WHAT IS ISOMERISM?
Isomerism refers to a phenomenon where two or more compounds have the same molecular formula, but they differ in connectivity (constitutional isomerism) or spatial arrangement (stereoisomerism) of atoms which they consist of, what may result in different physical, chemical or biological properties.
This is Why You Should Look Into Isomer Separation and Chiral Separation
• In recent decades, chromatographic techniques have advanced significantly, driven by regulatory guidelines in fields such as pharmaceuticals, materials science, agrochemicals, biotechnology, food, and environmental testing.
• The shift towards using single isomers instead of mixtures has highlighted the challenge of isomer separation, both in chemical analysis and industrial production. Traditional methods like crystallization and distillation often fail when isomers have similar physical properties.
• Compounds with multiple structural or stereoisomers further complicate separation, but differences in partition coefficients or solubility offer advantages for liquid-liquid chromatography.
• Centrifugal partition chromatography (CPC) is a major advancement in offering effective isomer separation, with superior efficiency, scalable chiral separation, preparative chiral separation, preparative isomer separation and environmental benefits. As technology evolves and industries realize its potential, CPC is poised to become essential in fields ranging from pharmaceuticals to environmental science.
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ISOMER SEPARATION WEBINAR
04. December 2024. Microsoft Teams
If you would like to learn more about isomerism, isomer purification or centrifugal partition chromatography, we invite you to join our FREE webinar and Q&A session on December 4.
You'll learn:
• The advantages of CPC over traditional methods (e.g. HPLC, SFC, CE), especially related to purifying notoriously difficult isomers.
• Exactly how you can implement and use CPC technology in your business and setup, especially for isomer purification.
• Real-life examples where CPC was used to purify isomers like vitamin E, nebivolol, amberketal, and voriconazole.
• Why CPC offers superior purity and quality for your final products than traditional methods.
• How CPC can make your isomer purification setup more sustainable and effective.
CPC vs HPLC: A cost-effective, Silica-Free Solution for Chiral Separation
Expensive
Highp-erformance liquid chromatography (HPLC) is the most widely used technique for chiral separations, but is also one of the most expensive ones.
Difficult Method Development
Method development may involve a lot of trial and error to find the best condition, and the use of chiral columns, high-purity HPLC-grade solvents and additives are required.
Column Degradation
Chiral columns tend to degrade faster than ordinary high-performance liquid chromatography columns, shortening their lifespan. They may provide limited selectivity due to the fact that not all enantiomers can be separated on one specific column.
CPC vs SFC: Separate Compounds Without Solubility Issues Caused by Low Polarity
Even More Expensive than HPLC
Instrumentation for supercritical fluid chromatography (SFC) is more complex and costly than HPLC systems.
Limitations of Highly Polar Compounds
Supercritical fluid chromatography is limited to separating highly polar compounds due to solubility issues arising from the low polarity of supercritical CO2.
BENEFITS OF CENTRIFUGAL PARTITION CHROMATOGRAPHY (CPC) FOR CHIRAL SEPARATION
Centrifugal partition chromatography (CPC) a liquid-liquid preparative chromatographic technique where separation takes place between two immiscible liquid solvent systems. It offers high efficiency and precision in separation of chiral compounds with liquid chromatography. CPC offers excellent scalability in separation of chiral compounds with liquid chromatography.
Liquid-Liquid Technique
CPC is a liquid-liquid preparative technique based on classical chromatography principles. It uses two immiscible liquid phases for separation, with a centrifugal force immobilizing the stationary phase.
No Silica Gel or Solid Support Required
Unlike traditional methods, CPC doesn’t require silica gel or solid supports, allowing for a broader range of solvents, including eco-friendly options like water.
Continuous Purification, Similar to Simulated Moving Bed
CPC, with its continuous purification possibilities offers purification setups similar to simulated moving bed (SMB) purification, and allows for chiral separation chromatography.
Cells and Rotors, No 'Columns'
In a CPC device, traditional chromatographic columns are replaced by cells connected in series by ducts and attached to a rotor filled with the liquid stationary phase. A strong centrifugal force is used to immobilize the liquid within these cells, enabling the separation process, for example chiral separation techniques.
Separation, Isolation and Remediation
Chromatography is commonly used for separation, isolation and remediation. Centrifugal partition chromatography can perform all three.
From Lab-Scale To Continuous Operation
A major advantage of CPC is its ease of scalability. CPC platforms offer linear scalability, allowing smooth transitions from benchtop and lab-scale instruments to large-scale, continuous purification systems. This enables businesses to use a single purification solution for both development and production, streamlining processes and reducing the need for multiple systems.
CPC cells, rotors and ascending-descending modes visualized.
Case Study: Enrichment and Isolation of Vitamin E Structural Isomers
CPC-UV Chromatogram of the separation.
Tocopherols (1a-d) and tocotrienols (2a-d) are the main naturally occurring forms of vitamin E, often extracted from palm oil. As antioxidants, they help neutralize free radicals, which may contribute to certain diseases. Different isomers of tocopherols and tocotrienols can trigger various biological activities, making it essential to study them individually. Some isomers are used as food additives (e.g., E307, E308, E309), while others are found in sunscreens and lotions for UV protection and skin repair.
Structurally, all isomers share the same chromane head (hydrophilic) but differ in their isoprenoid side chains (lipophilic). Tocopherols have a saturated phytyl chain with 3 asymmetric carbons, while tocotrienols have an unsaturated chain with 3 trans double bonds and only one chiral center. These structural differences create 8 isomers: 4 tocopherols and 4 tocotrienols, known as α-, β-, γ-, and δ-isomers.
Enriching Tocotrienol in Vitamin E Oil
The study aimed to enrich the tocotrienol content in raw vitamin E oil (palm oil containing triglycerides) and isolate each tocotrienol with. Initial solvent screening showed that anhydrous biphasic systems offered better partition coefficients due to the hydrophobic nature of the target compounds.
Analysis indicated that the tocotrienol content was 90% pure after normalization, with a total content of 719 mg/g. When 5 g of crude material was injected, 2534 mg of product was obtained, yielding 72% tocotrienol content and a 97% overall yield.
How CPC Performed in the Study
The separation process was first assessed using a laboratory-scale CPC instrument in descending elution mode. The maximum injectable quantity was 1 g of raw material dissolved in 12 mL of the upper phase. The chromatogram showed the effective separation of tocotrienols, highlighting three main components, in elution order. Analysis indicated that the tocotrienol content reached 90% purity. From the injection of 5 g of crude material, a total of 2534 mg of product was obtained, yielding 72% tocotrienol content with a 97% yield.
Separation Of chiral Chromane Epoxide, Nebivolol
Nebivolol is a selective β1-receptor antagonist and β-blocker commonly prescribed for hypertension. The original patented synthesis produces a racemic mixture of its two enantiomers. Although the molecule has four asymmetric carbons, allowing for 16 possible stereoisomers, an exoskeletal symmetry plane through the central nitrogen atom reduces this number to 10.
Producing the active pharmaceutical ingredient (API), nebivolol hydrochloride, requires a highly stereocontrolled synthesis or targeted purification method. However, this process typically involves expensive, eco-friendly transition metal catalysts, making it less feasible for industrial production.
An alternative approach is to enrich the desired stereoisomers during synthesis through various separation or purification methods, which is particularly useful for producing key intermediates in complex drug synthesis. For instance, specific chromane epoxides are crucial synthons in nebivolol synthesis. Increasing the proportion of necessary stereoisomers early in the synthesis enhances diastereomeric excess, leading to cleaner reactions and simpler purification.
Solvent System and Purification Results
This study focused on purifying synthetic crude by isolating each isomer from a mixture of the two chromane epoxide diastereomers. Screening identified a ternary CPC solvent system as optimal for separating the diastereomeric pair, achieving partition coefficients of 1.5 for isomer 3 and 0.8 for isomer 4, resulting in a selectivity factor of 1.87 in descending mode. Under these conditions, lab-scale CPC experiments yielded purities of over 99% for both diastereomers, with respective yields of 99.6% and 74%.
The chromatogram and the details of the separation of the diastereomers of Nebilovol’s precursor
Purification Of A Highly Valued Synthetic Odorant For Perfumery Purposes
Chromatogram of the separation of amberketal and iso-amberketal on a lab-scale instrument.
Amberketal is a synthetic odorant that mimics the highly valued natural ambergris and is found in the bark of the western white pine tree (Pinus monticola). Its sustainable synthetic production is increasingly important due to environmental concerns.
Structurally, amberketal is a polycyclic labdane-type terpenoid with five asymmetric carbon centers, allowing for numerous stereoisomers. Typically, only amberketal and its diastereomer, iso-amberketal, are produced under common synthetic conditions.
Challenges
During the synthesis of these compounds and their precursors, racemic polycyclic intermediates are generated, necessitating effective purification methods. Previous attempts using column chromatography and fractional distillation were not cost-effective for this separation challenge.
Solutions
To address this, several ternary and quaternary CPC solvent systems were screened for optimal solubility, partition, and selectivity for the diastereomers. The selected ternary biphasic solvent system achieved a high selectivity factor (α = 1.65) for separating the diastereomeric pair, with partition coefficients of 2.40 for amberketal and 3.96 for iso-amberketal. This solvent system was selected for further CPC testing in descending elution mode, allowing for high flow rates that reduce run times.
By applying the optimized method on a pilot-scale CPC instrument, 30 g/h throughput can be achieved resulting amberketal with its purity above 95% and adequate 81% yield.
Separation of the Diastereomer of a Nucleotide Prodrug, Remdesivir
Recently, the stereoselective synthesis and purification of chiral phosphorus compounds have gained significant attention. These compounds serve as crucial intermediates in synthesizing nucleotide-based pharmaceuticals like oligonucleotides, mRNAs, and DNAs. A notable example is Remdesivir, an FDA-approved phosphoramidite prodrug used to treat COVID-19, originally developed for Ebola. This broad-spectrum antiviral inhibits the viral RNA-dependent RNA polymerase, blocking SARS-CoV-2 replication. Remdesivir belongs to the ProTide prodrug family, which directly targets the virus.
Structurally, Remdesivir is a 1’-cyano substituted adenosine nucleotide analogue with a phosphorus stereocenter, allowing for the formation of SP- and RP-diastereomers. Currently, only the SP-diastereomer is clinically approved, although the RP-diastereomer is also a potential antiviral. Both diastereomers are challenging to purify due to complex and expensive synthetic procedures with poor stereoselectivity.
Method Development
The initial isolation method involved preparative HPLC, which is costly and difficult to scale. The investigation began by screening over 200 binary, ternary, and quaternary solvent systems to find the optimal balance of solubility, selectivity, and partitioning for both compounds. Fine-tuning the solvent system was critical, particularly in selecting cosolvents and adjusting pH, given Remdesivir's poor water solubility.
Finding the Right Solvent System
After extensive optimization, a quaternary CPC solvent system was identified as the most effective, achieving a selectivity of 1.29 with partition coefficients of 1.54 for the RP-diastereomer and 1.99 for the SP-diastereomer.
Using the Continuous CPC in multiple dual-mode (MDM), successful isolation of both diastereomers was achieved, yielding 66% for the SP-diastereomer and 58% for the RP-diastereomer, with purities of 95.1% and 95.0%, respectively, in just 50 minutes using a dual-rotor setup (2 x 2.1 L rotor volume, DSC-ASC-DSC cycle).
Moreover, the challenging RP-diastereomer was successfully isolated using the trapping-MDM technique, achieving less than 99% purity and a 39% isolated yield within 180 minutes. This novel preparative method for purifying and isolating the Remdesivir diastereomers resulted in a two-fold increase in productivity compared to previously reported liquid-liquid chromatography (LLC) methods.
MDM CPC chromatogram of the separation of Remdesivir diastereomers.
Trapping MDM CPC chromatogram of the separation of the RP-diastereomer of Remdesivir.
Preparative Enantioseparation Of An Azole Type Racemate – Isolation Of An Antifungal API, Voriconazole
Separation of the two enantiomers of voriconazole by lab-scale CPC.
Voriconazole is a widely used third-generation antifungal azole effective against fungal and yeast infections. The molecule contains two asymmetric carbons, leading to four stereoisomers: two enantiomers from diastereomeric pairs. The preferred synthesis of voriconazole results in a racemic mixture of the (2R,3S)-enantiomer, known as the eutomer (the active pharmaceutical ingredient, or API), and its enantiomer, the (2S,3R) stereoisomer, which is the distomer (less potent or potentially toxic).
Industrial-scale purification of voriconazole, mainly through optical resolution or chromatography, poses challenges due to high costs, environmental concerns, time consumption, and low yields. Therefore, centrifugal partition chromatography emerged as a promising, cost-efficient, and environmentally friendly alternative.
For enantioselective separations, chiral selectors are necessary. During our investigations, various cyclodextrin derivatives were screened as chiral selectors across multiple two-phase solvent systems. In total, nearly 90 biphasic solvent systems with varying concentrations of cyclodextrins in the aqueous phase were examined to optimize the partition coefficients (Kd) for each enantiomer. Ultimately, a specific CPC solvent system was chosen, resulting in partition coefficients of 1.53 for the (2S,3R)-enantiomer and 0.50 for the (2R,3S)-enantiomer, achieving a selectivity of 3.04 without pH adjustment.
Lab-Scale CPC Separation
Based on these results, a lab-scale CPC separation was conducted in ascending mode using a 200 mg solution of racemic voriconazole in 20 mL of the upper phase, eluting at a flow rate of 20 mL/min. A notable advantage of this method is that during ascending elution, the more pharmacologically significant eutomer elutes first, followed by the distomer. Baseline separation was achieved within 20 minutes, yielding a pure (>99%) enantiomer with a 97% yield.
ABOUT ROTACHROM
RotaChrom Brings Industrial-Scale CPC Solutions to Businesses, Researchers, CROs and CDMOs.
RotaChrom Technologies developed the world’s first industrial scale centrifugal partition chromatography technology solution. These liquid chromatography machines revolutionized purification by providing innovative, sustainable, and efficient industrial scale LC-CPC chromatography to customers all over the world in various industries, including pharmaceutical, nutraceutical and botanical extracts.
• RotaChrom’s proven GMP compliant CPC designs are effective solutions when looking for a high-purity, high-capacity, cost-effective and environmentally conscious solution. RotaChrom, with its in-house research laboratory employing world class PhD chemists, engineers and leading business professionals on multiple continents, is the best partner in purification.
• Our portfolio includes several products, which can help you scale your business from research to industry. Select between lab scale, pilot scale or industrial scale purification:
• The rCPC platform for pilot-scale purification tasks within a laboratory environment. It is ideal for all batch sizes, smaller or larger, or campaign type of research work, and continuous-batch production.
• The iCPC platform, which is the largest commercially available centrifugal partition Chromatography instrument in the world. It is designed for industrial-scale purification projects in continuous batch operation mode.
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PRODUCT PORTFOLIO
CPC Modeler: Purification method development platform
The CPC Modeler is a desktop-sized CPC platform, which combines an intuitive method development engine with tailor-made semi-preparative chromatographic equipment. The CPC Modeler predicts, tests, and validates the most effective purification method of different Compounds of Interest (CoI), while also offering the possibility to scale-up production. The
platform offers a mg to g/cycle loading capacity and can purify all the compounds that the rCPC can, including pharmaceutical APIs and natural extracts.
rCPC: Pilot-Scale Purification
The rCPC platform is designed to execute lab-scale purification tasks with CPC technology. With its 10 g to 100 g/cycle loading capacity and potential throughput up to 2 kg/day, it is ideal for all batch sizes, smaller or larger, or campaign types of research work, and is the foundation of our continuous-batch production processes. The device is also available for pharma GMP environment and in NRTL/UL-compliant equipment design.
iCPC: Industrial-Scale CPC Purification
The iCPC is the largest serialized CPC instrument in the world, designed for industrial-scale purification projects. This device is by far the most cost-effective industrial-scale chromatography platform available on the market. With its kg/cycle loading capacity, it has the capacity to process up to tens of kg of crude input material per day. The iCPC, also a core element of the pCPC platform, always comes with solvent preparation and recovery and regeneration systems.
Continuous CPC: Maximized Productivity With Small Ecological and Economical Footprint
The Continuous CPC delivers purification and polishing solutions for difficult matrices, where the difference between individual constituents is negligible. The platform can operate in multiple-dual mode, which greatly increase production output. The dual-rotor design enhances productivity and further reduces the relative ecological and economical footprint using two rotors working together under a single control unit. The platform can be extended with a tailor-made solvent handling system on-demand.