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There’s been a lot of CCC activity at Zheijiang University in Hangzhou China. Recently, a group from the chemistry department described a comparison of elution-extrusion CCC (EECCC) and back-extrusion CCC (BECCC).

In both EECCC and BECCC the columns are first filled with upper phase, which is the stationary phase for the first part of the run. The lower phase, which is also the mobile phase is now pumped through the column.  The compounds with the highest affinity for the mobile phase are eluted first, however, some compounds with higher affinities for the stationary phase remain in the column.  The compounds still present in the stationary phase may be separated, but would required wasteful amounts of mobile phase to be eluted. EECCC, avoids wasting time and mobile phase by extruding the stationary phase with one column volume of upper phase.  The stationary phase is fractionated during the extrusion process.

In BECCC the elution step is exactly the same, however the flow direction is changed for the extrusion step and mobile phase is pumped into the column rather than stationary phase.   EECCC is more amenable to high-throughput fractionation because each injection cycle begins and ends in the same state (filled with upper phase).  In BECCC, each injection cycle ends with the column filled with the opposite phase as the previous cycle.  One advantage to BECCC is that it avoids producing a large surplus of one phase over another, but otherwise I doesn’t see much use in our lab.

An original Ito apparatus “a coil planet centrifuge with one 140-mL coil and a counter-weight” was used for the both the EECCC and the BECCC experiments.  My first CCC separation was on an Ito apparatus which was what got me “hooked,” on CCC in the first place. The column I learned CCC on, now 25 years old, is still fully operational! If well cared-for, CCC columns can have a virtually endless life.  For solvent system selection, they describe the use of an “analytical-scale integrated parallel CCC separation system manufactured by the Zhejiang University machine shop”.  The column has three parallel 40 ml columns.

Earlier this year another research group from Zheijiang U, in the Research Center of Siyuan Natural Pharmacy and Biotoxicology Department, reported their efforts to develop a “new multichannel CCC method and apparatus for the high-throughput fractionation of natural products for drug discovery.” Their method makes use of a single CCC solvent system (hexane:ethanol:water 6:5:1) for all investigated samples (all of which were crude defatted EtOAc extracts), followed by purification of active fractions with preparative HPLC.  The authors state that this regime was developed as a response to the intrinsic challenges of determining an ideal solvent system for any given target compound.  Also, they note that the shorter CCC columns provide lower resolution than what is obtained with a comparable single-channel instrument. Innovative features of the apparatus include temperature control from 20-60C and three independent 300 ml columns, each with separate dedicated pump, detector, and fraction collector.   They note that senior engineer Yucheng Wu, who is not listed as an author, fabricated the instrument at Zheijiang U.

The authors provide a succinct review of high-throughput purification methods for natural product compound libraries and summarize with the following quote:  “These throughput purification techniques mentioned above, including FC, HPLC and SFC, are revolutionizing the process of natural product discovery and provide high-quality compounds or compound mixtures for biological screening. However, these techniques always use a solid support matrix, resulting in irreversible adsorptive sample loss and deactivation, tailing of solute peaks, and contamination.”

The authors conclude with “it is essential to take into account of the lower resolution compromise for the fabrication of new multi-channel CCC devices in the future.”

We find the technology quite promising, particularly if efforts to develop regimes of orthogonal CCC solvent systems are incorporated into their method (e.g. following Brent Friesen’s work on solvent system families).  Use of solid adsorbents in a sense defeats the stated purpose of avoiding absorptive loss, deactivation and contamination.

Link to original articles

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It is often difficult for a new technology to compete in the same sector as a well established one, even if it has major advantages over the prevalent technology. Consider for a moment: the microwave oven. This staple of modern life started as a crude, expensive, and demanding cooking device. Initially only those that truly understood the advantages of the technology, and how to best apply those advantages to their cooking could benefit from it. And because the microwave was so fundamentally different from other cooking technologies, there was a learning steep learning curve that added to its expense.

The market for chemical separation instrumentation is in a similar position with the introduction of CCC. Conventional solid-phase techniques have the advantage of time and many educated users — CCC lacks this.  But, unlike the microwave which mainly just shortend cooking times, CCC is able to achieve things that are extremely difficult and often impossible with other methods.