The biggest drawback of “elution mode” chromatography, which I am using to refer to methods that rely solely on the differential attraction of analytes to the mobile phase versus the stationary phase for separation, is that they often require low loading. On an analytical scale, these problems are not significant. When your working on a preparative scale this is when high loading capacity is critical.

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.

As biofuels occupies a larger and larger portion of our planet’s total fuel consumption, vast amounts of biomass will be converted into more useful forms (e.g. biodiesel). What is often missed is that biomass can contain some interesting and valuable phytochemicals, like flavonoids and saponins.

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

One of the most compelling reasons for adopting CCC is it’s ability to achieve gentle, lossless separations - where conventional techniques fail. The examples are endless, but I found this one to be a particularly interesting application to marine research (preparative isolation of tunichrome B-1 pigment from sea squirts).

I was initially exposed to this work via this blog entry. But I strongly urge those interested to obtain the original article (which can be found here) - an interesting read. The problem and solution is summed up quite simply in this quote from Nakanishi et al. (1986):

The tunichromes readily decompose on hplc and hence could not be separated on a scale larger than analytical. Luckily, a prototype CCCC instrument became available in our laboratory, and it was only through this chromatographic method that further semiprep scale purification of tunichromes was achieved.

Nakanishi was lucky enough to have access to an early prototype instrument to complete his work. I should note that more than twenty years later, current instrumentation and knowledge would yield much faster runs with much greater ease.