Separation of cannabinoids for clinical use

PrepChrom C-700: Isolation of cannabidiol (CBD) and tetrahydrocannabinol (THC) from other cannabinoids extracted from cannabis

PrepChrom WordPressThe separation of cannabinoids is an important process which is necessary for clinical studies. The aim of these studies is to investigate the effect of the isolated cannabinoids on patients suffering from chronic illnesses and side effects caused by chemotherapy. The relaxation of laws concerning medical use of cannabis all around the world, especially the United States, has increased the interest in determining the effect of cannabinoids on the human body. Clinical studies require large amount of highly pure separated cannabnnoids. This can easily be achieved with the PrepChrom C-700.

Introduction

Cannabis is an extremely versatile plant and has been used for many purposes throughout history. Some specific sorts are used for fibers in the textile and paper industry because of their long stems. It is a useful source of foodstuffs, such as hemp oil and seed. Due to the cannabinoids, cannabis is also a popular recreational drug. These substances can cause mental and physical effects when consumed. The impact of the cannabinoids on the human body is of great interest for the pharmaceutical sector. The research emphasis lies in treating the side effects of chemotherapy, inflammatory diseases like multiple sclerosis or degenerative illnesses such as Parkinson’s disease.[3] AI FAME GmbH, a Swiss company, is a pioneering company to extract the plant-based active substances and make them water-soluble for improved, further processing.

Here, we aim to separate three cannabinoids, i.e. cannabidiol, tetrahydrocannabinol and tetrahydrocannabinolic acid in high purity from the extract.

Experimental

Equipment: PrepChrom C-700

Sample: Water-soluble cannabis extract. Cultivated, extracted and provided by AI FAME GmbH

Preparation: The chromatographic separation is performed with 1 mL of the water-soluble cannabis extract diluted in 1 mL methanol : water (1:1)

Separation: Method parameters
Column:  Sepacore® C-18 80 g
Particle size:  40-63 μm
Flow rate:  40 mL/min
Sample loop:  2 mL
Detection:  253, 270 nm and SCAN 200-600 nm

Gradient: methanol (A) : water(B)
50 % – 90 % methanol (A) in 20 min
90 % – 90 % methanol (A) in 10 min
90 % – 95 % methanol (A) in 5 min
95 % – 95 % methanol (A) in 10 min

Results

Cannabidiol (CBD) bearing a 1,3-diol functional group on its benzene ring, is the first substance to be eluted when applying reversed phase conditions (Figure 1), hence, is the most polar cannabinoid in the sample. The second compound, eluting after 27 minutes, is tetrahydrocannabinol (THC). The last substance to be eluted is tetrahydrocannabinolic acid (THCA), a form of cannabinoid which easily decarboxilates in to THC when exposed to light or heat. Assignment of the signals and identification was done by comparison with reference HPLC data provided by AI FAME GmbH.

Cannabinoide SN finalFigure 1: Methanol : water separation of the extracted cannabinoids using the PrepChrom C-700. Detection at 270 nm, 254 nm, and 200-600 nm SCAN.

 

 

Conclusion

The three cannabinoids extracted by AI FAME GmbH could easily be separated and separately collected with the PrepChrom C-700.

The water-solubility of the extract enables the use of aqueous solvents that have inherent economic and ecologic advantages over common organic solvents.

The growing interest in the clinical use of cannabinoids is unbreakable. Furthermore, cannabis is decriminalized and it is allowed to exploit the potential of its valuable substances. BUCHI offers straight forward solutions to separate and purify small to large size batches of the precious extracts.

Acknowledgment

AI FAME GmbH, Switzerland, is thanked for providing the extract and for the fruitful analytical discussion.

References

[1] Sutton, IR. Daeniken, P. (2006). Cannabinoids in the management of intractable chemotherapy-induced nausea and vomiting and cancer-related pain. The Journal of Supportive Oncology.

[2] State Medical Marijuana Laws. National Conference of State Legislatures. (16.3.2015). http://www.ncsl.org/research/health/state-medical-marijuana-laws.aspx

[3] Kogan, NM. Mechoulam, R. (2007) Cannabinoids in health and disease. Dialogues Clin Neurosci. 2007 Dec; 9(4): 413–430.

>> See additional Application Notes

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NEW Product Announcement

BUCHI PrepChrom C-700: bridging the gap between flash chromatography and preparative HPLC

BUCHI introduces the innovative PrepChrom C-700 chromatography system, combining flash chromatography with preparative HPLC. Bridging the gap between them, the C-700 sets a new standard for purification of synthesis mixtures or complex natural extracts.

C-700_C-650_left_313345-1258

The space saving and easy to use system allows organic chemists to perform the complete purification process, using the interactive user interface specially designed for preparative chromatography applications.

The high quality pumping system features a quaternary gradient module and delivers accurate flow rates up to 250 mL/min at a pressure up to 100 bar (1450 psi). Any type of flash column as well as preparative HPLC columns with a diameter up to 50 mm filled with silica having a particle size down to 10 µm can be used.

Combining high-end hardware technology together with the software designed for easy purification, the PrepChrom C-700 is the ideal solution to solve any purification bottleneck in the research of new active compounds.

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Live Webinar: From TLC to Flash to Prep-HPLC on BUCHI’s new all-in-one solution – PrepChrom C-700

07_313345-1038

Wednesday, June 18, 2014
11:00am Eastern Time


What if there were a way to combine flash chromatography and preparative HPLC into one solution that took up a quarter of the space? Learn about how you can go from TLC to purified product without multiple instruments and without being an expert in chromatography. A test application will show how one product can give you the results you need quickly and easily.

Key Learning Objectives:
• Learn about your options to purify compounds and reaction mixtures.
• Learn how several techniques involved in preparative chromatography can be combined into one solution.
• See interesting applications optimized from TLC to flash and from flash to HPLC fractions.

Click to register


New BUCHI Pre-Packed Flash Chromatography Cartridges

New Sepacore Flash Chromatography Cartridges

New Sepacore Flash Chromatography Cartridges

BUCHI Corporation, a leading supplier of quality high-performance scientific laboratory products, has expanded its portfolio of Sepacore® flash chromatography offerings to include an extensive line of pre-packed disposable flash cartridges. The new Sepacore® Flash cartridges are designed to enable fast, cost-effective, and reproducible isolation of high purity compounds using any flash chromatography instrument.

The combination of a broad range of sorbents and the large choice of cartridge sizes provides a separation solution for just about any purification requirement. Sepacore® flash cartridges range from 4g up to 330g in size, and are available with the choice of Silica (normal-phase) , C18 (reversed-phase), Amine, Cyano, Diol and SCX-2 cation exchange packing materials. And for the more difficult separations, the available Silica HP cartridges provide smaller particles with higher resolution capabilities. For larger-scale purifications 750g and 1500g cartridges are available with both Silica and C18 sorbent materials.

The high quality Silica packing material used in the new Sepacore® cartridges is specially selected to provide efficient and reproducible separations. Its narrow particle size distribution, low metal ion content, neutral pH, and controlled water content enable the overall purification process to be performed faster and at a lower cost than most conventional cartridges.

While these new Sepacore® cartridges work equally well on all flash chromatography instruments, their full advantage is most conveniently realized when used together with the BUCHI Sepacore® flash chromatography system. This system provides optimal scale-up capability with a maximal flow rate of 250mL/min. It enables high resolution separations even at the highest flow rates with a working pressure up to 50bars (725psi).

Comprised of the new pre-packed cartridges, modular instrumentation systems, glass column, and the unique Cartridger ® self-packing device, the Sepacore® range from BUCHI represents a complete solution for flash chromatography.

For over 50 years, BUCHI has been known as the market leader, inventor and innovator of lab instruments based on Evaporation and Vacuum technologies, and as the supplier of the Rotavapor® products worldwide. In addition, BUCHI Corporation is a proven North American provider of spray dryers for pharmaceutical and food agglomeration and microencapsulation, Kjeldahl and solvent extraction equipment for environmental and food analysis, NIR spectroscopy instruments for pharmaceutical and food Quality Control, modular flash chromatography systems, and other related laboratory equipment. Headquartered in New Castle, Delaware, BUCHI Corporation is an affiliate of BUCHI Labortechnik AG (Flawil, Switzerland).

BUCHI 7th Annual Technical Seminar & Workshop

BUCHI is hosting their 7th annual technical seminar and workshop today at the Holiday Inn in Somerset, NJ. Included here are some pictures at the event, please visit our website for any information on any of the laboratory equipment mentioned below.

BUCHI not only manufactures market leading instruments, but we also provide method development and application support to our customers. For more than 70 years, BUCHI has developed techniques and equipment designed to simplify your workflow and make your lab and facility more productive.

Attendees of this FREE one day seminar are learning essential technical information and participating in open discussions on implementation, application and method development among users and peers.

Agenda:
9:00 Registration with continental breakfast – Intro to BUCHI

9:15 BUCHI introduction and product overview

9:30 Sepacore® – Preparative and Flash Chromatography – Extended pressure range up to 725psi using innovative three piston pulsation free pumps – unmatched for Flash. Presented by Jeff Reid, BUCHI Corporation

•Benefits of a modular approach
•Various configurations, options, and setups
•SepacoreControl software and SepacoreRecord for data acquisition
•Unique Cartridger – high performing columns you can pack

10:15 Break

10:30 SpeedExtractor – Pressurized Solvent Extraction System, and Syncore® Concentration. Presented by William Ickes, BUCHI Corporation

•Design improvements simultaneously extract up to 6 samples in 20 minutes!
•Precise pressure and temperature control and reporting
•Unique design features for better reproducibility and accuracy
•Streamlined concentration with solvent recovery – Syncore and Multivapor™ together

11:15 Demonstration of Sepacore® and the SpeedExtractor. Open discussion and hands-on operation of instruments. Explore the flexibility of the Sepacore modular chromatography system and try samples on the world’s most innovative pressurized solvent extractor the SpeedExtractor.

12:00 Lunch – provided by BUCHI

1:00 Introduction to FTNIR and NIRCal®
Presented by Dr. Ron Rubinovitz, BUCHI Corporation

•Comparison of FTNIR to FTIR and Raman
•Pharma/Biotech – BUCHI’s unique fully validated ID library
•Nutraceuticals – Meet GMP requirements for incoming Material Identification
•Unique ChromaDex® Standards Data Base
•Steps for a successful NIR implementation

1:45 FTNIR – NEW developments
Presented by Dr. Ron Rubinovitz, BUCHI Corporation

•New NIRMaster Analyzer – World’s first “hardened” FTNIR – can be hosed down in process
•New software networking capabilities/continuous monitoring capabilities
•Checkmaster – Full automation at the Tablet Press – content uniformity and physical properties

2:30 Demonstration of N-500 FTNIR NIRFlex® Analyzer and NIRCal® Software. Open discussion and hands-on operation of instruments.

*The N-500 and the NIRMaster are in operation and a NIRCal software demo will be done this afternoon.

Separation of Plant Extracts Using Sepacore Flash Chromatography System

Natural Plant ExtractsNatural compounds are more and more evaluated as alternatives to classical drugs and therefore the needs for the separation of such complex mixtures are also growing. Flash chromatography is a very valuable technique in the field of natural compounds research because it provides a fast and economical way to separate the main components of complex plant extracts. Download this flash chromatography study and view our flash chromatography applications for other related studies.

Introduction:
During the last decade, several systems for rapid preparative chromatography with pre-packed cartridges have been commercialized. Pre-packed cartridges ensure rapid separation cycles and ease of use.

Flash chromatography systems were initially developed for rapid and easy purification of synthetic products, and numerous applications are documented in the experimental section of publications in synthetic chemistry. In contrast, application of such systems in the separation of complex natural product mixtures such as extracts has been neglected.

The aim of this work was to explore the potential and limitations of cartridges for the purification of natural product extracts. Empirical rules have been established for the determination of the separation conditions by preliminary TLC and HPLC analyses.

The influence of solid introduction compared to liquid injection has been also investigated. The performance of the cartridges was compared to that of classical selfpacked MPLC glass columns for the separation of complex plant extracts of medicinal importance.

Instrumentation:
Preparative separations were performed on a Sepacore® chromatography system (Büchi Labortechnik) consisting of two C 605 pump modules, a C 620 control unit, a C 635 UV detector and a C 660 fraction collector. The system was controlled by the software SepacoreControl 1.0.

Flash chromatography separations were performed on pre-packed silica gel (40 – 63 μm) and RP18ec (40 – 63 μm) polypropylene cartridges (12 x 150 or 40 x 150 mm, Büchi) at a flow rate of 10 mL (12 x 150 mm cartridges) and 30 mL/min (40 x 150 mm cartridges), resp. Medium pressure liquid chromatography (MPLC) separations were carried out on a glass column (26 x 460 mm) packed with silica gel Si60 (15 – 40 μm) or LiChroprep RP18 (25 – 40 μm) (Merck), at a flow rate of 10 mL/min. Liquid injection was carried out through a 6-way valve with a 20 ml loop. Solid introduction was performed by means of a PrepElut cartridge (Flash chromatography) or a glass precolumn (MPLC) connected to the top of the cartridge or the column, resp. The samples were adsorbed to silica gel Si60 or LiChroprep RP-18, resp., prior to introduction.

Conclusions:
• Reversed phase HPLC separations can be transposed by increasing the gradient time
by a factor 2-4.

• For normal phase separations, solvent compositions resulting in Rf values of 0.15-0.2 on TLC for the most lipophilic and the most hydrophilic constituents, respectively, should be selected as gradient endpoints.

• Sepacore® cartridges enabled a good separation of compounds with a broad range of polarity, as typically found in plant extracts. The chromatographic resolution remained, however, lower than that achieved by MPLC on columns packed with material of smaller particle size. For poorly soluble extracts, solid introduction gave better results than liquid injection.

• Despite lower resolution as compared to MPLC, pre-packed cartridges are an attractive alternative for the purification of extracts and crude fractions due to their ease of use and speed of separation.

Short Note #60: Cleaning Up Of alpha-Methylstyrene by Flash Chromatography

This short note details the isolation of alpha-Methylstyrene from a crude reaction mixture. This may sound easy, but the reaction mixture contains some dimethyl sulfoxide and the oily wetting agent from the sodium hydride. Both of them are not UV-active and therefore in the TLC invisible. Download or view this Flash Chromatography short note and visit our Chromatography Applications page for other synthetic reaction mixture notes.

Sepacore configuration:
40 X 150mm Cartridge prepacked with silica gel 60
2 C-605 Pump Modules
C-660 Fraction Collector
C-620 Control Unit with SepacoreControl Software
C-635 UV Photometer

Separation Conditions:
Eluent: n-hexane with 0%, 2% und 15% ethyl acetate, step gradient
Flow rate: 100ml/min
Sample: 3 g crude mixture, dissolved in toluene
Injection volume: 4.5 ml (although the mixture is liquid, the sample is not fully miscible with n-hexane and must be dissolved in toluene and
injected directly onto the cartridge)

Sepacore Flash Chromatography System

Sepacore Flash Chromatography System