Countercurrent chromatography
The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases.
A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation.
In reversed-phase chromatography, for example, the stationary phase can be regarded as a liquid which is immobilized by chemical bonding to a micro-porous silica solid support.
When gas chromatography or HPLC is carried out with large volumes, resolution is lost due to issues with surface-to-volume ratios and flow dynamics; this is avoided when both phases are liquid.
Vigorous mixing of the phases is critical in order to maximize the interfacial area between them and enhance mass transfer.
The settling time is a property of the solvent system and the sample matrix, both of which greatly influence stationary phase retention.
[18] To most process chemists, the term "countercurrent" implies two immiscible liquids moving in opposing directions, as typically occurs in large centrifugal extractor units.
[19] Several researchers have proposed renaming both CCC & CPC to liquid-liquid chromatography,[20] but others feel the term "countercurrent" itself is a misnomer.
A wide array of biphasic solvent mixtures are available to the CCC practitioner including the combination n-hexane (or heptane), ethyl acetate, methanol and water in different proportions.
The original complex matrix will have been fractionated into discrete narrow polarity bands, which may then be assayed for chemical composition or bioactivity.
DCCC enjoyed some success with natural product separations but was largely eclipsed by the rapid development of high-speed countercurrent chromatography.
[34] The main limitation of DCCC is that flow rates are low, and poor mixing is achieved for most binary solvent systems.
[36] Much development was needed to engineer the instrument so that required planetary motion could be sustained while the phases were being pumped through the coil(s).
[37] Parameters such as the relative rotation of the two axes (synchronous or non-synchronous), the direction of flow through the coil, and the rotor angles were investigated.
More recently instrument derivatives have been offered with rotating seals for various hydrodynamic CCC designs, instead of flying leads, either as custom or standard options.
[52] Hydrostatic CCC or centrifugal partition chromatography (CPC) was invented in the 1980s by the Japanese company Sanki Engineering Ltd, whose president was Kanichi Nunogaki.
More recently, in France and UK, non-stacked disc CPC configurations have been developed with PTFE, stainless steel or titanium rotors.
These have been designed to overcome possible leakages between the stacked discs of the original concept, and to allow steam cleaning for good manufacturing practice.
Contrary to hydrodynamic CCC, the rotation speed is not directly proportional to the retention volume ratio of the stationary phase.
Like DCCC, CPC can be operated in either descending or ascending mode, where the direction is relative to the force generated by the rotor rather than gravity.
[58] The aforementioned hydrodynamic and hydrostatic instruments may be employed in a variety of ways, or modes of operation, in order to address the particular separation needs of the scientist.
This is not possible with all biphasic solvent systems, due to excessive loss of stationary phase created by disruption the equilibrium conditions within the column.
Dual-flow, also known as dual, countercurrent chromatography occurs when both phases are flowing in opposite directions inside the column.
This mode may accommodate continuous or sequential separations with the sample being introduced in the middle of the column or between two bobbins in a hydrodynamic instrument.
[72] The process of re-separating selected fractions from one chromatography experiment with another chromatographic method has long been practiced by scientists.
For example, 6 oxindole alkaloids were isolated from a 4.5g sample of Gelsemium elegans stem extract with a biphasic solvent system composed of hexane–ethyl acetate–methanol–water (3:7:1:9, v/v) where 10 mM triethylamine (TEA) was added to the upper organic stationary phase as a retainer and 10 mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter.
[77] Ion-exchange modes such as pH-zone-refining have tremendous potential because high sample loads can be achieved without sacrificing separation power.
[78] Countercurrent chromatography and related liquid-liquid separation techniques have been used on both industrial and laboratory scale to purify a wide variety of chemical substances.
Separation realizations include proteins,[79] DNA,[80] Cannabidiol (CBD) from Cannabis Sativa[81] antibiotics,[82] vitamins,[83] natural products,[31] pharmaceuticals,[52] metal ions,[84] pesticides,[85] enantiomers,[86] polyaromatic hydrocarbons from environmental samples,[87] active enzymes,[88] and carbon nanotubes.
[89] Countercurrent chromatography is known for its high dynamic range of scalability: milligram to kilogram quantities purified chemical components may be obtained with this technique.
