula:

where εr is the relative permittivity of the medium, εo is the permittivity of vacuum, R is the universal gas constant, T is the absolute temperature, c is the molar concentration, and F is the Faraday constant

When an electric field is applied to the fluid (usually via electrodes placed at inlets and outlets), the net charge in the electrical double layer is induced to move by the resulting Coulomb force. The resulting flow is termed electroosmotic flow. In CEC positive ions of the electrolyte added along with the analyte accumulate in the electrical double layer of the particles of the column packing on application of an electric field they move towards the cathode and drag the liquid mobile phase with them.

The relationship between the linear velocity u of the liquid in the capillary and the applied electric field is given by the Smoluchowski equation as

where ζ is the potential across the Stern layer (zeta potential), E is the electric field strength, and η is the viscosity of the solvent.

Separation of components in CEC is based on interactions between the stationary phase and differential electrophoretic migration of solutes.

Instrumentation

The components of a capillary electrochromatograph are a sample vial, source and destination vials, a packed capillary, electrodes, a high voltage power supply, a detector, and a data output and handling device. The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution. The capillary is packed with stationary phase. To introduce the sample, the capillary inlet is placed into a vial containing the sample and then returned to the source vial (sample is introduced into the capillary via capillary action, pressure, or siphoning). The migration of the analytes is then initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high-voltage power supply. The analytes separate as they migrate due to their electrophoretic mobility, and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer. The data is then displayed as an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different migration times in an electropherogram.

Advantages

Avoiding the use of pressure to introduce the mobile phase into the column, results in a number of important advantages. Firstly, the pressure driven flow rate across a column depends directly on the square of the particle diameter and inversely on the length of the column. This restricts the length of the column and size of the particle, particle size is seldom less than 3 micrometer and the length of the column is restricted to 25 cm. Electrically driven flow rate is independent of length of column and size. A second advantage of using electroosmosis to pass the mobile phase into the column is the plug-like flow velocity profile of EOF, which reduces the solute dispersion in the column, increasing column efficiency.

See also

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc, which have been dissolved into liquid solutions.

<span class="mw-page-title-main">Electro-osmosis</span> Movement of liquid through a conduit due to electric potential

In chemistry, electro-osmotic flow is the motion of liquid induced by an applied potential across a porous material, capillary tube, membrane, microchannel, or any other fluid conduit. Because electro-osmotic velocities are independent of conduit size, as long as the electrical double layer is much smaller than the characteristic length scale of the channel, electro-osmotic flow will have little effect. Electro-osmotic flow is most significant when in small channels, and is an essential component in chemical separation techniques, notably capillary electrophoresis. Electro-osmotic flow can occur in natural unfiltered water, as well as buffered solutions.

<span class="mw-page-title-main">Gas chromatography</span> Type of chromatography

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

Micellar electrokinetic chromatography (MEKC) is a chromatography technique used in analytical chemistry. It is a modification of capillary electrophoresis (CE), extending its functionality to neutral analytes, where the samples are separated by differential partitioning between micelles and a surrounding aqueous buffer solution.

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 electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" by means of gradients in conductivity and pH.

<span class="mw-page-title-main">Paper chromatography</span> Separation of coloured chemicals on paper

Paper chromatography is an analytical method used to separate coloured chemicals or substances. It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Liquid chromatography–mass spectrometry</span> Analytical chemistry technique

Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography – MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC–MS is also appropriate for metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Since the early 2000s, LC–MS has also begun to be used in clinical applications.

Chiral column chromatography is a variant of column chromatography that is employed for the separation of chiral compounds, i.e. enantiomers, in mixtures such as racemates or related compounds. The chiral stationary phase (CSP) is made of a support, usually silica based, on which a chiral reagent or a macromolecule with numerous chiral centers is bonded or immobilized.

See Patented via Nth Cycle for metal electro-extraction process.

Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.

<span class="mw-page-title-main">Hydrophilic interaction chromatography</span> Type of chromatography

Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC); however, SFC typically utilizes carbon dioxide as the mobile phase. Therefore, the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state whereby bulk liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The idea of liquid and gas properties convergence was first envisioned by Giddings.

Micellar liquid chromatography (MLC) is a form of reversed phase liquid chromatography that uses an aqueous micellar solutions as the mobile phase.

Electrochromatography is a chemical separation technique in analytical chemistry, biochemistry and molecular biology used to resolve and separate mostly large biomolecules such as proteins. It is a combination of size exclusion chromatography and gel electrophoresis. These separation mechanisms operate essentially in superposition along the length of a gel filtration column to which an axial electric field gradient has been added. The molecules are separated by size due to the gel filtration mechanism and by electrophoretic mobility due to the gel electrophoresis mechanism. Additionally there are secondary chromatographic solute retention mechanisms.

<span class="mw-page-title-main">Thermospray</span>

Thermospray is a soft ionization source by which a solvent flow of liquid sample passes through a very thin heated column to become a spray of fine liquid droplets. As a form of atmospheric pressure ionization in mass spectrometry these droplets are then ionized via a low-current discharge electrode to create a solvent ion plasma. A repeller then directs these charged particles through the skimmer and acceleration region to introduce the aerosolized sample to a mass spectrometer. It is particularly useful in liquid chromatography-mass spectrometry (LC-MS).

<span class="mw-page-title-main">Elution</span> Extraction of a material by washing with a solvent

In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.

A monolithic HPLC column, or monolithic column, is a column used in high-performance liquid chromatography (HPLC). The internal structure of the monolithic column is created in such a way that many channels form inside the column. The material inside the column which separates the channels can be porous and functionalized. In contrast, most HPLC configurations use particulate packed columns; in these configurations, tiny beads of an inert substance, typically a modified silica, are used inside the column. Monolithic columns can be broken down into two categories, silica-based and polymer-based monoliths. Silica-based monoliths are known for their efficiency in separating smaller molecules while, polymer-based are known for separating large protein molecules.

In mass spectrometry, liquid junction interface is an ion source or set-up that couples peripheric devices, such as capillary electrophoresis, to mass spectrometry.

References

  1. Dittmann, Monika M.; Rozing, Gerard P. (1996). "Capillary electrochromatography — a high-efficiency micro-separation technique". Journal of Chromatography A. 744 (1–2): 63–74. doi:10.1016/0021-9673(96)00382-2. ISSN   0021-9673.
  2. Cikalo, Maria G.; Bartle, Keith D.; Robson, Mark M.; Myers, Peter; Euerby, Melvin R. (1998). "Capillary electrochromatography". The Analyst. 123 (7): 87–102. Bibcode:1998Ana...123...87C. doi:10.1039/a801148f. ISSN   0003-2654.

Further reading