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ONLINE SFE-SFC FOR MONITORING BATCH EXTRACTION PROCESSES Shawn Helmueller, Jason Hill Waters Corporation, Milford MA, USA, 01757

INTRODUCTION Online SFE-SFC instrumentation is used to generate real-

time analytical information during batch extractions.

Strategies for optimizing extraction cycles, vessel

switching, and scaling to bulk processes are discussed.

Routine analysis is essential during each stage of the

cannabis production workflow. However, many cannabis

production facilities contract out their analyses to third-

party analytical testing laboratories. Since each sample

carries a significant price tag, production facilities are

selective in the samples they submit for analytical testing.

This lack of analytical information produces a knowledge

gap with regard to basic quality control checkpoints and

formulation research and development that results in

workflow inefficiencies and inconsistent products. In

addition, samples that are submitted for testing can take a

week or more for labs to return results. This means acute

issues linger until issues are identified and corrective

actions are made. Moving analysis in-house significantly

reduces turn-around time for receiving feedback about a

particular process, resulting in increased productivity and

improved batch-to-batch quality.

Here, this idea of fast, continuous feedback is taken a step

further. Extract chromatographic analysis is moved online,

at the moment of extraction, using online SFE-SFC

systems. This real-time extraction analytical information

has the potential to quickly identify and correct

inefficiencies in the extraction workflow, while streamlining

the tedious optimization/re-optimization processes involved

in developing targeted extraction outcomes.

SYSTEM CONFIGURATION

Acknowledgments:

The authors would like to thank Chris Hudalla, ProVerde Laboratories, and Sylvain Cormier, Waters Corporation, for their collaboration in portions of this work.

Initial investigations were done utilizing a manufactured mixture of

acetophenone and trihydroxyacetophenone spiked onto an inert matrix of

diatomaceous earth. This allows for a simplification of the complex sample-

matrix interactions that take place in natural products, allowing for focus on the

system interface and system characteristics. Figure 3 (above) shows an initial

extraction method that has three stages over the course of 40 minutes. The

extraction starts out at low pressure and neat CO2, increases the pressure, then

adds a polar modifier to complete the extraction of the more polar compound.

Normally the analyst is blind to the effect each one of these method stages has

on the extraction of the target analytes, but here operators can clearly see what

is extracted when and adjust their method accordingly.

Figure 4 (above) shows an optimized extraction developed from information

gained in figure 3. The ability to develop and optimize extraction methods at the

analytical or semi-preparative scale provides a huge savings in time and sample

consumption for research and development.

Figure 5 (left) shows scaling at the analytical scale is influenced significantly by

the solvent exchange rate in the extraction vessel. As long as the exchange rate

is held constant, 1 column volume per minute in this case, extractions performed

at the 5 mL and 10 mL scale are nearly identical. Provided enough control of

system set points, those optimized methods can be scaled up to the production

scale system relatively easily. However, analytical and preparative scale systems

can operate in vastly different flow rate regimes (with much different exchange

rates). When developing preparative SFE methods at the analytical scale, it is

important to start with the end goal in mind. Questions to ask are:

1.) What is the flow rate on the prep system?

2.) What is the vessel size on the prep system?

Next, the proper analytical configuration can be selected for doing development

work for the target production system. Figure 6 (left) shows an optimized

analytical scale extraction performed in the same flow rate regime as a 5L prep-

scale extraction at 200 g/min; the analytical system utilized a 100mL vessel and a

flow rate of 4mL/min. Development work at this scale used 50 times less sample

per run than if the same development work were performed using the 5L prep

system. The next step would be to employ the method parameters developed at

the analytical scale in the preparative extraction system.

This online SFE-SFC approach was implemented on a production SFE system to

monitor the cannabinoid extraction process (Figure 7, below). Under mild

extraction conditions (150 bar, 50oC) the less polar, lower molecular weight

neutral cannabinoids (black) are readily extracted and their extraction rate

steadily decreases throughout the duration of the run, while extraction rate of

the heavier, more polar acidic cannabinoids (red) is constant. Increasing

pressure corresponds to an increase in extraction of the acidic cannabinoids.

Detailed extraction conditions and figure details can be found in the figure

caption.

Analytical feedback is needed at every stage of the cannabis

processing workflow

Analytical and preparative SFE systems were coupled with UPC2 for

real-time analytical feedback on batch extraction cycles

Method optimization and scaling parameters were investigated at the analytical scale using a manufactured mixture of acetophenone and

trihydroxyacetophenone

Sample runtime was decreased from 40 minutes to 15 minutes using

online SFC feedback

Vessel exchange rate was identified as an important scaling parameter and strategies for scaling from analytical to preparative

SFE are being investigated

SFE-SFC instrumentation was deployed to begin studying targeted

natural products extractions for cannabis

Figure 1. SFE-UPC2 system interface for monitoring extraction

cycles. Depending on a number of factors, a split of the extraction flow or full extraction flow can be taken to the chromatographic system.

Online AnalysisExtraction

ABPR

Split

PDA

Waste/

Collection

2D inject

valve

UPC2

Isolation

Valve

Extraction

Vessel

Valve

Figure 2. 2D data generated from the SFE-SFC set up in figure 2. The blue trace is a multi-injection UPC

2 chromatogram of extraction

effluent and the red trace is a UV “extractogram” at 228nm.

Figure 3. SFE-SFC data generated for the extraction of acetophenone and trihydroxy-acetophenone using Waters MV-10ASFE and UPC

2. Bottom shows real-time UV data of

the extraction effluent. Top shows SFC analyses of extraction effluent every 2 minutes throughout the extraction cycle. Peaks 1 and 2 correspond to acetphenone and trihydroxyacetophenone, respectively.

RESULTS AND DISCUSSION

Figure 5. Scaling vessel size from 5mL (red) to 10mL (black) at 1.0 column volumes per minute. Notice how the extraction profiles are the same as long as the exchange rate is held constant.

Figure 6. Analytical scale method development for preparative scale SFE. Extraction of the acetophenone mixture using a 100mL extraction vessel and flow rate of 4mL min. Exchange rate (0.04 CV/min) mimics 5L prep SFE system flowing at 200 mL/min.

Figure 4. Optimized extraction developed from information gained from figure 3. Exchange rate and extraction temperature were held constant for both extractions (1 CV/min). Extraction pressure and duration of method steps were optimized resulting in decreased run-time, while maintaining good analyte selectivity.

Figure 7. Batch extraction monitoring using SFE-SFC approach. Extraction conditions are as follows: Pressure variable from 150-200 bar, temperature 50

oC,

flow rate 170 g/min. The black trace corresponds to chromatographic data collected at 228nm and shows extraction of the neutral cannabinoids. The red trace corresponds to data collected at 270nm and shows extraction of the acidic cannabinoids. The inset shows peak identifications for the injection made at the 20 minute mark. Inject-to-inject cycle time was 1 minute, and a total of 60 individual, real-time analyses are shown.

CONCLUSION

In a traditional extraction workflow, extract analysis takes place after the extraction is complete. In a batch production environment these results inform on the next extraction, but it is often too late to affect that particular batch. Here, extract analysis is accomplished at the point of extract generation, even before fraction collection. This gives real-time feedback about the currently running extraction cycle and allows processors to make decisions with regard to a number of extraction parameters such as temperature, pressure, flow rate, duration, etc. Figure 1 (above) shows a block diagram of the 2D system interface.

Two SFE-SFC systems were developed. One consists of Waters 5L Bio-Botanical Extraction System (BBES) and Waters UPC

2 . The

other utilizes Waters MV-10 ASFE and UPC2; the general layout and

operation is largely the same. Full-flow or a split is taken from the main extraction flow and sent to the chromatographic system for analysis. Prior to extraction, an analytical method is developed for the analysis of interest. During extraction, multiple injections are made during a single analytical run. As soon as the previous injection finishes separating, the next sample is loaded and injected. An example of the data collected is shown in Figure 2 (below). Process resolution, or the time between analytical injections, is determined by the analytical cycle time, so fast separations are desirable.