David Keay, md of Dynamic Extractions, offers an example of how the latest generation of countercurrent chromatography technology is being used to separate potential plant-based drug actives rapidly at a suitable scale for drug studies
Natural products were once considered redundant to the lead identification process in pharmaceutical development. However, they are now making a resurgence as a starting point for identifying potential blockbuster molecules. In particular, many traditional Chinese medicines (TCMs), where the starting materials are plant-derived, are being extensively tested for their pharmacological activity.
But this work has been limited due to the scarcity of purified components in sufficient quantity for identification, characterisation, toxicity testing and clinical trial phases.1 The problem of how to purify these complex mixtures of compounds is being addressed by an old solution in a 21st century guise.
Due to the complexity of TCM extracts and their low levels of target compounds, purification by solid phase chromatography techniques, such as High Performance Liquid Chromatography (HPLC) and flash chromatography, is possible but expensive. Since the early 1980s there has been a liquid-liquid chromatography technique, known as countercurrent chromatography (CCC), that uses liquid rather than a solid stationary phase. Despite offering very high recoveries, the viability of the technique had been undermined by the performance of the first machines.
However, the development of a new generation of high performance CCC instrumentation (HPCCC) is having a beneficial impact on natural product purification.
There are a number of contributing factors that make CCC useful in natural product separation. First is the high volume of liquid stationary phase available: typically higher than 60% against the 5%-10% available for all solid phase techniques. This allows very high sample loadings per injection and hence enables sensible quantities of purified material to be produced from these complex extracts and their low levels of target compounds.
Second, recoveries are extremely high - typically in excess of 90-95% - which compares favourably with solid phase techniques where recoveries are at best 25-30%. This ensures that companies don't lose more of the valuable compound than is purified.
Third, CCC is a gentle process that guarantees compounds are not denatured while being purified.
CCC is simply liquid-liquid partitioning of a sample between two immiscible liquid phases. The operating
principle of CCC equipment requires a column, comprised of a tube that is coiled around a bobbin. The bobbin is then rotated in a double-axis gyratory motion (a cardioid), which causes a variable gravity (G) field to act on the column during each rotation (see
diagram left). This motion causes the column to see one partitioning step per revolution and components of the sample separate in the column due to their partitioning coefficient between the two immiscible liquid phases used.2
Another consequence of the column's motion is that the heavier of the two liquid phases will naturally be pushed to the outer end of the column (the periphery) and the lighter phase to the centre. Through column design this effect is used to keep one liquid phase stationary while the other becomes the mobile phase.
However, in the early machines, known as high speed CCC (HSCCC), the speed of rotation was slow and the G-level generated was low, at only 60- 80G. This limited the rate at which you could flow the mobile phase, causing the separations to take many hours to perform. This low performance, combined with the industry's move away at that time from natural products, caused a minimal adoption of the technique.
During the past five years HPCCC, defined by achieving high resolution separations at high sample loadings in minutes rather than hours, has been developed that is capable of generating 240G. This development provides two key benefits.
The first is that both liquid phases are now being acted on by a far higher force, increasing the mobile phase flow rate by up to 10 times what it was before. Therefore separations that used to take hours now take between 20 and 40 minutes.
The second benefit is that partitioning happens between the two immiscible liquid phases, but only up to a certain sample loading. HPCCC instruments achieve three times the G-level of HSCCC machines and this allows sample loadings to be increased by a factor of three compared with the earlier instrumentation. This loading capacity, combined with the shorter separation times, greatly increases the productivity of the CCC technology.
This increase in the mass of sample that can be injected onto the column enhances throughput and makes the technique viable for the chemist and chromatographer to use when isolating natural products at large-scale.
Two natural product separations, well documented in scientific papers illustrate the benefits this new instrumentation brings to natural product separations. The first example is the purification of glucoraphanin from an extract prepared from broccoli seeds, which is being investigated for its chemo preventative properties,3 and the second is the separation of two isomers,
honokiol and magnolol, the main bioactive constituents of the Houpu plant. The latter is a TCM where it is known that honokiol has a stronger pharmacological effect compared with magnolol.1
i. Selectivity and time of separation
If we look at the structures of the principal contaminants involved in these purifications (shown left), we can see that we are dealing with very similar molecules. The difference in structure between glucoraphanin and gluco-iberin is one methyl group; honokiol and magnolol (top left) are isomers and their difference is the position of one hydroxyl.
In both cases purification of the crude is achieved and the target compound purified to greater than 98.5%; both separations took less than 35 minutes to perform.1,2
ii. Loaded sample concentration and sample injection mass
Table 1 presents comparison data for the honokiol and magnolol purification when comparing CCC with HPLC instrumentation. The most obvious factor that stands out between these two data sets is the difference in sample loading that was achieved.3 This demonstrates the advantage that CCC brings to these types of purifications.
Further data is presented1 that shows the sample concentration loaded was 7mg/ml and concludes this is significantly higher than could be achieved on liquid-solid forms of chromatography. This data is typical for HPCCC instruments and the loading conditions on the glucoraphanin sample, processed on a similar instrument, was 17g per injection.4
The other practical advantage that CCC offers is high yields from the processed sample. There is no possibility of irreversible adsorption, because we are dealing with two liquid phases; both can be pumped off at the end of the purification ensuring complete recovery of the entire sample. A conservative estimate of the recoveries in CCC would be 90% plus, although cases of 95% and greater have been reported.5
iii. The simplicity of scale-up
In the introduction, it was indicated that it was necessary not only to be able to separate these compounds, but also very important to do this at sufficient scale so that kilos of these materials can be provided for various test procedures.
Work on both of these separations has been performed at milligram, gram and kilo scales of sample injection, but the method for the purifications has been developed and optimised only once. The performance, in terms of purity, yield and time of separation have been replicated and repeated.
This is possible because we are simply dealing with two liquid phases and it is now possible to engineer HPCCC instruments at larger scales to recreate the column conditions of the smaller size of instrument. The only purification conditions that must be changed are the sample injection volume and mobile phase flow rate, which are multiplied by the ratio of the two column volumes one wishes to scale between. CCC offers simple volumetric scale-up.6,7
In summary, the large-scale purification of natural products has always been possible, but has often been time consuming and has involved substantial waste of material. Although CCC instrumentation has offered a solution it has always been somewhat impractical and, therefore, has seen only a low level of adoption by industry.
Now that HPCCC instrumentation is available that allows high resolution, high capacity purifications at all scales, the technique is being added to the purification armoury because of the new options that it provides.