Courtesy of Roland Tanglao

The Algae industry has seen its fair share of hype in recent years. One reason for this, is that it is by far the most promising agricultural production method for bio-diesel. But, bio-diesel, when compared to other algae products, has a relatively low value. Accessory pigments, omega-3 fatty acids and squalene, just to name a few, have high value once sufficiently purified.

Caretinoids, which are sought after for their anti-oxidant properties as well as for use as natural food colorants, and phycobiliproteins like allophycocyanin (APC), which are useful for a variety biochemical applications because of their florescent properties, can all be purified on almost any scale using CCC. Additionally, the triterpene squalene and the two most popular omega-3 fatty acids, DHA and EPA, are also high value algae products in the rapidly growing nutriceutical market. In short, despite the bio-diesel hype, the low hanging fruit in the world of algae products seem to be high accessory pigments, Omega-3s and squalene among others.

Because of the messy nature of algae, purification of any of these compounds can be complicated without a robust purification technique like CCC. While the non-polar caratinoids and squalene can be purified on both CPC and HSCCC type systems, the water soluble phycobiliproteins can only be effectively purified using CPC type instruments.

Many of the compounds I mentioned appear in the literature precedents listed below, however, I was unable to turn up any publications on phycobiliprotein purifications using CCC. However, because phycobiliproteins are very polar they would require polar solvent systems that would only be suitable for CPC systems such as aqueous two phased systems or butanol based systems.

Preparative squalene purification from microalgae via HSCCC using n-hexane-methanol (2:1, v/v)

“Preparative separation and purification of squalene from the microalga Thraustochytrium ATCC 26185 by high-speed counter-current chromatography” Hai-Tao Lu, Yue Jiang, Feng Chen Journal of Chromatography A, 994 (2003) 37-43

Preparative astaxanthin purification from microalgae via HSCCC using n-hexane-ethyl acetate-ethanol-water (5:5:6.5:3 v/v)

“Preparative isolation and purification of astaxanthin from the microalga Chlorococcum sp. by high-speed counter-current chromatography” Hua-Bin Li, Feng Chen Journal of Chromatography A, 925 (2001) 133-137

Preparative lutein purification from microalgae via HSCCC using n-hexane-ethanol-water (4:1:1 v/v)

“Preparative isolation and purification of lutein from the microalga Chlorella vulgaris by high-speed counter-current chromatography” Hua-Bin Li, Feng Chen, Tian-You Zhang, Fu-Quan Yang, Guo-Qing Xu Journal of Chromatography A, 905 (2001) 151-155

Preparative zeaxanthin purification from microalgae via HSCCC using n-hexane-ethyl acetate-ethanol-water (8:2:7:3 v/v)

“Isolation and purification of the bioactive carotenoid zeaxanthin from the microalga Microcystis aeruginosa by high-speed counter-current chromatography” Feng Chen, Hua-Bin Li, Ricky Ngok-Shun Wong, Bo Ji, Yue Jiang Journal of Chromatography A, 1064 (2005) 183–186

Semi-peparative purification of hexadecatrienoic acid, octadecatetraenoic acid, docosahexanoic acid and eicosapentaenoic acid via HSCCC using heptane-acetonitrile-water (ratios not provided) and heptane-methanol-water (ratios not provided)

“Counter-current chromatographic separation of polyunsaturated fatty acids” Olivier Bousquet, Francois Le Goffic Journal of Chromatography A, 704 (1995) 21-216

In our previous article, I mentioned that isocratic-type CCC runs, have the advantage over gradient CCC runs in that you can track compound elution volumes based off of their k-values not only from run to run, but also from instrument to instrument. K-values (K) are related to elution volumes (Vr) by the following formula:

Vr=Vm + KVs

where Vm is the mobile phase volume in the column and Vs is the stationary phase volume in the column.  K-values, according to Pauli and Friesen, are the most appropriate x-axis for CCC chromatograms because they are a physiochemical properties of particular analytes in particular systems, whereas elution volumes are not and can change dramatically with stationary phase variation.  Taking this idea a step further, Friesen and Pauli developed Reciprocal Symmetry plots (ReS), where all values from zero to infinity are displayed and K and 1/K are positioned on either side of a line of symmetry on the CCC plot.

To find out more about ReS plots using K-values, read J. Brent Friesen and Guido F. Pauli’s 2007 Journal of Analytical Chemistry article “Reciprocal Symmetry Plots as A Representation of Countercurrent Chromatographs”

Gradient LC (liquid chromography) methods for CCC, HPLC, MPLC, Flash, and other solid stationary phase columns all typically increase the polarity range compared to isocratic runs. Contrary to isocratic LC methods, where the MP (mobile phase) composition remains static throughout the run, gradient methods gradually adjust the mobile phase composition over the course of the run.

For gradient methods, the solvent strength of the MP increases during the run. The term “solvent strength” refers to the ability of the MP to elute a particular solute or compound from the stationary phase. For example, non-polar compounds in reverse-phase methods are more soluble in methanol than water, making methanol the stronger solvent. Conversely, for normal-phase methods the solvent with the highest polarity in the mobile phase gradient would have to be a stronger solvent because the solutes that stick tightly to the column are more polar than those that pass through quickly. So, for mobile phase solvent mixtures the stronger solvent always increases in proportion to the weaker solvent over the course of the run.

While developing column chromatography (HPLC, MPLC, Flash, etc…) gradient methods the mobile phase gradient has no effect on the stationary phase, however, with CCC the liquid stationary phase is altered by the mobile phase gradient. If a method is not properly designed for CCC, the volume and composition of the stationary phase will change in detrimental ways. When developing a CCC gradient method from scratch, start by finding a solvent system with a favorable K value for your target compound, somewhere between 0.5 and 2. Next, you will choose a component solvent whose composition will be altered during the run to create a gradient; the solvent with the highest solvent strength will be the correct choice and will also partition the best between the upper phase and lower phase. In the case of the HEMWat system, Methanol is the strong solvent with LP mobile and Ethyl Acetate is the strong solvent with UP mobile.  Also, according to Alain Berthod in Countercurrent Chromatography: The Support-Free Liquid Stationary Phase (2002)  ”the gradient solvent will be the one that produces a reduction in stationary phase volume. Otherwise, if the stationary phase volume increases during the gradient elution, wash-off of stationary phase will be observed. This may be detrimental for the separation, and may render the continuous detection difficult or impossible.”  Remember to always check the literature before designing a method from scratch, here are some examples of gradient articles:

Fractionation of Grape Anthocyanin Classes Using Multilayer Coil Countercurrent Chromatography with Step Gradient Elution Stéphane Vidal, Yoji Hayasaka, Emmanuelle Meudec, Véronique Cheynier, and George Skouroumounis Journal of Agricultural and Food Chemistry 2004 52 (4), 713-719

Salting-out gradients in centrifugal partition chromatography for the isolation of chlorogenic acids from green coffee beans, Roman R. Romero-Gonzalez, Robert Verpoorte, Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4245-4251

Flow rate gradient high-speed counter-current chromatography separation of five diterpenoids from Triperygium wilfordii and scale-up, Aihua Peng, Rui Li, Jia Hu, Lijuan Chen, Xia Zhao, Houding Luo, Haoyu Ye, Yuan Yuan, Yuquan Wei, Journal of Chromatography A, Volume 1200, Issue 2, 25 July 2008, Pages 129-135

Completed preparative separation of alkaloids from Cephaltaxus fortunine by step-pH-gradient high-speed counter-current chromatography, Zhonghua Liu, Qizhen Du, Kuiwu Wang, Lili Xiu, Guanglei Song,Journal of Chromatography A, Volume 1216, Issue 22, 29 May 2009, Pages 4663-4667

Isolation of Limonoids from seeds of Carapa guianensis Aublet (Meliaceae) by high-speed countercurrent chromatography Vagner Pereira da Silva¹, Rodrigo Rodrigues Oliveira^2,*, Maria Raquel Figueiredo Volume 20, Issue 1, pages 77–81, January/February 2009

Solvent gradient elution for comprehensive separation of constituents with wide range of polarity in Apocynum venetum leaves by high-speed counter-current chromatography Yuchi Zhang, Chunming Liu, Zhengkun Zhang, Yanjuan Qi, Guimei Wu, Sainan Li Volume 33, Issue 17-18,pages 2743–2748, September 2010

Preparative isolation and purification of coumarins from Peucedanum praeruptorum Dunn by high-speed counter-current chromatography, Renmin Liu, Lei Feng, Ailing Sun, Lingyi Kong, , Journal of Chromatography A, Volume 1057, Issues 1-2, 19 November 2004, Pages 89-94

ISOCRATIC RUN ADVANTAGES

• K-value based elution volume prediction
• Only one pump head is required
• Simple

GRADIENT RUN ADVANTAGES

It’s been some time since my last post, but things have been very busy here at Cherry Instruments.  We have been heavily engaged in a research and development initiative to develop our own complete CCC system which will automate many of the most demanding aspects of CCC runs.  We are very excited to unveil our system’s many breakthrough features in the coming months, so make sure you stop by again soon.

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.

I’ve just read about an exciting new application for CCC. Our friends at Abbott, Jill Hochlowski, Jeffrey Pan, Philip Searle, Wayne Buck and Stephen Spanton, have taken advantage of one of the key benefits of the liquid stationary phase for ADME studies (absorption, distribution, metabolism, excretion). ADME studies involve looking for minor constituents in very complex biological matrices. A major obstacle for HPLC in doing ADME studies is that there is no assurance of complete recovery of the starting sample, on the contrary you will almost certainly lose some material due to permanent absorption on the silica (not to mention possible loss of material that can occur while you are preparing the sample for injection). CCC on the other hand has the advantages of requiring little to no sample prep and most importantly 100% recovery of even the most minor constituents.

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!!

On a rainy fourth of July weekend, my brother Sam and I were hard at work in the lab working on our latest project. CCC flavor separation.  Admittedly, we were not happy about missing out on bbq, beer and fireworks, but we were both excited about getting this project started. Our ultimate goal? To put the power of flavor separation in the hands of chefs, which is a power that is now largely restricted to flavor houses.

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“

I am happy to announce that Cherry Instruments will be exhibiting at PREP 2009 on July 19th through the 21st.  We are excited to participate. PREP is an international symposium, exhibit and workshop on Preparative and Process Chromatography.

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.

Tauto Biotech, our Chinese friends and producers of our line of type J instruments, have been using CCC to make quality reference standards for many years.  In fact, our entire line of reference standards are produced by Tauto Biotech HSCCC instruments.

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

Dr Chadwick and I have been preparing material for two online presentations. Next Thursday the 14th we will present “Introduction to Countercurrent Chromatography,” at noon and “Choosing CCC Solvent Systems,” at one.   Both webinars will consist of thirty minute presentations, after which Dr. Chadwick will be available for questions. If you are interested please send me an e-mail.