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Why use CCC
CCC complimentary HPLC

Applications 2005

Applications 1
Natural Product Bioactitives
Isocratic vs Step Gradient
Novel Alkaloids

Applications 2
Engine Oil Additives, Isocratic
Effect Coil Volume
Why need Gradients in CCC
Comparison to Flash Chrom
Which Biphasic Solvents
Linear Gradients
pH Modification Part Coeff
Step Gradient
Linear Gradient RP
Comparison NP to RP
Unknown Bioactivites
Engine Oil Additives :Gradient

Retail Prices
CCC Intro
Company Intro
Lab Scale
Pilot Process
Rio CCC 2001

PLEASE CLICK ON ONE OF THE FOLLOWING UNDERLINED TO GO TO OUR NEW WEBSITE for counter current chromatography , countercurrent chromatography and centrifugal partition chromatography as this website is presently being redesigned.



Fifeteen years of AECS-QuikPrep™ research has resulted in the latest modular, minimal footprint Quattro CCC™ Mk 6 QuikPrep™.

AECS-QuikPrep™ novel Seqential CCC-HPLC preparative strategies could achieve for your company, huge process etc cost savings. Laboratory sized method development optimisation studies based on our QuikPrep™ CCC and HPLC / Flash Chromatography products can easily be scaled to multiple tonne pre annum production.

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                     Dr Les Brown                               


Counter Current Chromatography ( sometimes referred to as centrifugal partition chromatography ) separates compounds on the basis of their partition or distribution between two immiscible solvents. One of the solvents is stationary, whilst the other flows. The counter current chromatography instrument is designed to create a fluctuating gravity field, such that as the mobile solvent flows, it is periodically mixed with, then settled from the stationary liquid.

Counter Current Chromatography is therefore a Liquid- Liquid Chromatographic Technique.


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The maximum pressures used, vary between 100 to 350 psi for PTFE columns, but much higher pressures can be obtained when using stainless steel coils and appropriate high-pressure fittings plus flying leads. The exact pressure limit depends on solvent type, flow rate, fitting type, coil internal diameter and / or material; see specification sheet or request custom specifications to suit your needs.

As there is no solid support in Counter Current Chromatography, there is no risk of infinite retention ( irreversible bonding ) and hugely reduced risk of compound transformation ( hydrolysis etc ) such as can occur on solid chromatographic media found in HPLC or flash chromatography; both solid liquid chromatography ( SLC ) systems.

Counter Current chromatography should therefore be the separation system of first choice, if preparation without sample loss or change of composition / bioactivity is required for sensitive samples.
Partition Coefficient ( k ) = concentration in upper phase
                                        concentration in lower phase

With a QUATTRO CCC™ a sample injected with the mobile phase could experience up to 100,000 mixing and settling steps an hour as it passes through the coil, alternately experiencing areas of high relative ' g ' ( settling ) and low relative ' g ' ( mixing ), owing to the planetary motion induced ' g ' force variable field. It is the variable ' g ' field, which controls the sequential mixing and settling, and hence compound partitioning between the two immiscible phases in Counter Current Chromatography


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It is, however, the Archimedean Screw Principle, caused by the rotation of the coil, which causes the selective retention of one of the immiscible solvent phases; the stationary phase, when the coil is filled with an appropriate solvent and then rotated.

Assuming correct fitment of flying lead to pump ( see below ), for as long as rotation is maintained at sufficient velocity, a percentage of the stationary phase will be retained in the coil, as an immiscible mobile phase is pumped through.

On cessation of rotation, both phases will be pumped out together.
For reverse phase operation ( upper stationary and lower mobile ), compounds with a high ( over k = 3 ) partition coefficient will be retained in the stationary phase, whilst those with partition coefficients between 0 and 1 will elute between the solvent front ( one bed volume of mobile phase ) and k = 1; stationary phase volume.. The k = 0.5 to 1.5 is a good position to aim to elute your component (s).

In isocratic Counter Current Chromatography, K = 3 or over will take a long time and is likely to lead to excessive band-spreading.

This problem is resolved in Counter Current Chromatography, as with HPLC, by the use of step or linear etc gradients. The gradient procedure will progressively increase the eluting power of the mobile phase to bring off previously infinitely retained components.

One major advantage of Counter Current Chromatography is that stopping the rotation will cause the Archimedean Screw Principle, previously retaining the stationary phase, to stop.

This will cause both the mobile and stationary phase to elute at the ratios present in the coil, as the incoming solvent pushes out both phases.
There is almost no discernable band spreading of the liquids in the coil during wash-off, thus previous resolution of held compounds is maintained and their elution profile can be interpreted to decide on a more appropriate solvent system ( See Applications 1 & 2 ).

The critical elements of Counter Current Chromatography, are the coil planet centrifuge, a means of pumping the liquid ( typically a HPLC pump ), and an injector ( typically a HPLC Rheodyne etc ) but both low-pressure pumps and injectors may be used , thus achieving a considerable cost saving.
A fraction collector and ' on-line ' detector are optional.

The Quattro CCC™ Counter Current Chromatography system should therefore be viewed simply as a mechanical replacement liquid / liquid alternative to a preparative HPLC or Flash Chromatography SLC……preparative column.

Counter Current Chromatography systems therefore use all the standard ancillary equipment of SLC.

Simulated Moving Bed ( SMB ) scenarios are also feasible when considering process scale-up in Counter Current Chromatography .

Unlike HPLC and Flash Chromatography, the active separation device is a coil of hollow tubing, such as a continuous length of PTFE or stainless steel tubing of appropriate internal bore diameter. Typical bore diameters in Counter Current Chromatography would range between less than 0.5 to more than 5.0 mm id.

This tubing is spirally wound on a drum in the direction of rotation, for preferential stationary phase retention characteristics and then the coil assembly is rotated in an epicylic planetary motion.

Thus Counter Current Chromatography systems are in effect epicyclic, planet centrifuges, which rotate coils filled with immiscible liquids in a planetary motion in order to generate a variable ' g ' field………….. not devices from some long lost magical art form, as many chromatographers seem to think.

Owing to the differential “ g “ field the coil will experience a fluctuating force field from high ' g ' to low ' g ' each rotation. The differential between the high and low is governed by the relativity of the sun and planet radii, this ratio are called the beta value.
The rotation speed, plus the sun and planet radii, governs the magnitude of all the 'g' forces.

At the sun and planet radii of a Quattro CCC™, typically speeds of 600 to 800 rpm would be required for tubing of 1mm and above to function.
Other competitor’s designs of Counter Current Chromatography instrumentation, with smaller sun / planet radii require up to 3000 + rpm, just to approach the ' g ' force differential capability of all Quattro CCC™ instruments.

The Quattro CCC™ QuikPrep™, LabPrep™, PilotPrep™ and ProcessPrep™ all share similar sun / planet dimensions and beta values, plus coil od / id options, thereby ensuring predictable scale up from mg to tonnes per annum.

There are a near infinite number of biphasic solvent system, and components to be resolved. Many of these, are critical to beta value, sun / planet radii, id of column or rotation speed. Others are extremely critical to one or more of these variables.

That is, even on a single Quattro CCC™, with all other parameters the same, excepting a marginal, normally non-critical increase of bore, separations have failed, which theoretical mathematical treatise would have predicted as being successful in scale-up Counter Current Chromatography.

Users should thus be cautious, if anyone suggests that all separations can be scaled-up by mathematical extrapolation, or methods may be transferred absolutely predictably between instruments of differing key parameters.

With a Quattro CCC™ scale-up is accomplished by trials based on the use of any model, as all have identical key parameters. The user will by experimentation optimise to appropriate biphasic solvent system / bore / speed / linear flow velocity etc. Scale-up is then linear and predictable for a given biphasic solvent system, flow rate and id bore of column………. no matter how critical the chromatographic system.

This absolute predictability of process scale-up is unique to the modular Quattro CCC™ design.

These results can then be applied to the modular Quattro CCC™ design to produce any production scale-up capacity required, from a single 3 litre stainless steel constructed ProcessPrep™ module to a 100+ such modules working in series / parallel or simulated moving bed configurations.

It is an approximate Rule of Thumb in Counter Current Chromatography that 0.5 to 2 grams may be prepared per 100ml capacity if working in standard mode, to up to 10 times this in certain pH Refining modes.

Multiple ' low cost ' intrinsically safe material ( stainless steel etc and no aluminum ) rotors, connected in series, parallel or as a simulated moving bed can give any required yield.

Process rotors can be supplied in their own stainless steel casework, or as bare rotors for placement in an intrinsically safe room.

In this latter configuration, any number of rotors can be run from a remote motor, and the intrinsically safe room can control temperature, solvent venting, and by limiting access when active, the user’s safety.

How would I operate a Quattro CCC™ Counter Current Chromatography instrument ?

To begin a preparation, first with no rotation, the coil is filled with the chosen stationary phase :-

For reverse phase : - upper phase ( typically organic in organic / aqueous solvent systems, excepting chloroform etc where the organic solvent is more dense than water )( and except in non-aqueous CCC, where both phases organic, ie hexane and acetonitrile ) will be the stationary phase and the instrument is configured such that solvent enters the HEAD ( H ) and flows to the ( TAIL )( T ).

For convenience, fill stationary phase from the ( H ) with no rotation. Then rotate the coil, typically to 600 to 800 rpm for a standard LabPrep etc, after rotation has stabilized, pump the lower phase into the ( H ).

A typical successful Counter Current Chromatography biphasic solvent would result in a stationary phase wash off of 50 to 5 % with, by definition stationary phase retention of 50 to 95 %. Lower retentions can still be used, but above 50 % and preferably above 70% is recommended.

For normal phase : - this procedure is reversed, with the pumping of the mobile upper phase ( when rotating ), being from ( T ) to ( H ) and thus for convenience the lower phase stationary phase can also be pumped from the tail, as there is no rotation and thus directional requirement, when filling with stationary phase.

To operate any Counter Current Chromatography successfully only requires an open mind and willingness to view the QUATTRO CCC™ mechanical unit as another separation column.

Once this is accepted, the near limitless potential of liquid-liquid chromatography is open to the user :-

1. Reverse or Normal Phase ( or even switch from one to other half way through a separation ).
2. Chiral separation without the need to bond chiral selector to a stationary phase.
3. Micelle or reverse micelle chromatography.
4. Ion exchange chromatography using liquid ion exchangers.
5. Separation without solid phase adsorption.
6. Separation without solid phase component degradation.
7. Ligand exchange for purification of precious metals, radio nucleotides, transition metals etc.
8. Even the possibility for custom Counter Current Chromatography units to inject slurry's, cell suspensions etc ( Not Appropriate For Standard Units )
9. On-column reaction, transformation and extraction in one system, using catalysts in the stationary phase, reacting with in-coming feedstock, to produce end product of different polarity which then extracted into the stationary phase
10. Your imagination !!




There are a bewildering number of biphasic solvent systems which may contain : - 2, 3, 4, 5 or more different solvents, in the literature ( Ref 2 ) ( web site :- bibliography and examples ).

The new QUATTRO CCC™ user is recommended to always start with the practical example quoted in the excellent reference book by Dr Walter Conway ( Ref 1 ).

The separation is of benzyl alcohol and phenyl ethanol, using a tertiary solvent mix of Heptane : Isopropanol : Water ( see Appendix 1, web site Applications / Isocratic for more detail ). This system is run in reverse phase, organic upper layer is stationary, aqueous mobile, and is run ( H ) to ( T ) as previously described in introduction.

Using the 100 ml Stainless Steel or PTFE coil 1.6 mm id bore, it is recommended that the new user first uses 2 ml / min to fill the coil with stationary phase and subsequently use 2 ml / min to run mobile phase after starting rotation.

Once familiar with the procedure and the first preparation is finished, a simple flow increase up to 6 ml / min ( noting the increase in backpressure is below the recommended maximum of 500 psi ) will cause more stationary phase to wash off. This amount of wash-off should be added to the original amount to calculate the new stationary phase retention. The injection should be repeated, and preparation will now be completed in a third of the original time.

With the stainless steel coil, still higher flow rates could be assessed for still greater time reductions

As with all preparative and process chromatography systems, the user will have to optimize for time, loading / over loading and or resolution as appropriate.

Other solvent systems, which are more viscous or less retention stable, will not be so tolerant to flow increase and the user must always be accurately aware of the pressure implications of their actions.

Equally tubing of smaller id will not retain stationary phase ( see below ref linear velocity ) and if PTFE coils are utilized user must always be aware that they may burst if too radical flow optimizations are attempted.

Having optimize flow rate……….A secondary optimization to look at column overload is then recommended.

This time rather than use ' on-line ' detectors which can overload and give spurious results, it is recommended to fraction collect and analysis the fractions ' off-line ' to define overlap.

This is one separation where theoretical predicted and actual scale-up are in close agreement.

The user should note that different bores will have different pressure characteristics, and the pressure limit for PTFE coils is much less than for Stainless Steel ( See Specifications ). Please note pressure suitability : - as burst coils caused by excessive flow, inappropriate flow of viscous solvents, precipitation in the coil and other misuses are not covered by the unit's guarantee.

The user should be aware than in addition to pressure considerations, the critical flow parameter in Counter Current Chromatography is not the pumped flow but linear velocity.



Experience has shown that settling times for biphasic solvent systems in a simple test tube……. shake test ……..of less than 30 to 60 seconds is of interest, but it is only a guide. Systems taking 120 seconds to settle may work whilst others, which settle in 20 seconds may not, this is dependent on the type of Counter Current Chromatograph and on the chosen biphasic solvent system.

This same anomaly applies to using test tube partition tests, very often they are useful, but other times they may be totally false and can lead to either useful systems being discarded, or a waste of time chasing an inappropriate system.

An example, is given on the web site ( Application 2 ), of a very successful optimization by test tube partition testing, others have been equally disappointing.

As describe in web site ( Application 2 ) : running a Acetonitrile : Water reverse phase ODS gradient from 1 to 99% Acetonitrile can similarly give an indication of compound polarity and hence a possible choice of solvent systems. This optimization system has its uses.

By far the best technique is to go to a step gradient ( see web site Applications 1 & 2 ) and run a separation, collecting the eluted fractions ( in Counter Current Chromatography on-line detection can be very misleading, but it can also on certain occasions be useful ). Then switch off rotation and continue to collect the wash-off until all stationary phase eluted and examine all the fractions off-line.

Based on this step gradient Counter Current Chromatography response, either a optimization of that system will be suggested or alternatives will be indicated ( free advice available to QUATTRO CCC™ users ).

Counter Current Chromatography does, however, have a large number of proven biphasic solvent in the literature. The user is recommended to perform a literature search to define an appropriate biphasic solvent system for their type of compound / mixture. Based on the literature biphasic system, an empirical step gradient optimization approach would be our preference. To achieve this, vary the aqueous phase mix for reverse phase and the organic phase mix for normal phase; from a ratio of 0.1 to the maximum allowed before mono-phasic conditions occur during the test step gradient ( detailed above ).



Most QUATTRO CCC™ 's only have one id , typically 1.6 mm id. This id gives an excellent compromise between resolution and reasonable linear flow tolerance, to allow quick preparations and good on-column loading ability. A typical loading would be between 500 and 2000 mg per 100 ml capacity. Many factors affect this, but primarily solubility and susceptibility to precipitation when running isocratic or gradients are the key considerations.

The chosen coil length / volume primarily will be govern by :

1. Speed, 20 ( smaller volume is not recommended with a 1.6 mm id bore ) or 50 ml 1.6 mm id columns, loading approximately 100 mg running at 4 ml /min could complete an isocratic run of K = 4, with typical 80 % stationary phase retention, in 10 to 20 min, including filling with stationary phase
2. Maximum resolution, requires maximum length, but users should be aware to always remember that a change of resolution by more appropriate solvent selection is invariably, more useful than using a coil over 200 ml in most lab scale preparations. Process scale preparations of 100 gram to multiple kilograms per run require multiple coils of often 1000 to 3000 ml.
3. Maximum resolution for low mass injections it is best to use 1 mm id tubing, but if PTFE tubing, care has to taken as this is a low back pressure tolerant material. Also the linear velocity of 1 mm id is such that solvent retention favors actual flows of only 0.5 to 1 ml /min hence increasing the preparation time.
4. Maximum yield per time. Whilst many advocate larger volume coils, we advocate using multiple coils in parallel or manifolded simulated moving bed systems working in parallel as the best way to maximize yield. That is, if require 100 grams, this would suggest a 10,000 ml coil. Such a coil would generate backpressure if run at say 1000 ml / min and would be cumbersome. Using 10 times 1000ml coils or 3 times 3333 ml each running at 100 ml /min ( this is a perfectly acceptable and proven flow for certain solvent systems on a stainless steel coiled QUATTRO CCC™ : PilotPrep™ or ProcessPrep™ the 100 grams is easily rapidly accommodated. At the same time this modular system allows additional flexibility so it could be used as independent coils for 10 or 3 different processes, or as a simulated moving bed system if required.



When considering a Hybrid QUATTRO CCC™ with coils of different id’s of bore, 1 mm id is best for maximum resolution at relatively low actual ( 0.5 to 1 ml /min ) flows. One mm id coils would usually be used for complex samples when only low mass to be injected.

Larger id's allow higher actual flow rates to be optimized prior to choice of a possible process instrument specification.

The Hybrid QUATTRO CCC™ coil system shown on the Home Page and in the Picture Gallery, is an extremely effective laboratory based research tool for defining ultimate process instrument parameters, whilst also allowing detailed, high-resolution research of small mass samples.

With bores of 3 to 5 mm id and over, tens and even hundreds of mls per minute become feasible in QUATTRO CCC™ Counter Current Chromatography instruments, thus allowing practical consideration of multiple tones per annum production.

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