Displacement chromatography and pH zone refining offer two different approaches that make high loading, and high purity possible on a preparative scale. In both cases, modifying agents are used to dramatically change the dynamics of separation within the column.

In displacement chromatography, which is a type of column chromatography, analytes are separated based on competitive binding of the analytes to biomolecules in the chromatography matrix.  This process works by using a displacer molecule, a molecule that has a very high affinity for the binding sites within the column, to compete with and displace the analytes in the column.  The analytes that have the weakest interactions are displaced faster than analytes that have stronger interaction with the column. The analytes come off of the column as rectangular peeks, with minimal overlap and high purity.

pH zone refining, which can be preformed on any countercurrent chromatography column, uses a series of “retainer,” acids are used to modify the hydrophobicity of ionizable analytes within the column sequentially, based on their pI.  That was a mouthful-in other words-the pH within the column is constantly changing during the process of separation. Modifying agents referred to as retainer acids (or conversely as eluter acids when running in reverse phase), buffer the pH of within the CCC column allowing stepwise elution of analytes based on pI. This creates very different results when compared to using a pH gradient alone. pH gradients produce chromatograms similar to those created by displacement chromatography. This image I pulled from Yoichiro Ito’s pH-zone-refining patent shows a chromatogram produces by a pH gradient.

separation of DNP amino acids using a pH gradient on CCC

When buffers are used to control the pH change within the CCC column, complete separation of the DNP-amino acids occur. The buffers allow the sequential elution of analytes to occur in discrete steps, rather.

separation of the same DNP amino acids with the addition of retainer acids (pH zone refining)

Although pH zone refining and displacement chromatography are similar in that they can both allow for high loading capacity and produce nice robust rectangular peeks, with minimal overlap in the case of displacement chromatography and no overlap in the case of pH zone refining. It is important to remember that the basis of separation is completely different because this allows you to quickly deduces which technology is more suitable for your application.  For example if you know you in advance that the species you are trying to separate are not ionizable, or have pI’s that have a difference of less than (0.3), displacement chromatography would be the best fit for your preparative needs. However, if you need to completely eliminate peek overlap pH zone refining is for you. Or if you know or suspect that the compounds you are working with have quite different pI’s look into pH zone refining.  Luckily, if you have a CCC column the only additional material required for pH zone refining are the retainer acids. However, with displacement chromatography you’re going to need to purchase a special displacement column and a specialized displacer agent.

For their application an automated CCC system was prepared with an on-line MS in addition to the standard UV detection.  This allowed for definitive identification of target material during the run.

The article “Purification of drugs from biological fluids by counter-current chromatography,” appears in the Journal of Chromatography A, 1216(2009)6162-6169.  Check it out!!

It  started a few weeks earlier. As I was impatiently waiting in outbound Chicago traffic, on my way home for the weekend, I get a call from Sam.  He wanted to talk with me an idea he had about using CCC for flavor separation.  In the kitchen CCC could be used to separate complex flavors into groups of flavor constituents.  A chef with the right CCC instrument and a grasp of the basic concepts could use the technology to either to isolate desired flavors from a mix or remove an undesirable flavor. It sounded great to me. I love food and I’m always game for fun and offbeat projects like this.

Despite many hours logged watching food network, I didn’t know enough about the culinary world to understand how CCC could fit into a kitchen.  I needed to consult and expert. Alton Brown came to mind, but since I didn’t think he’d be particularly easy to get in touch with I decided to call around to talk with some local chefs.  Many calls later with little progress I went to the net to look for culinary folks who were into integrating cool new technology into to kitchen. Enter Dave Arnold.

Food hacker and director of culinary tech at the French Culinary Institute

After talking to Dave Arnold, the director of culinary technology at the French Culinary Institute, I finally felt that I’d found the connection to the inside culinary knowledge that was required for the project.  Dave told me he spends the majority of his day thinking about new ways to tweak flavors. I knew I was talking to the right guy.

After telling Dave about Sam’s CCC flavor separation idea it was time to take this project to the next level.  Sam and I were on a mission to prove that flavor separation on CCC was feasible in a kitchen, which meant we needed a food safe solvent system and proof that it was easy enough to be a practical tool for a chef.

The first step was to design a solvent system that is food safe. It was fairly obvious where we needed to go for food safe supplies… the grocery store! We bought a variety of oils, distilled water and everclear.

A variety of oils and everclear (distilled water not shown)

For our solvent system to have any hope of working we needed the system to have a decent settling time. So we spent hours and hours mixing water, alcohol and different oils. Then we shook up all of our prospective systems and timed how long it took for them to separate.

Glass vials fill of prospective food safe solvent systems

Our solvent system evaluations lead us to adopt everclear and canola oil for our first attemts at flavor separation.

With our solvent system in place we needed some food samples to try out.

Find what our friends at the French Culinary Institute though of our preliminary flavor separation as the story continues on Dave Arnold’s blog see “Just another flavor separation technique“

PREP 2009 will be taking place at the Loews Hotel in Philadelphia. We will be at booth #31. For more information go to www.PREPSYMPOSIUM.org or contact us.

I recently asked Jack Jiang, a Tauto employee, about Tauto’s experience using CCC to produce reference standards.

1) How does the production of reference standards using HSCCC benefit
Tauto Biotech and how did it all start?

Since the year 1999 when Tauto was founded, the company has been devoting efforts on the development of the chemical database of natural monomers and the separation work of botanical reference materials by HSCCC. The separation with HSCCC technology can prove the efficiency of active components separation with the related HSCCC equipment, while it can also get the wanted active components with high purity and low cost.

2) Why use CCC rather than other techniques such as HPLC to produce
reference standards?

Based upon the features of CCC, it has its own advantages in separation of reference materials:

large injection volume, high resolution, high yield rate, good reproducibility,easy to scale-up, low cost in production, no consumption of consumables, low maintenance fee.

3) Are there any instances where HSCCC was particularly useful for
producing reference standards? (for example where the purity is
superior or where reference standards can be produce much more
efficiently?

Instance: Separation of Ginkgolide J, Huperzine B, Isomers of Gambogic Acid

4) What are the most important things to know for someone who is
considering adopting the use of HSCCC for the production of reference
standards?

a. Check the characteristics of the sample to see whether it is suitable for the separation by CCC.

b. The most important technology point is the selection of solvent system for HSCCC separation

We recently visited the Biological Engineering department at the University of Arkansas (UARK) to enhance the detection capabilities of their CPC system with an ELSD.  During our visit we learned that this group, led by Dr. Danielle Julie Carrier, is laying the groundwork to develop a process that could be seamlessly integrated into the current biochemical refineries.  The production of vegetable oils (the main component of biodiesel) leaves behind large quantities of potentially useful phytochemicals, many possessing valuable properties such as antioxidant or antimicrobial activity.  These chemicals could be easily extracted with pressurized hot water prior to the biofuel conversion process.

As an early adopter of countercurrent chromatography to this industry, Dr. Carrier’s group is using their CPC as an isolation/purification tool for investigating these valuable compounds.  She says quite simply, “CCC technology is allowing us to develop novel processing methods. ”

CPC is a wonderful tool for isolating compounds from a crude plant extract.  In a single run, with a crude extract solution from Milk thistle seeds, I was able to purify silydianin (a flavonolingan) up to 94.6% purity.  Another great feature is that since there is no solid stationary phase, the solvent system of the CPC can be modified in order to separate almost any compound.

There is immeasurable value in industrial waste streams, and CCC is likely to be the single most valuable tool for exploiting them.

Before we can use petroleum to power our transportation or produce products such as plastic, pesticides and pharmaceuticals, we have to get that black gold out of the ground and refine it.  Measuring trace elements in the oil is critical to downstream processes because some trace elements interfere with fluid catalytic cracking most importantly nickel and vanadium (from Wikipedia).

According to Maryutina et al. current methods for oil analysis require time consuming sample preparation and have poor detection limits as well as a restricted number of elements that can be tested for at one time.  “CCC gives a unique possibility of direct isolation and pre-concentration (without additional sample prep) of trace elements from oil.” (Maryutina et al) This is just one example of the benefit of CCC’s ability to handle raw samples! Believe it or not, crude oil can actually be used as a mobile phase in CCC.

Check out their paper Counter-current chromatography for oil analysis: Retention features and kinetic effects

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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