Countercurrent chromatography

Countercurrent chromatography (CCC, also known as counter-current or counter current chromatography) is an analytical chemistry technique that is used to separate, identify, and quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography encompasses a collection of related liquid chromatography techniques that employ two immiscible liquid phases without a solid support. The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. 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.

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, DNA, antibiotics,McAlpine, James B.; Friesen, J. Brent; Pauli, Guido F. (2012). "Separation of Natural Products by Countercurrent Chromatography". Natural Products Isolation. Vol. 864. Totowa, NJ: Humana Press. pp. 221–254. ISBN 978-1-61779-623-4 978-1-61779-624-1. {{cite book}}: |access-date= requires |url= (help); Check |isbn= value: length (help); External link in |chapterurl= (help); Unknown parameter |chapterurl= ignored (|chapter-url= suggested) (help); Unknown parameter |editors= ignored (|editor= suggested) (help) vitamins,Kurumaya, Katsuyuki; Sakamoto, Tetsuto; Okada, Yoshihito; Kajiwara, Masahiro (1988-01). "Application of droplet counter-current chromatography to the isolation of vitamin B12". Journal of Chromatography A. 435: 235–240. doi:10.1016/S0021-9673(01)82181-6. ISSN 0021-9673. Retrieved 2016-02-21. {{cite journal}}: Check date values in: |date= (help) natural products,Friesen, J. Brent; McAlpine, James B.; Chen, Shao-Nong; Pauli, Guido F. (2015-07-24). "Countercurrent Separation of Natural Products: An Update". Journal of Natural Products. 78 (7): 1765–1796. doi:10.1021/np501065h. ISSN 1520-6025 0163-3864, 1520-6025. Retrieved 2016-02-21. {{cite journal}}: Check |issn= value (help) pharmaceuticals,Sumner, Neil (2011-09). "Developing counter current chromatography to meet the needs of pharmaceutical discovery". Journal of Chromatography A. 1218 (36): 6107–6113. doi:10.1016/j.chroma.2011.05.001. ISSN 0021-9673. Retrieved 2016-02-21. {{cite journal}}: Check date values in: |date= (help) metal ions, pesticides, and carbon nanotubes.

Countercurrent chromatography is known for its high dynamic range of scalability: milligram to kilogram quantities purified chemical components may be obtained with this technique. It also has the advantage of accommodating chemically complex samples with undissolved particulates.

Some types of countercurrent chromatography, such as dual flow CCC, feature a true countercurrent process where the two immiscible phases flow past each other and exit at opposite ends of the column.Ito, Yuko; Goto, Tomomi; Yamada, Sadaji; Matsumoto, Hiroshi; Oka, Hisao; Takahashi, Nobuyuki; Nakazawa, Hiroyuki; Nagase, Hisamitsu; Ito, Yoichiro (2006-03). "Application of dual counter-current chromatography for rapid sample preparation of N-methylcarbamate pesticides in vegetable oil and citrus fruit". Journal of Chromatography A. 1108 (1): 20–25. doi:10.1016/j.chroma.2005.12.070. ISSN 0021-9673. Retrieved 2016-02-21. {{cite journal}}: Check date values in: |date= (help) More often, however, one liquid acts as the stationary phase and is retained in the column while the mobile phase is pumped through it. The liquid stationary phase is held in place by gravity or by centrifugal force. An example of a gravity method is called droplet counter current chromatography (DCCC). There are two modes by which the stationary phase is retained by centrifugal force: hydrostatic and hydrodynamic. In the hydrostatic method, the column, a series of chambers connected by channels, is rotated about a central axis. Hydrostatic instruments are marketed under the name centrifugal partition chromatography (CPC).Marchal, Luc; Legrand, Jack; Foucault, Alain (2003-07). "Centrifugal partition chromatography: A survey of its history, and our recent advances in the field". The Chemical Record. 3 (3): 133–143. doi:10.1002/tcr.10057. ISSN 1528-0691 1527-8999, 1528-0691. Retrieved 2016-02-21. {{cite journal}}: Check |issn= value (help); Check date values in: |date= (help) [1][3] Hydrodynamic instruments are often marketed as high-speed or high-performance countercurrent chromatography (HSCCC and HPCCC respectively) instruments which rely on the Archimedes' screw force in a helical coil to retain the stationary phase in the column.

The components of a CCC system are similar to most liquid chromatography configurations such as HPLC. One or more pumps deliver the phases to the column which is the CCC instrument itself. Samples are introduced into the column through a sample loop filled with as automated or manual syringe. The outflow is monitored with various detectors such as UV-vis or Mass Spectrometry. The operation of the pumps, CCC instrument, sample injection, and detection may controlled manually or with a microprocessor.

Support-free liquid chromatography

Standard column chromatography uses an apparent solid stationary phase and a liquid mobile phase, while gas chromatography (GC) uses a solid or liquid stationary phase on a solid support and a gaseous mobile phase. By contrast, in liquid-liquid chromatography, both the mobile and stationary phases are liquid. The contrast is, however not as stark as it first appears. In reverse phase HPLC, the phases, can in many cases be regarded as liquids which are immobilized by chemical bonding to a solid support, such as micro-porous silica. As opposed to CCC/CPC where mechanical/gravitational forces are used to achieve to stationary liquid layer instead of bonding to a solid support. By eliminating solid supports, permanent adsorption of the analyte onto the column is avoided, and a high recovery of the analyte can be achieved. The instrument is also easily switched between normal-phase and reversed-phase modes of operation simply by changing the mobile and stationary phases. With liquid chromatography, operation is limited by the composition of the columns and media commercially available for the instrument. Nearly any pair of immiscible solutions can be used in liquid-liquid chromatography provided that the stationary phase can be successfully retained.

Solvent costs are also generally lower than for high-performance liquid chromatography (HPLC), and the cost of purchasing and disposing of solid adsorbents is eliminated. Another advantage is that experiments conducted in the laboratory can be scaled to industrial volumes. When GC 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.

Partitioning coefficient (Kd)

The CCC separation process can be thought of as occurring in three stages: mixing, settling, and separation of the two phases (although they often occur continuously). Vigorous mixing of the phases is critical in order to maximise the interfacial area between them, and the analyte can distribute between the phases according to its partition coefficient.

The mobile phase mixes with, then settles from the stationary phase throughout the column. The degree of stationary phase retention (inversely proportional to the amount of stationary phase loss or "bleed" in the course of a separation) is a crucial parameter. Common factors that influence stationary phase retention are flow rate, solvent composition of the biphasic solvent system, and the G-force created by rotation. The settling time is a property of the solvent system and the sample matrix, both of which greatly influence stationary phase retention.

To most process chemists, counter current chromatography implies two biphasic liquids moving in opposing directions, as typically occurs in large process extraction units. With the exception of Dual-Flow (see below), most CCC & CPC applications have a stationary phase and a mobile phase. Even in this situation, counter current flows occur within the coil/rotor counter. Several researchers have proposed renaming both CCC & CPC to liquid-liquid chromatography, but others feel the name chromatography itself is a misnomer. CPC, or centrifugal partition chromatography, is arguably a more appropriate name, but CPC has historically applied only to sun centrifuges, and not planetary centrifuges, which are still called counter current chromatographs. This confusion was possibly added to, rather than resolved, when the CCC/CPC community chose to classify planetary CCC as HYDRODYNAMIC CCC and sun centrifuges (CPC) as HYDROSTATIC CCC.

Typically most modern commercial CCC and CPC can inject 5 to 40 g per liter capacity. The range is so large, even for a specific instrument, let alone all instrument options, as the type of target, matrix and available biphasic solvent vary so much. Approximately 10 g per liter would be a more typical value, that the majority of applications could use as a base value.

Unlike Flash Chromatography and HPLC, CCC & CPC can inject large volumes relative to coil/rotor/column volume. Typically 5 to 10% of coil volume can be injected. In some cases this can be increased to as high as 15 to 20% of the coil etc., volume.

In comparison to flash chromatography/preparative/process HPLC, flows and total solvent usage can in most CCC/CPC be reduced by half and even up to a tenth of the needs of Flash Chromatography/Preparative/Process HPLC.

An example of a major application of CCC/CPC is to take an extremely complex matrix, run on a generic biphasic with elution (possibly step gradient) and then extrusion, and fractionate original complex matrix into discrete narrow polarity bands, which could then be assayed for chemical composition or bioactivity. Working this scenario in conjunction with other chromatographic and non chromatographic techniques has the potential for rapid advances in compositional recognition of extremely complex matrices.

Droplet

Droplet CCC (DCCC) is the oldest form of CCC. It uses only gravity to move the mobile phase through the stationary phase. In descending mode, droplets of the denser mobile phase and sample are allowed to fall through a column of the lighter stationary phase using only gravity.

If a less dense mobile phase is used it will rise through the stationary phase; this is called ascending mode. The eluent from one column is transferred to another; the more columns that are used, the more theoretical plates can be achieved. The disadvantage of DCCC is that flow rates are low, and poor mixing is achieved for most binary solvent systems, which makes this technique both time-consuming and inflexible.

Hydrodynamic

High-speed

The modern era of CCC began with the development by Dr. Yoichiro Ito of the planetary centrifuge and the many possible column geometries it can support. Peter Carmeci (deceased) initially commercialized the Ito "J" Type as the PC Inc CCC which utilized a single bobbin (onto which the coil is wound) and a counter balance, plus a set of "Flying Leads". Dr. Walter Conway & others later evolved the bobbin design such that multiple coils, even coils of different id, could be had on the single bobbin. Dr Edward Chou (deceased) later evolved and commercialized a triple bobbin design as the Pharmatech CCC which had a de-twist mechanism for leads between the three bobbins. The Quattro CCC released in 1993 further evolved the "J" type centrifuge by utilizing a novel mirror image, twin bobbin design that did not need the de-twist mechanism of the Pharmatech between the multiple bobbins, so could still accommodate multiple bobbins on the same instrument. Hydrodynamic CCC are now available with up to 4 coils per instrument. These coils can be in PTFE, PEEK, PVDF, or Stainless Steel tubing. The 2, 3 or 4 coils can all be of the same bore to facilitate "2D" CCC (see below) or the use of one instrument to do four same eluent, or four different eluent preparations at the same time, on same instrument. The coils can also be of different ids, on one instrument, ranging from 1 to 2 to 3.7 to 6 mm on one instrument, thus allowing a single instrument to optimize from mg to kilos per day. Other instrument designs have since copied this mirror image multiple bobbin concept. More recently instrument derivatives have started to be offered with rotating seals for various Hydrodynamic CCC designs, instead of flying leads, either as custom options or as standard.

The flying lead devices make non-rotating connections between the stator and the rotor of a centrifuge, via twist/de-twist mechanisms.

High-performance

An Example of a HPCCC system

The operating principle of CCC equipment requires a column consisting of a tube coiled around a bobbin. The bobbin is rotated in a double-axis gyratory motion (a cardioid), which causes a variable gravity (G) field to act on the column during each rotation. This motion causes the column to see one partitioning step per revolution and components of the sample separate in the column due to their partitioning coefficient between the two immiscible liquid phases used.

High-performance countercurrent chromatography (HPCCC) works in much the same way as HSCCC but with one vital difference. A seven-year R&D process that has produced HPCCC instruments that generated 240 G's, compared to the 80 G's of the HSCCC machines. This increase in G-level and larger bore of the column has enabled a tenfold increase in through put, due to improved mobile phase flow rates and a much higher stationary phase retention.

Countercurrent chromatography is a preparative liquid chromatography technique, however with the advent of the higher-G HPCCC instruments it is now possible to operate instruments with sample loadings as low as a few milligrams, whereas in the past 100s of milligrams had been necessary.

Major application areas for this technique include natural products purification and also drug development.

Hydrostatic

Hydrostatic CCC or centrifugal partition chromatography (CPC) was invented in the eighties by the Japanese company Sanki Engineering Ltd, whose president was the late Kanichi Nunogaki. CPC has been extensively developed in France starting from the late nineties, which initially optimized the stacked disc concept initiated by Sanki. More recently in France and UK non-stacked disc CPC, 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 GMP. The volumes ranging from a 100 ml to 12 liters are available in many rotor materials. The 25 liters per rotor CPC has a Titanium rotor. CPC uses centrifugal force to speed separation and achieves higher flow rates than DCCC (which relies on gravity). This technique is sometimes sold under the name FCPC or SCPC.

Principle

This technique is still based on the principles of liquid/liquid partitioning chromatography: two non-miscible liquid phases are mixed together to form a two-phase system, and are then separated multiple times. The individual solutes are isolated based on the different partitioning coefficients of each compound in this two-phase system. One of the liquid phases of the two-phase system is used as a stationary liquid phase: it is fed into the column (the rotor) while the latter is spinning at moderate rotational speed. The stationary phase is retained inside the rotor by the centrifugal force generated. The second phase of the two-phase system is used as the mobile phase containing the solutes to be extracted. It is fed under pressure into the rotor and pumped through the stationary phase. Both phases are mixed together. It is at that time that the exchange of molecules between the two phases occurs. The separation of the solutes is achieved as a function of the specific partitioning coefficient (Kd) of each solute between the mobile and stationary phases. The mobile phase then decants at each cell outlet thus entering the next cell. The eluted fractions of the mobile and stationary phases are collected over a period of several minutes to several hours. These fractions, or eluates, will contain the individual purified solutes.

Realization

The centrifugal partition chromatograph is constituted with a unique rotor (=column). This rotor rotates on its central axis (while HSCCC column rotates on its planetary axis and simultaneously rotates eccentrically about another solar axis). With less vibrations and noise, the CPC offers a wider rotation speed range (from 500 to 2000 rpm) than HSCCC. That allows a better decantation and retention for unstable biphasic system (e.g., aqueous aqueous systems or butanol/water systems).

The CPC rotor is constituted by the superposition of disks engraved with small cells connected by head / tail ducts. These cells, where the chromatographic separation takes place, can be compared to lined-up separate funnels.

The rotor is filled with the stationary phase, which stays inside the rotor thanks to the rotation speed, while the mobile phase is pumped through. 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. According to the fast and permanent evolution of the cells design, the efficiency and flow rate with low back pressure are improved.

Advantages

The CPC offers now the direct scale up from the analytical apparatuses (few milliliters) to industrial apparatuses (some liters) for fast batch production.

Modes of operation

Normal-phase

In normal phase (organic mobile phase), the non-aqueous phase is pumped through as the mobile phase, with a more polar stationary phase. As in many sciences, the cause of original nomenclature is often forgot, but understanding it is relevant. As original forms of paper chromatography (the term chromatography is itself a misnomer as refers to colors being separated, rather than concept resolution of target columns by partitioning between stationary and mobile phase) were superseded by more efficient means, eventually diatomaceous earths (natural micro-porous silica), and then modern synthesized micro-porous silica, the phase was polar (OH groups) and maximum retention was achieved with non-polar solvents such as n-hexane. Progressively more polar eluents were then used to elute non-eluting targets. The evolution of alkane bonded phases where variously non-polar C2/C4/C8/C12/C18/C22 etc., were tried. Historically with C18 being most popular. All are chemically bonded to the silica, and a reversal of the elution trend occurred. Thus a polar stationary became normal phase chromatography, and the non-polar stationary phase chromatography became REVERSE PHASE CHROMATOGRAPHY. In much the same way that nobody is proposing changes to the names, chromatography to reflect the processes non-dependence on color, or changes to normal and reverse phase, the many misnames in CCC/CPC, pH Zone refining etc., are now possibly so well set, that in respect to originators they should be left alone.

Reverse-phase

In reverse-phase (e.g. aqueous mobile phase), the aqueous phase is used as the mobile phase with a less polar stationary phase.

Dual-mode

The mobile and stationary phases are reversed part way through the run.

Gradient mode

The concentration of one or more components in the mobile phase is varied throughout the run to achieve optimal resolution across a wider range of polarities. For example, a methanol-water gradient may be employed using pure heptane as the stationary phase. This is not possible with all binary systems, due to excessive loss of stationary phase. It will be found that linear gradients can be reproduced, as the bleeding of stationary phase as biphasic mix changes is usually reproducible. It may, however, for CCC & CPC be best to consider step gradients, where bleed of stationary phase quickly re-stabilizes after each gradient step.

Elution extrusion mode

In elution extrusion mode (EECCC), the washing off of stationary phase by either stopping rotation whilst flowing a solvent or changing from mobile to stationary phase whilst maintaining rotation was used by various chromatographers before the term EECCC was suggested. The mobile phase is extruded after a certain point by switching the phase being pumped into the system. For example, during the elution portion of a separation using an ethyl acetate-water system running head to tail, the aqueous mobile phase is being pumped into the system. In order to switch to extrusion mode, organic phase is pumped into the system. This can be accomplished either with a valve on the inlet of single pump, or ideally with an integrated system of two or three pumps, each dedicated either to a single phase of a binary mixture, or to an intermediate wash solvent. This also allows for good resolution of compounds with high mobile-phase affinities. It requires only one column volume of solvent and leaves the column full of fresh stationary phase.

pH Zone refining

Acidic and basic solvents are used to elute analytes based on their pKa. As with much of CCC/CPC terminology the use of pH-Zone refining term, which suggests a unique to CCC/CPC process, confuses many chromatographers. pH gradients, occasionally referred to as Frontal Chromatography, where ionization induced mobilization effects which occur during pH gradients, can, at appropriate eluting compound concentrations, in effect buffer the attempted pH gradient, by large amounts of often single eluting product. This may cause a period where the major "buffering component" may become the major or only component. This chromatographic phenomena had been a common, often used technique in Open Column, Flash Chromatography and HPLC, plus well understood, for a long period, prior to the use of the term pH-zone refining was chosen for CCC/CPC. This has caused confusion, and possibly it might have been better to use the terms that this previously described phenomena, was recognized as in other chromatography forms. But as discussed above, provided researchers are aware that the name is a popular term in CCC/CPC and refers to well recognized phenomena common in Open Column, Flash & HPLC, then in respect to originator it should be left alone.

Dual-flow

Dual-flow or true counter-current flow occurs when during a portion of the CCC or CPC both phases are flowing in opposite directions. Instruments are available for dual-flow operation for both Hydrodynamic CCC and hydrostatic CCC (CPC). Typically in certain instruments the CCC coil or CPC rotor can have 4 inlets/outlet options. If A) one end of coil or rotor, is original inlet for stationary phase in appropriate head to tail or tail to head operation in CCC, or ascending or descending mode in CPC, then D) will be exit at far end for stationary phase. A) Will be used to initially fill coil with stationary phase, when B) & C) will be closed, so stationary phase exits at D), until coil is full of stationary phase. B) (typically close to A)) will then become new entrance for stationary phase, and typically travel 3/4rds or more of coil to D). C) will become the mobile phase new inlet, and will flow typically 3/4rds or more of coil to A). Between the sections B) and C) true dual-flow, counter-current mixing and settling will occur. The efficiency of such instruments usually exceeds more traditional dual flowing industrial process countercurrent plant.

Sequential (2D) CCC/CCC

CPC/CPC or heart-cut column Switching, is possible in CCC/CPC instruments which have up to 4 coils of the same id in CCC or 2 or more rotors with same diameter chambers, plus multiple flow path capability. It is possible to start run with initial injection into a given biphasic solvent system, and then with appropriate switching valves, HEART-CUT up to 3 sections (if 4 coils/rotors) of the initial chromatograph into up to 3 different biphasic systems, each optimized for better resolution of appropriate polarity band than the first GENERAL BIPHASIC for the POLARITY BAND of the HEART-CUT SECTION. It is anticipated that this technique will be particularly useful with elution extrusion, where later eluting, extrusion peaks have a totally non-optimal reaction to initial general biphasic system and would be infinitely retained where it not for coil/rotor extrusion.

Ionic liquid-liquid chromatography

Historically it was thought that no commercial CCC or CPC could cope with the high viscosities of IONIC LIQUIDS. Recent inventions in CCC/CPC have shown that not to be the case, and instruments that can utilize 30 to 70+ % IONIC LIQUIDS (and potentially 100% IONIC LIQUID, IF BOTH PHASES ARE SUITABLY CUSTOMIZED IONIC LIQUIDS) have become available. The future may well see ILLC become a new, totally novel science, where customized one or two IONIC LIQUID containing biphasic mixes are synthesized to give the CCC/CPC appropriate PARTITRION COEFFICIENT, rather the historic procedure of mixing various traditional, or sub / super critical fluids in a manner to achieve CCC/CPC appropriate PARTITION COEFFICIENTS. IONIC LIQUIDS can be customized for polar / non-polar organic, achiral and chiral compounds, bio-molecule, and inorganic separations, as IONIC LIQUIDS can be customized to have extraordinary solvency and specificity.

See also

• History of chromatography
• Supercritical fluid chromatography