The detailed work-flow for your first CCC run (and every run thereafter) is highly dependent your CCC system hardware and software. In all cases, the following choices must be made:

A) solvent system:  the essence of a successful CCC separation is the solvent system.   There are virtually limitless possibilities, but we recommend starting with one of the more well-established solvent system families such as heptane-MeOH-acetonitrile, hexane-EtOAc-MeOH-water ("HEMWat" or "AriZona" systems), (EtOAc)-butanol-acetic acid-water, depending on the solubility of your sample.  This choice is by far the most intimidating aspect of CCC for the new user.  After some experience, however, you learn how the various solvent system families relate to each other and can rapidly choose 'orthogonal' systems to develop rapid purification regimes.  For more information on solvent system selection, consider attending one of our webinars.  For more info email This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

B) mobile/stationary phase:  in CCC, switching between 'normal' and 'reverse' phase is as simple as flipping a switch.  If rapid desolventization of eluted fractions is important, choose the organic phase as the mobile phase.  If you wish to minimize the amount of organic solvent vapor in the lab, choose the aqueous phase as the mobile phase.  Another factor to consider is the propensity for crystalline solids to form in CCC fractions.  Because CCC fractions are generally highly concentrated compared with adsorption chromatography fractions, it is not uncommon for crystals to form in the hours or days after the fractions elute.  To "force" crystallization, choose the mobile phase appropriately (usually the aqueous phase is more likely to generate crystals).

C) sample preparation and loading:  Dissolve the sample in a mixture of the two bulk phases of the solvent system.  Sample concentration should be chosen such that, when dissolved in a mixture of the two phases, the sample can be shaken gently and still settle back into two phases in 30 seconds or less.  Sample volume load can be 10% of the total column volume or higher (e.g. up to 30 ml loop on a 300 ml instrument); for optimum resolution we recommend loading 2-5% of total column volume.  Depending on solubility, it may be sufficient to dissolve the sample only in either the mobile or stationary phase, but save those optimization studies for your second CCC run or later! 

D) instrument operation parameters:  unless you purchase a top-of-the-line system, you will need to know how to set the valves, rotation speed, flow rates, elution volume (typically one column volume), and extrusion volume (typically 1.2 column volumes).  We are more than happy to walk you through these parameters to optimize settings for your particular CCC system.  Contact This e-mail address is being protected from spambots. You need JavaScript enabled to view it for more info. Please be prepared to provide information about your CCC column and peripherals (pump, detectors, controllers, etc.).

The following step-by-step procedure assumes that A-D above have been chosen as follows:  

A) solvent system = hexane-EtOAc-MeOH-water (1:1:1:1).

B) mobile phase = lower phase

C) sample dissolved in a mixture of upper and lower phase (200 mg sample in1 ml upper phase and 1 ml lower phase). It was confirmed that at this concentration (100 mg/ml), the sample could be shaken gently and settle into two distinct phases in under 30 seconds.

D) operational parameters:  Elution-extrusion CCC (EECCC) - elute for 1 column volume, extrude for 1.2 column volumes. Column volume = 20 ml.

PROCEDURE

Step 2.  fill the entire system, including the sample loop, with stationary phase (upper phase). The loop is included in this step in order to assure that there is no rinse solvent (e.g. MeOH) left in the loop from a previous run.

Step 3.  load the sample loop

Step 4. start spinning the CCC instrument

Step 5.  ELUTION:  start pumping mobile phase (lower phase), and pump one column volume of mobile phase.

Step 6.  EXTRUSION: switch to upper phase and pump 1.2 column volumes of upper phase.

Step 7.  The instrument is now filled again with stationary phase and ready for another sample injection (step 3).  

Step 8.  Always leave the instrument filled with wash solvent (e.g. 20% aq. MeOH) if it will not be used for several days.

NOTE:  For current top-of-the-line CCC systems, the user needs to make choices A-C.  For typical CCC systems, each operational parameter can (and should) be optimized. Again, we are happy to help you optimize your system.