Sustainable cannabinoids purification through twin-column recycling chromatography and green solvents.

In the present study, twin-column recycling chromatography has been employed for the purification of a Cannabis extract by using a green solvent, ethanol, as the mobile phase. In particular, the complete removal of the psychoactive tetrahydrocannabinol (THC) from a Cannabis extract rich in cannabidiol (CBD) was achieved under continuous conditions. The performance of the method, in terms of compound purity, recovery, productivity and solvent consumption, was compared to that of traditional batch operations showing the potential of the twin-column recycling approach. The employment of a theoretical model to predict the band profiles of the two compounds during the recycling process has facilitated method development, thus further contributing to process sustainability by avoiding trial and error attempts or at least decreasing the number of steps significantly.

Enhancing the purification of crocin-I from saffron through the combination of multicolumn countercurrent chromatography and green solvents.

Crocin-I, a valuable natural compound found in saffron (Crocus sativus L.), is the most abundant among the various crocin structures. Developing a cost-effective and scalable purification process to produce high-purity crocin-I is of great interest for future investigations into its biological properties and its potential applications in the treatment of neurological disorders. However purifying crocin-I through single-column preparative chromatography (batch) poses a yield-purity trade-off due to structural similarities among crocins, meaning that the choice of the collection window sacrifices either yield in benefit of higher purity or vice versa. This study demonstrates how the continuous countercurrent operating mode resolves this dilemma. Herein, a twin-column MCSGP (multicolumn countercurrent solvent gradient purification) process was employed to purify crocin-I. This study involved an environmentally friendly ethanolic extraction of saffron stigma, followed by an investigation into the stability of the crocin-I within the feed under varying storage conditions to ensure a stable feed composition during the purification. Then, the batch purification process was initially designed, optimized, and subsequently followed by the scale-up to the MCSGP process. To ensure a fair comparison, both processes were evaluated under similar conditions (e.g., similar total column volume). The results showed that, at a purity grade of 99.7%, the MCSGP technique demonstrated significant results, namely + 334% increase in recovery + 307% increase in productivity, and - 92% reduction in solvent consumption. To make the purification process even greener, the only organic solvent employed was ethanol, without the addition of any additive. In conclusion, this study presents the MCSGP as a reliable, simple, and economical technique for purifying crocin-I from saffron extract, demonstrating for the first time that it can be effectively applied as a powerful approach for process intensification in the purification of natural products from complex matrices.

Novel insights into the dependence of adsorption-desorption kinetics on particle geometry in chiral chromatography.

The existence of slow adsorption-desorption kinetics in chiral liquid chromatography is common knowledge. This may significantly contribute to worsening the efficiency and kinetic performance of a chromatographic run, especially when high flow rates are employed. Many attempts and protocols have been proposed to access this term, the so-called c ads , but they are based on different (theoretical) assumptions. As a consequence, no official method is available for the estimation of the adsorption-desorption kinetics term. In this work, a novel approach to access c ads is presented. This procedure combines experimental results obtained with kinetic and thermodynamic measurements. The investigations have been performed on two zwitterionic teicoplanin chiral stationary phases (CSPs) based on 1.9 µ m fully porous and 2.0 µ m superficially porous particles (FPPs and SPPs), using Z-D,L-Methionine as probe molecule. Kinetic studies have been performed through the combination of both stop-flow and dynamic measurements, while adsorption isotherms have been calculated through Inverse Method. This study has confirmed that, on both particle formats, analyte diffusion on the surface of the particle is negligible, meaning that adsorption is localized, and it has been demonstrated that adsorption-desorption kinetics is strongly dependent on particle geometry and, in particular, on the loading of chiral selector. These findings are fundamental not only to unravel novel aspects of the complex enantiorecognition mechanism but also to optimize the employment of CSPs for ultra-fast and preparative applications.

The influence of drying and storage conditions on the volatilome and cannabinoid content of Cannabis sativa L. inflorescences.

The increasing interest in hemp and cannabis poses new questions about the influence of drying and storage conditions on the overall aroma and cannabinoids profile of these products. Cannabis inflorescences are subjected to drying shortly after harvest and then to storage in different containers. These steps may cause a process of rapid deterioration with consequent changes in precious secondary metabolite content, negatively impacting on the product quality and potency. In this context, in this work, the investigation of the effects of freeze vs tray drying and three storage conditions on the preservation of cannabis compounds has been performed. A multi-trait approach, combining both solid-phase microextraction (SPME) two-dimensional gas chromatography coupled to mass spectrometry (SPME-GC × GC-MS) and high-performance liquid chromatography (HPLC), is presented for the first time. This approach has permitted to obtain the detailed characterisation of the whole cannabis matrix in terms of volatile compounds and cannabinoids. Moreover, multivariate statistical analyses were performed on the obtained data, helping to show that freeze drying conditions is useful to preserve cannabinoid content, preventing decarboxylation of acid cannabinoids, but leads to a loss of volatile compounds which are responsible for the cannabis aroma. Furthermore, among storage conditions, storage in glass bottle seems more beneficial for the retention of the initial VOC profile compared to open to air dry tray and closed high-density polyethylene box. However, the glass bottle storage condition causes formation of neutral cannabinoids at the expenses of the highly priced acid forms. This work will contribute to help define optimal storage conditions useful to produce highly valuable and high-quality products.

Understanding the Transition from High-Selective to High-Efficient Chiral Separations by Changing the Organic Modifier with Zwitterionic-Teicoplanin Chiral Stationary Phase.

The retention behavior of small molecules and N-protected amino acids on a zwitterionic teicoplanin chiral stationary phase (CSP), prepared on superficially porous particles (SPPs) of 2.0 µm particle diameter, has shown that efficiency and enantioselectivity, and so enantioresolution, dramatically change depending on the employed organic modifier. In particular, it was found that while methanol permits the boost of enantioselectivity and resolution of the amino acids, at the cost of efficiency, acetonitrile allows for the ability to reach extraordinary efficiency even at high flow rates (with reduced plate height <2 and up to 300,000 plates/m at the optimum flow rate). To understand these features, an approach based on the investigation of mass transfer through the CSP, the estimation of the binding constants of amino acids on the CSP, and the assessment of compositional properties of the interfacial region between bulk mobile phase and solid surface has been adopted.

Investigating the effect of polarity of stationary and mobile phases on retention of cannabinoids in normal phase liquid chromatography.

This work reports about a screening of four adsorbents with different polarity employed for the separation of the main phytocannabinoids contained in Cannabis sativa L., under normal phase liquid chromatography (NPLC). The effect of polarity and type of interaction mechanisms of the adsorbents (namely Si-, CN-, Diol-, and NH2-based SPs) on retention has been investigated under a variety of conditions either by using different combinations of apolar solvents (heptane or hexane) and alcohols (ethanol or isopropanol). The columns have also been employed for the separation of a real cannabis sample. Graphical Abstract Investigating the effect of polarity of stationary and mobile phases on retention of cannabinoids in normal phase liquid chromatography.

Detailed investigation of the composition and transformations of phenolic compounds in fresh and fermented Vaccinium floribundum berry extracts by high-resolution mass spectrometry and bioinformatics.

INTRODUCTION: Blueberries are known for their very high content of biologically active phenolic compounds; nonetheless, differently from the North American and European species of blueberries, Neotropical blueberries have not been extensively studied yet. OBJECTIVES: In the present paper, the phenolic composition of Vaccinium floribundum Kunth, which is endemic to the Andean regions and grows 1,600 to 4,500 meters above sea level, was investigated by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Native and fermented berries were compared in terms of phenolic composition as well as antioxidant activity, total phenolic content, and total anthocyanin content. MATERIALS AND METHODS: V. floribundum native and fermented berries were extracted and analyzed by UHPLC-HRMS. The acquired datasets were processed by Compound Discoverer 3.1 using a dedicated data analysis workflow that was specifically set up for phenolic compound identification. RESULTS: In total, 309 compounds were tentatively identified, including anthocyanins, flavonoids, phenolic acids, and proanthocyanidins. The molecular transformations of phenolic compounds during fermentation were comprehensively investigated for the first time, and by a customized data processing workflow, 13 quinones and quinone methides were tentatively identified in the fermented samples. Compared to other species of the genus Vaccinium, a peculiar phenolic profile is observed, with low abundance of highly methylated compounds. CONCLUSION: Andean berries are a rich source of a wide variety of phenolic compounds. Untargeted MS analyses coupled to a dedicated data processing workflow allowed expanding the current knowledge on these berries, improving our understanding of the fate of phenolic compounds after fermentation.

Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography.

The market of biomolecules with therapeutic scopes, including peptides, is continuously expanding. The interest towards this class of pharmaceuticals is stimulated by the broad range of bioactivities that peptides can trigger in the human body. The main production methods to obtain peptides are enzymatic hydrolysis, microbial fermentation, recombinant approach and, especially, chemical synthesis. None of these methods, however, produce exclusively the target product. Other species represent impurities that, for safety and pharmaceutical quality reasons, must be removed. The remarkable production volumes of peptide mixtures have generated a strong interest towards the purification procedures, particularly due to their relevant impact on the manufacturing costs. The purification method of choice is mainly preparative liquid chromatography, because of its flexibility, which allows one to choose case-by-case the experimental conditions that most suitably fit that particular purification problem. Different modes of chromatography that can cover almost every separation case are reviewed in this article. Additionally, an outlook to a very recent continuous chromatographic process (namely Multicolumn Countercurrent Solvent Gradient Purification, MCSGP) and future perspectives regarding purification strategies will be considered at the end of this review.

Modern trends in downstream processing of biotherapeutics through continuous chromatography: The potential of Multicolumn Countercurrent Solvent Gradient Purification.

Single-column (batch) preparative chromatography is the technique of choice for purification of biotherapeutics but it is often characterized by an intrinsic limitation in terms of yield-purity trade-off, especially for separations containing a larger number of product-related impurities. This drawback can be alleviated by employing multicolumn continuous chromatography. Among the different methods working in continuous mode, in this paper we will focus in particular on Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) which has been specifically designed for challenging separations of target biomolecules from their product-related impurities. The improvements come from the automatic internal recycling of the impure fractions inside the chromatographic system, which results in an increased yield without compromising the purity of the pool. In this article, steps of the manufacturing process of biopharmaceuticals will be described, as well as the advantages of continuous chromatography over batch processes, by particularly focusing on MCSGP.

Investigation of mass transfer properties and kinetic performance of high-efficiency columns packed with C18 sub-2 µm fully and superficially porous particles.

Three columns packed with 2.0 µm superficially porous particles, 1.7 µm fully porous particles, and monodisperse 1.9 µm fully porous particles with narrow particle size distribution have been deeply characterized from a kinetic point of view. The 1.9 µm column showed excellent kinetic performance, comparable to that of the superficially porous one. These two columns also exhibit flatter c-branches of the van Deemter curve compared to the 1.7 µm fully porous particles column, resulting in smaller loss of efficiency when they are operated at higher flow rates than the optimal ones. The independent evaluation of each contribution to band broadening has revealed that the difference in kinetic performance comes from the very small eddy dispersion contribution on the 1.9 µm column, surprisingly even lower than that of the superficially porous one. This finding suggests a very good packing of the monodisperse 1.9 µm column. On the other hand, the potential of 1.7 µm fully porous particles is completely broken down by the strong frictional heating effect already arising at relatively low flow rates.

New frontiers and cutting edge applications in ultra high performance liquid chromatography through latest generation superficially porous particles with particular emphasis to the field of chiral separations.

About ten years after their introduction to the market (happened in 2006), the so-called second generation superficially porous particles (SPPs) have undoubtedly become the benchmark as well as, very often, the preferred choice for many applications in liquid chromatography (LC), when high efficiency and fast separations are required. This trend has interested practically all kinds of separations, with the only exception of chiral chromatography (at least so far). The technology of production of base SPPs is advanced, relatively simple and widely available. The deep investigation of mass transfer mechanisms under reversed-phase (RP) and normal-phase (NP) conditions for achiral separations has shown the advantages in the use of these particles over their fully porous counterparts. In addition, it has been demonstrated that SPPs are extremely suitable for the preparation of efficient packed beds through slurry packing techniques. However, the research in this field is in continual evolution. In this article, some of the most advanced concepts and modern applications based on the use of SPPs, embracing in particular ultrafast chiral chromatography and the design of SPPs with engineered pore structures or very reduced particle diameter, are revised. We describe modern trends in these fields and focus on those aspect where further innovation and research will be required. Graphical Abstract Word cloud of cutting edge applications of superficially porous particles in liquid chromatography.

The Way to Ultrafast, High-Throughput Enantioseparations of Bioactive Compounds in Liquid and Supercritical Fluid Chromatography.

Until less than 10 years ago, chiral separations were carried out with columns packed with 5 or 3 µ m fully porous particles (FPPs). Times to resolve enantiomeric mixtures were easily larger than 30 min, or so. Pushed especially by stringent requirements from medicinal and pharmaceutical industries, during the last years the field of chiral separations by liquid chromatography has undergone what can be defined a "true revolution". With the purpose of developing ever faster and efficient method of separations, indeed, very efficient particle formats, such as superficially porous particles (SPPs) or.

Recent advancements and future directions of superficially porous chiral stationary phases for ultrafast high-performance enantioseparations.

This review focuses on the use of superficially porous particles (SPPs) as chiral stationary phases for ultra-high performance liquid enantioseparations. In contrast to what happened in achiral separations where core-shell particles invaded the market, the introduction of SPPs in chiral liquid chromatography (LC) has been relatively recent. This is due in part to the technical difficulties in the preparation of these phases, and in part to scarce understanding of mass transfer phenomena in chiral chromatography. As a matter of fact, nowadays, the development of superficially porous CSPs is still in its infancy. This paper covers the most recent advancements in the field of core-shell technology applied to chiral separations. We review the kinds of chiral selectors that have been used for the preparation of these phases, by discussing the advantages of chiral SPPs over their fully-porous counterparts for high efficient high throughput enantioseparations. Notwithstanding the apparently obvious advantages in terms of the mass transfer of chiral SPPs, some critical aspects that could impact their development are presented.

Integrated multidimensional chromatography on preparative scale for oligonucleotides purification.

Therapeutic oligonucleotides represent a recent breakthrough in the pharmaceutical industry due to their ability to regulate gene expression with great specificity. This aspect allows treatment of a wide range of diseases. However, since oligonucleotides are used for therapeutic purposes, the Active Pharmaceutical Ingredient (API) must fulfill strict purity levels which require intensive purification steps. For oligonucleotides, and biomolecules in general, preparative liquid chromatography is the technique of choice to perform large scale purifications, typically in batch mode, i.e. using a single column. Specifically, since ONs are mainly large, hydrophilic and charged molecules, Anion Exchange chromatography (AEX) and Ion Pair Reversed Phase chromatography (IP-RP) are the preferred chromatographic modes for their downstream processing. Nevertheless, these approaches suffer from a purity-yield trade-off, and for this reason, more than one purification step is usually required. The two chromatographic modes can therefore be used consequently to remove different groups of impurities, thanks to their orthogonality. In this work, a multidimensional and orthogonal approach on a (semi)preparative scale, namely "Integrated Batch process", was applied for the purification of a single-stranded DNA oligonucleotide. This process combines two chromatographic steps without any hold step, operator intervention or sampling of the first step. The performance parameters of the Integrated Batch were compared to those obtained in the single batch runs under different experimental conditions (chromatographic mode, eluent systems), showing the potential of this integrated approach. This proof-of-concept study illustrates how this technique can considerably reduce overall production time and how it allows to increase the robustness and reproducibility of the method, since the process is highly automated.

A Look into Ocular Diseases: The Pivotal Role of Omics Sciences in Ophthalmology Research.

Precision medicine is a new medical approach which considers both population characteristics and individual variability to provide customized healthcare. The transition from traditional reactive medicine to personalized medicine is based on a biomarker-driven process and a deep knowledge of biological mechanisms according to which the development of diseases occurs. In this context, the advancements in high-throughput omics technologies represent a unique opportunity to discover novel biomarkers and to provide an unbiased picture of the biological system. One of the medical fields in which omics science has started to be recently applied is that of ophthalmology. Ocular diseases are very common, and some of them could be highly disabling, thus leading to vision loss and blindness. The pathogenic mechanism of most ocular diseases may be dependent on various genetic and environmental factors, whose effect has not been yet completely understood. In this context, large-scale omics approaches are fundamental to have a comprehensive evaluation of the whole system and represent an essential tool for the development of novel therapies. This Review summarizes the recent advancements in omics science applied to ophthalmology in the last ten years, in particular by focusing on proteomics, metabolomics and lipidomics applications from an analytical perspective. The role of high-efficiency separation techniques coupled to (high-resolution) mass spectrometry ((HR)MS) is also discussed, as well as the impact of sampling, sample preparation and data analysis as integrating parts of the analytical workflow.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part II: Purification of a therapeutic peptide.

Preparative liquid chromatography in reversed phase conditions (RPLC) is the most common approach adopted in the downstream processing for the purification of therapeutic peptides at industrial level. Due to the strict requirements on the quality imposed by the Regulatory Agencies, routinary methods based on the use of aqueous buffers and acetonitrile (ACN) as organic modifier are commonly used, where ACN is practically the only available choice for the purification of peptide derivatives. However, ACN is known to suffers of many shortcomings, such as drastic shortage in the market, high costs and, most importantly, it shows unwanted toxicity for human health and environment, which led it among the less environmentally friendly ones. For this reason, the selection of a suitable alternative becomes crucial for the sustainable downstream processing of peptides and biopharmaceuticals in general. In this paper, a promising green solvent, namely dimethyl carbonate (DMC) has been used for the separation of a peptide not only in linear conditions but also for its purification through non-linear overloaded chromatography. The performance of the process has been compared to that achievable with the common method where ACN is used as organic modifier and to that obtained with two additional solvents (namely ethanol and isopropanol), already used as greener alternatives to ACN. This proof-of-concept study showed that, thanks to its higher elution strength, DMC can be considered a green alternative to ACN, since it allows to reduce method duration while reaching good purities and recoveries. Indeed, at a target purity fixed to 98.5 %, DMC led to the best productivity with respect to all the other solvents tested, confirming its suitability as a sustainable alternative to ACN for the purification of complex biopharmaceutical products.

Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part I: Separation of small molecules.

Nowadays, environmental problems are drawing the attention of governments and international organisations, which are therefore encouraging the transition to green industrial processes and approaches. In this context, chemists can help indicate a suitable direction. Beside the efforts focused on greening synthetic approaches, currently also analytical techniques and separations are under observation, especially those employing large volumes of organic solvents, such as reversed-phase liquid chromatography (RPLC). Acetonitrile has always been considered the best performing organic modifier for RPLC applications, due to its chemical features (complete miscibility in water, UV transparency, low viscosity etc); nevertheless, it suffers of severe shortcomings, and most importantly, it does not fully comply with Environmental, Health and Safety (EHS) requirements. For these reasons, alternative greener solvents are being investigated, especially easily available alcohols. In this work, chromatographic performance of the most common solvents used in reversed-phase chromatography, i.e., acetonitrile, ethanol and isopropanol, have been compared to a scarcely used solvent, dimethyl carbonate (DMC). The analytes of interest were two small molecules, caffeine and paracetamol, whose kinetics and retention behaviour obtained with the four solvents have been compared, and all contributions to band broadening have been assessed. Results about kinetic performance are very promising, indicating that a small amount (7 % v/v) of DMC is able to produce the same efficiency as a 2.5-times larger ACN volume (18 % v/v), and larger efficiency than alcohols. This paper reports, for the first time, fundamental studies concerning the mass transfer phenomena when DMC is used as an organic solvent in RPLC, and, together with the companion paper, represents the results of a research whose final aim was to discover whether DMC is suitable for chromatographic applications both in linear and preparative conditions.

Investigation of retention behavior of natural cannabinoids on differently substituted polysaccharide-based chiral stationary phases under reversed-phase liquid chromatographic conditions.

The growing popularity of cannabis products and recent legalization of cannabis for recreational purposes have contributed to the increase of the demand for analytical methods able to give a detailed characterization of cannabis samples and derivatives. In this context, one of the aspects that is strongly emerging is about the hazardous potential of uncharacterised minor cannabinoids, including chiral ones, for which achiral potency testing methods currently employed do not give any information. For this reason, there is a growing interest towards the development of liquid chromatographic methods for the enantioseparation of cannabinoids. Much work is needed in this field where one of the major limitations is the lack of optically pure standards. This manuscript reports about the chromatographic behavior of five popular cannabinoids (including the cannabichromene racemate, CBC) on nine immobilised polysaccharide-based chiral stationary phases (CSPs) differently substituted, under reversed phase conditions. Results showed that chemo-selectivity of CSPs is not affected by changes in mobile phase composition, in the range of mobile phase investigated. In addition, the presence of electron withdrawing groups on the CSPs systematically leads to shorter retention times compared to when electron donating groups are present. An application of separation of cannabinoids from a real hemp extract on two of the chiral columns employed in this work revealed the presence of both CBC enantiomers in the sample.

Boosting the enantioresolution of zwitterionic-teicoplanin chiral stationary phases by moving to wide-pore core-shell particles.

A novel zwitterionic-teicoplanin chiral stationary phase (CSP), based on superficially porous particles (SPPs) of 2.7 µm particle diameter and 160 Å pore size, has been prepared and evaluated towards the enantioseparation of important classes of compounds, including chiral drugs, pesticides, and N-derivatized amino acids. The comparison with two analogous CSPs prepared on SPPs with 2.7 and 2.0 µm particle diameter and 90 Å pore size has revealed that the use of large-pore particles allows to dramatically improve both the enantioselectivity and the resolution-per-analysis-time, at the point that the column prepared with the new CSP outperformed the one packed with the finest particles. On the novel wide-pore CSP, the separation of fifteen racemates of pratical importance was significantly improved in terms of both enantioselectivity and resolution-per-analysis time-compared to the CSPs based on SPPs with smaller pores (90 Å). Such a CSP would be suitable for very fast enantioseparations allowing the saving of solvent for greener high-efficiency/high-throughput applications.

Potency testing of cannabinoids by liquid and supercritical fluid chromatography: Where we are, what we need.

Hemp and cannabis industry is undergoing a renewed interest due to legalization of marijuana (a topic that all countries are discussing, especially in recent years) and the growing importance of therapeutic properties of cannabinoids. Together with an increment in the production of hemp and recreational cannabis, there has been an increasing demand for accurate potency testing of products (i.e. quantification of main cannabinoids present in the plant in terms of weight percentage) prior commercialization. This translates in an urgent need of reliable analytical methods to characterize cannabis and hemp samples. Cannabis and hemp preparations are commercialized under various forms (e.g., flowers, oils, candies or even baked goods) usually containing a large number of often very similar compounds making their separation very challenging. Strictly connected to this, another emerging topic concerns the need for the developing of large scale separation techniques for the purification of cannabinoids from complex matrices and for the preparation of analytical-grade standards (including the chiral ones). This paper reviews the most recent achievements in both these aspects. Cutting-edge applications and novel opportunities in potency testing by high performance liquid chromatography (HPLC) with UV detection (which is becoming the golden standard, according to several pharmacopeias, for this kind of measurements) are discussed. The focus has been given to the very important topic of enantio-discrimination of chiral cannabinoids, for which supercritical fluid chromatography (SFC) appears to be particularly suitable. The last part of the work covers the purification of cannabinoids through preparative chromatography. In this regard, particular attention has been given to the most innovative multi-column techniques allowing for the continuous purification of target molecules. The most recent advancements and future challenges in this field are discussed.