Modern Countercurrent Chromatography

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Since exactly the same liquid system can be used on larger CCC columns, the scaling up is straightforward being almost geometrical: an x time larger column can separate an x time larger amount of sample in the same time if the flow rate is increased x times [ 6 ]. It was even found that the phase mixing being better in larger CCC column, it is possible to inject more than the amount predicted by simple geometrical scaling up [ 26 ].

The purification of an industrial pest control agent with the trade name spinetoram Dow Chemical was done by LC on C 18 silica stationary phases. As derivatives of biologically produced compounds, spinetorams pose less environmental risk than chlorinated pesticides or other totally synthetic ones [ 27 ].

The biological activity of pure J or L forms is far superior to that of the mixture justifying the separation. The common way to separate the J and L spinetoram forms is using reversed phase liquid chromatography. This is completely unrealistic. A different system dedicated to preparative purification is used.

The flow rate was 0. Using a protocol similar to the one described for the P. In rotating bobbins or spools with coiled tubes, the thread of the tube produces a force that pushes the contained liquid toward one end of the tube or the other.


This force is called an Archimedean screw. Its intensity and direction depend on the rotational speed and direction and on the way the coil was made. When two immiscible liquid phases are contained in the coiled tube, the denser liquid is subjected to a stronger force than the lighter liquid phase pushing it to gather at one end of the coil called head. This motion produces an inversion of the centrifugal field direction, hence the mingling decantation of the two liquid phases.

The number of channels in a CPC hydrostatic machine that are needed to produce one theoretical plate. This is s measure of the efficiency of the liquid system and chromatographic efficiency. One of the principal instrumental parameters in CCC is the total volume of the column, which is important when understanding analyte elution and when calculating countercurrent chromatograms.

Total column volume V C also determines the load capacity of a particular machine. It is recommended to use the experimental column volume obtained by measuring the retention volume of a compound injected into the column containing a single liquid phase. A hydrodynamic CCC machine containing one or several spools or bobbins of coiled tube mounted on a rotor, first introduced by Ito [ 1 ]. Various forms of coil planet centrifuge are possible. A kind of hydrodynamic CCC machine containing two spools of coiled tube mounted on a rotor in such a way that the axis of rotation of the spools is at right angle to the central axis of rotation of the rotor.

It was never commercialized as it is mechanically very fragile. The first hydrostatic CCC column working with gravity only. See Section 2. A particular way to use a CCC column to elute strongly retained solutes. After elution of a given volume of mobile phase, the phase role is interchanged.

Factors having an impact on the CCC separation

The mobile phase becomes the stationary phase and vice versa. Since the density difference is reversed, the flowing direction of the phases is also reversed. Dual countercurrent chromatography performs genuine countercurrent chromatography in which both upper and lower phases move through a coiled column in the opposite direction. Dual CCC has been achieved with both a multilayer coiled separation column and a spiral disk.

Chromatography – VI: Counter Current Extraction, Ultra Centrifugation

The force or field of forces exercised to hold the stationary phase in the CCC column. This field is calculated as a function of rotational speed rpm and rotational radius. In hydrodynamic CCC, the end of the coil where the liquid or any small light object such a bubble or dense object such a bead is pushed by the Archimedean screw action when the rotor is spinning.

A CCC machine containing planetary spools or bobbins, on which tubing is wound. Two axes of rotation and a gear arrangement produce a periodically changing centrifugal acceleration field. The machine generates the centrifugal field needed to retain the liquid stationary phase.

During a run, the machine can be considered a column containing a volume V S of liquid stationary phase and V M of mobile phase. Most hydrodynamic machines do not have any rotary seal. A CCC machine containing channels, chambers, or geometrical units interconnected according to a repetitive pattern in a centrifuge rotor and working with a constant centrifugal field one axis of rotation. These machines are also called centrifugal partition chromatographs, a term that was once a trademark. CPC instruments use rotating seals and generally operate at higher back pressures than hydrodynamic CCC columns.

The DCCC is a hydrostatic column without centrifuge. A hydrodynamic CCC system that uses a multilayer coil separation column rotating in a planetary motion introduced by Ito [ 2 , 10 ]. The term high speed was used because the new hydrodynamic CCC columns were able to separate mixtures in several hours, while several days were needed with gravitational hydrostatic droplet CCC columns. Planetary motion involves two axes. Both the rotor and the coil s rotate in the same direction, and a simple gear arrangement produces a coil rotation exactly double the rotor rotation. It is used exclusively in modern CCC hydrodynamic instruments.

Several spiral disks piled together in one unit essentially replace the coil of a traditional CCC instrument [ 28 ]. In hydrodynamic CCC, the tail is the end of the coil opposite to the head. A hydrostatic CCC device in which the geometrical pattern is simply a turn of tubing arranged at a fixed radius in a uniform centrifugal force field. In brief, carefully selected volumes of the effluent from the first CCC column are introduced into a second CCC column with a different biphasic liquid system to enhance the separation of target analytes [ 29 ].

Term used in hydrostatic CCC columns or centrifugal partition chromatographs indicating that the mobile phase is entering through the bottom of the column and leaving it out of the top. The mobile phase is the lighter liquid, while the stationary phase is the denser one.

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The term used in hydrostatic CCC columns indicating that the mobile phase is entering through the top of the column and leaving it out of the bottom. The mobile phase is the denser liquid, while the stationary phase is the lighter one. EECCC is a recently developed and fully parameterized CCC method that takes advantage of the liquid nature of the stationary phase by combining classical elution and extrusion in a single run.

After an initial elution stage in classical mode, extrusion of the stationary phase is achieved by switching the supply of the flowing liquid from the mobile phase to the originally stationary phase, while maintaining the centrifugal force through continued rotation. The point at which extrusion is begun called the switch volume, V CM can be adjusted to optimize the resolution of target analytes and minimize the runtime.

Countercurrent chromatography - Wikiwand

After performing classical elution for a certain period of time, the noneluted analytes migrate inside the column. Extrusion provides access to these analytes without the need to reach the elution volume, and this can be achieved by pumping stationary phase into the column.

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In gradient CCC, the composition of the mobile phase is adjusted during the CCC run to help hasten the elution of highly retained compounds from the column. The composition of the liquid stationary phase also changes [ 14 ]. The term indicating that the mobile phase is entering a hydrodynamic column through its head and leaving it out of the tail.

The mobile phase in this configuration is the denser liquid, while the stationary phase is the lighter one. This mode corresponds to the descending mode for hydrodynamic columns. The volume of mobile phase needed to elute a particular analyte. Retention volumes are often calculated by multiplying the retention time and the flow rate.

V R depends on the liquid phase ratio inside the CCC column and on the solute liquid—liquid distribution ratio Equation 3. This is the major difference between the CCC techniques and all other chromatographic techniques: the liquid stationary phase volume is not a constant. It depends on the experimental conditions so that the special parameter, S f , is needed to quantitate it. S f is the ratio of the volume of the stationary phase retained in the CCC machine to that of the machine volume Equation 1.

Note : S f is often expressed as a percentage. The term indicates that the mobile phase is entering a hydrodynamic CCC column through its tail and leaving it out of the head. In classical mode, the mobile phase in this configuration is the lighter liquid, while the stationary phase is the denser one.

Modern Countercurrent Chromatography

This mode corresponds to the ascending mode for hydrostatic columns. A biphasic liquid system in which both phases are aqueous solutions e. It will ionize first the solutes and, last, the retainer contained in the stationary phase. Notes: A good synonym for K is partition ratio. The distribution constant is proportional to the CCC retention time if the solute can exist in only a single definite form no ionization, no complexation, no chemical reaction possible. The distribution constant of a particular solute depends only on the physicochemical properties of the liquid system used, composition and temperature, but not on the CCC column. The ratio of the total analytical concentration of a solute in the liquid stationary phase, regardless of its chemical form, to its total analytical concentration in the mobile phase. Notes: The term ratio is preferable to coefficient.

Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography Modern Countercurrent Chromatography
Modern Countercurrent Chromatography

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