Multiple Dual Mode Research Articles

Evaluation of advanced liquid–liquid chromatography operating modes for the tetrahydrocannabinol-remediation of hemp extracts

The presence of tetrahydrocannabinol (THC) in cannabinoid-based materials, such as hemp flower extracts, can pose a severe commercialization burden. In this study, various operating modes of liquid–liquid chromatography (LLC), i.e., batch mode (conventional), trapping multiple dual mode (semi-continuous), and sequential mode (continuous), were evaluated for the THC-remediation of hemp extracts. The biphasic system composition, the feed concentration range, and the maximum flow rate were selected in preliminary experiments. The operating parameters were chosen using short-cut methods, whereas simulations were based on the equilibrium-cell model. Batch elution provided good productivity, easy operation, and yielded a THC-free hemp extract. Trapping multiple dual mode showed a lower productivity than batch, whereas the sequential mode afforded a THC-free extract with a similar composition to the batch mode and offered the advantage of continuous operation and superior productivity. The results of this work constitute a solid basis for the model-based design and optimization of LLC processes for the industrial remediation of hemp extracts.

Liquid-liquid chromatography separation of hemp terpenes with repellent properties against Hyalomma marginatum: A multi-methodological approach

Tick-borne diseases raise serious health concerns for both humans and animals. Tick control and disease prevention largely depend on synthetic acaricides and repellents, often incriminated for high toxicity. To find alternative, safe, and efficient agents against ticks, research on plant-based volatile terpenes constitutes a promising direction. This study aimed to separate and identify hemp flower compounds with repellent properties against Hyalomma marginatum (Acari: Ixodidae). Produced by supercritical CO2 extraction under different pressure (100–110 bar) and temperature (40–60 °C) conditions, the hemp extracts were tested in a moving object bioassay. The extract with the highest repellent power was further subjected to batch liquid-liquid chromatography (LLC). The biphasic solvent system (n-heptane/methanol/acetonitrile 10/5/10, v/v/v) for separating the major hemp terpenes was selected by combining the COSMO-RS-based distribution coefficient screening with experimental validation. Bisabolol (>99%), caryophyllene oxide (>98%), and caryophyllene/humulene (3.5/1, w/w, >95% purity) were thus isolated and assessed in the H. marginatum bioassay. The activity of bisabolol (20% w/w in hexane) was comparable to the synthetic repellent DEET at the same concentration. For the large-scale purification of bisabolol, trapping multiple dual mode, a semi-continuous flow-reversal LLC operating mode, was proposed and evaluated. Bisabolol was obtained with a solvent consumption 2.6-times lower and productivity 2.7-times higher than the batch process. In conclusion, using a multi-methodological computer-aided approach, bisabolol was efficiently identified and isolated as a good tick repellent agent from hemp flowers.

Separation of minor cannabinoids from hemp extract with trapping multiple dual mode liquid-liquid chromatography

Aside from Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), other less common cannabinoids have recently gained an increasing popularity, mostly due to their promising biological potential. However, time-saving and cost-effective methods for their preparative purification are missing. In this study, trapping multiple dual mode (MDM), a flow-reversal liquid-liquid chromatography (LLC) operating mode, was used for the separation of different minor cannabinoids from a hemp extract. Separation task specific biphasic solvent systems were selected for the purification of the target constituents, as follows: n-hexane/methanol/water 10/6.5/3.5 for cannabielsoin (CBE); n-hexane/methanol/water 10/7/3 for cannabidivarin (CBDV) and cannabigerol (CBG); n-hexane/methanol/water 10/8/2 for cannabinol (CBN) and n-hexane/methanol/water 10/9/1 for cannabichromene (CBC) and cannabicylol (CBL). For each separation task, the concentration of the hemp extract in the feed stream and mobile phase flow rate were selected by shake-flask and stationary phase retention experiments, respectively. For the determination of the trapping MDM operating parameters, the short-cut method was implemented and followed by equilibrium-cell model-based simulations. The trapping MDM allowed the separation of the targeted cannabinoids with purities of 93-99%, yields of 73-95%, solvent consumption 2-4-fold lower and productivities almost double than those obtained using batch separation.

Easy Computation of the Various Topologies and Modes of Liquid–Liquid Partition Chromatography by the Theory of Random Walks

The article revisits the discrete recurrence method to model the instruments of liquid–liquid partition chromatography as counter-current chromatography (CCC) and centrifugal partition chromatography (CPC). The purpose is to simplify the computation of the concentration profiles without supplementary approximations, rather by going back to the seminal model of binomial random walks, associated with the stochastic master equation that generates simple discrete recurrence relations. It fits the model of the prototype of liquid–liquid chromatography: the Craig’s apparatus. Three emblematic separation technique group cases are computed in batch injection, batch multiple dual mode (MDM), and continuous injection by the “True Moving Bed” (TMB) in CPC.

Trapping multiple dual mode liquid-liquid chromatography: Preparative separation of nootkatone from a natural product extract

Trapping multiple dual mode (trapping MDM) is a preparative liquid-liquid chromatography (LLC) technique well-suited to difficult separations of intermediately-eluting components from similarly structured impurities. In this demonstrative study, a design approach for high process throughput is applied for the trapping MDM separation of a target component, nootkatone (NK), initially comprising 16.7% of an industrial side stream mixture with over 90 impurities. This design approach, previously developed and validated using ternary mixtures of model solutes, is applied to a complex real mixture for the first time. The approach consists of five steps: (1) determination of the maximum starting mixture concentration for feed preparation; (2) determination of the maximum flow rate for maintenance of the pre-set stationary phase fraction; (3) determination of the partition coefficients of the target and main impurities; (4) selection of step durations and number of cycles using an established short-cut method; (5) execution of the trapping MDM separation. The target, NK, was obtained along with a co-eluting component at 78.7% purity and 84.6% yield, demonstrating the effectiveness of trapping MDM for the separation of intermediately-eluting natural product target components from complex starting mixtures.

Hydrophilic Doped Quantum Dots “Ink” and Their Inkjet‐Printed Patterns for Dual Mode Anticounterfeiting by Reversible Cation Exchange Mechanism

AbstractLuminescent quantum dots (QDs) patterns have attracted great attention in anticounterfeiting applications because they can emit fluorescence upon excitation. Here, different from traditional luminescent patterns for anticounterfeiting purposes, a novel strategy based on the reversible cation exchange enabled Ag doped CdS quantum dots (CdS:Ag QDs) is reported to provide a higher security for anticounterfeiting. First, the reversibility of cation exchange enabled doped QDs crystal engineering have been revealed. The switch from nonluminescence to luminescence for several cycles by such a mechanism has been achieved. Second, the hydrophilic doped QDs “ink” can be prepared by surface modification, and then flexible bulk‐sized patterns can be printed on different substrates by inkjet printing technology, such as the parchment paper, PET foil, and paper currency substrates. Third, owing to the characteristic of stable crystal structure of CdS:Ag QDs, the patterns of such doped QDs on substrates can be switched by in situ cation exchange to achieve conversion of the disappearance and recovery of luminescent signals, from which a multiple dual mode anticounterfeiting verification can be provided.

Operating mode selection for the separation of intermediately-eluting components with countercurrent and centrifugal partition chromatography

Two operating modes that can be used for the separation of intermediately-eluting target components from complex mixtures using solid support-free liquid-liquid chromatography (LLC) include batch injections and trapping multiple dual mode (trapping MDM). Batch injections offer the advantage of simpler equipment and process design, while the trapping MDM process, although more complex, can provide improved resolution at low separation factors. In this study, a thorough comparison of batch injection and trapping MDM performance with respect to productivity is made using simulations for a wide range of separation tasks (target component/impurity separation factors; minimum purity and yield requirements). For each separation task, the maximum productivities obtained by the two operating modes were separately determined with respect to their independent parameters. The results indicate that trapping MDM can be used to extend the application of LLC down to separation factors as low as 1.1, with its use being most advantageous for separation factors of approximately 1.3. However, the simpler batch injection process remains preferable for separation factors around or greater than 1.5.

An easy-to-use calculating machine to simulate steady state and non-steady-state preparative separations by multiple dual mode counter-current chromatography with semi-continuous loading of feed mixtures

Multiple dual mode counter-current chromatography (MDM CCC) separation processes with semi-continuous large sample loading consist of a succession of two counter-current steps: with “x” phase (first step) and “y” phase (second step) flow periods. A feed mixture dissolved in the “x” phase is continuously loaded into a CCC machine at the beginning of the first step of each cycle over a constant time with the volumetric rate equal to the flow rate of the pure “x” phase. An easy-to-use calculating machine is developed to simulate the chromatograms and the amounts of solutes eluted with the phases at each cycle for steady-state (the duration of the flow periods of the phases is kept constant for all the cycles) and non-steady-state (with variable duration of alternating phase elution steps) separations. Using the calculating machine, the separation of mixtures containing up to five components can be simulated and designed. Examples of the application of the calculating machine for the simulation of MDM CCC processes are discussed.

Continuous Synthesis and Purification by Coupling a Multistep Flow Reaction with Centrifugal Partition Chromatography.

Continuous‐flow multistep synthesis is combined with quasi‐continuous final‐product purification to produce pure products from crude reaction mixtures. In the nucleophilic aromatic substitution of 2,4‐difluoronitrobenzene with morpholine followed by a heterogeneous catalytic hydrogenation, the desired monosubstituted product can be continuously separated from the co‐ and by‐products in a purity of over 99 % by coupling a flow reactor sequence to a multiple dual‐mode (MDM) centrifugal partition chromatography (CPC) device. This purification technique has many advantages over HPLC, such as higher resolution and no need for column replacement or silica recycling, and it does not suffer from irreversible adsorption.

Continuous Synthesis and Purification by Coupling a Multistep Flow Reaction with Centrifugal Partition Chromatography

AbstractContinuous‐flow multistep synthesis is combined with quasi‐continuous final‐product purification to produce pure products from crude reaction mixtures. In the nucleophilic aromatic substitution of 2,4‐difluoronitrobenzene with morpholine followed by a heterogeneous catalytic hydrogenation, the desired monosubstituted product can be continuously separated from the co‐ and by‐products in a purity of over 99 % by coupling a flow reactor sequence to a multiple dual‐mode (MDM) centrifugal partition chromatography (CPC) device. This purification technique has many advantages over HPLC, such as higher resolution and no need for column replacement or silica recycling, and it does not suffer from irreversible adsorption.

Trapping multiple dual mode centrifugal partition chromatography for the separation of intermediately-eluting components: Throughput maximization strategy

Trapping multiple dual mode centrifugal partition chromatography (trapping MDM CPC) is an alternative to isocratic pulse injections for the separation of intermediately-eluting components from complex mixtures using liquid-liquid chromatography. In this work, a throughput maximization strategy is developed and validated to investigate the full potential of trapping MDM CPC as a preparative technique. In the proposed approach, shake flask and stationary phase retention experiments are used to determine the maximum feed concentration and flow rate, respectively. A model-based parameter selection process combining a mathematical short-cut method and simulations based on the equilibrium cell model is used to obtain the column loading and step durations resulting in maximized process throughput. The proposed throughput maximization strategy is experimentally validated for the separation of a ternary model mixture of parabens. A preliminary comparison of trapping MDM CPC separation performance to that of stacked pulse injections is also made.

Trapping multiple dual mode centrifugal partition chromatography for the separation of intermediately-eluting components: Operating parameter selection

The preparative separation of intermediately-eluting components in liquid-liquid chromatography is commonly performed with isocratic batch injections, a technique which often leads to low yield and/or purity as a result of peak overlap. Two-column trapping multiple dual mode centrifugal partition chromatography, an alternative discontinuous method for the separation of a mixture into three product fractions (early-, intermediately-, and late-eluting components) at full recovery, is presented in this work. A mathematical shortcut method based on equilibrium theory assumptions is derived for the determination of the key operating parameters (i.e., step durations and number of steps). The feasibility of the technique and the accompanying short-cut method is demonstrated by proof-of-concept experiments for the separation of two paraben model mixtures.

Purification of vandaterosides from Vanda teres (Orchidaceae) by stepwise gradient centrifugal partition chromatography.

Vandaterosides are polar glucosyloxybenzyl eucomate derivatives found in Vanda teres (Orchidaceae), which display biological activities that slow the skin ageing process. In order to obtain larger quantities to allow us to go further in the bioassays, the hydroalcoholic extract of aerial parts (leaves and stems) of V. teres were fractionated by centrifugal partition chromatography, combining isocratic, gradient, and dual elution modes. The first fractionation was performed on the extract maintained in the stationary phase as water saturated in butanol, while increasing the polarity of the mobile phase by changing the proportions of ethyl acetate/1-butanol/water, in order to obtain two enriched fractions. Vandateroside I was then purified by isocratic mode with ethyl acetate/ethanol/water (46:14:40), while vandateroside II was obtained by combining isocratic elution with ethyl acetate/isopropanol/water (30:20:50) followed by a multiple dual mode with ethyl acetate/ethanol/water (46:14:40). In this manner, hundreds of milligrams of vandateroside I and II were recovered from 10 g of V. teres extract.

Steady state preparative multiple dual mode counter-current chromatography: Productivity and selectivity. Theory and experimental verification

In the steady state (SS) multiple dual mode (MDM) counter-current chromatography (CCC), at the beginning of the first step of every cycle the sample dissolved in one of the phases is continuously fed into a CCC device over a constant time, not exceeding the run time of the first step. After a certain number of cycles, the steady state regime is achieved, where concentrations vary over time during each cycle, however, the concentration profiles of solutes eluted with both phases remain constant in all subsequent cycles. The objective of this work was to develop analytical expressions to describe the SS MDM CCC separation processes, which can be helpful to simulate and design these processes and select a suitable compromise between the productivity and the selectivity in the preparative and production CCC separations. Experiments carried out using model mixtures of compounds from the GUESSmix with solvent system hexane/ethyl acetate/methanol/water demonstrated a reasonable agreement between the predictions of the theory and the experimental results.

Multiple dual mode counter-current chromatography with periodic sample injection: Steady-state and non-steady-state operation

The multiple dual mode (MDM) counter-current chromatography (CCC) separation consists of a succession of two isocratic counter-current steps and is carried out in series alternating between normal phase and reversed phase operation. The performance of the MDM technique can be improved by the sample re-injection between each of the dual-mode steps. The objective of this work was to develop analytical expressions to describe the MDM CCC with periodic sample injection, which can be used to simulate these processes and select optimal operating conditions for the separation of a given feed mixture. Two possible methods of the MDM separation with periodic sample injection are considered: 1 – steady-state separation: the duration of the flow periods of the phases is kept constant for all the cycles and the steady-state regime is achieved after a certain number of cycles. 2 – non-steady-state separation with variable duration of phase elution steps. It is shown that proper selection of the duration of phase flow times allows to reach complete separation of solutes in continuous steady-state operation mode even in a low efficiency column. This mode of operation provides both high productivity and high resolution. The non-steady-state method with variable duration of phase elution steps offers several options to split a mixture into groups of compounds and/or concentrate target compounds.

Multiple dual mode counter-current chromatography with variable duration of alternating phase elution steps

The multiple dual mode (MDM) counter-current chromatography separation processes consist of a succession of two isocratic counter-current steps and are characterized by the shuttle (forward and back) transport of the sample in chromatographic columns. In this paper, the improved MDM method based on variable duration of alternating phase elution steps has been developed and validated. The MDM separation processes with variable duration of phase elution steps are analyzed. Basing on the cell model, analytical solutions are developed for impulse and non-impulse sample loading at the beginning of the column. Using the analytical solutions, a calculation program is presented to facilitate the simulation of MDM with variable duration of phase elution steps, which can be used to select optimal process conditions for the separation of a given feed mixture. Two options of the MDM separation are analyzed: 1 – with one-step solute elution: the separation is conducted so, that the sample is transferred forward and back with upper and lower phases inside the column until the desired separation of the components is reached, and then each individual component elutes entirely within one step; 2 – with multi-step solute elution, when the fractions of individual components are collected in over several steps. It is demonstrated that proper selection of the duration of individual cycles (phase flow times) can greatly increase the separation efficiency of CCC columns. Experiments were carried out using model mixtures of compounds from the GUESSmix with solvent systems hexane/ethyl acetate/methanol/water. The experimental results are compared to the predictions of the theory. A good agreement between theory and experiment has been demonstrated.

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter-current chromatography in chiral separations. Two aromatic acids, 2-(6-methoxy-2-naphthyl)propionic acid (NAP) and 2-phenylpropionic acid (2-PPA), were enantioseparated by MDM counter-current chromatography using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phase solvent systems consisting of n-hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP-β-CD (7.5:2.5:10 for NAP and 7:3:10 for 2-PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first-phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2-PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter-current chromatography, MDM elution greatly improved peak resolution in chiral separations.

Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography

Commercial samples of Coomassie Brilliant Blue G-250 (CBB) were not pure enough to give reliable results when used as indicator of amine content in biological material. The polar and apolar impurities produce unacceptable biases in the results. Counter current chromatography (CCC) was used to purify significant amounts of CBB. The liquid system heptane/1-butanol/water 2:3:4 (v/v) was appropriate to separate crude CBB in three groups of components: polar, partitioning in the aqueous lower phase, intermediate, partitioning well between the aqueous and organic phases, and apolar, preferring greatly the organic phase. The dual-mode way of using a CCC chromatograph was found appropriate for the separation injecting the crude CBB in the middle of a two coil CCC instrument. A multi dual-mode purification was performed allowing to eliminate the polar impurities in the aqueous phase at the column tail and the apolar ones in the organic phase at the column head, trapping the purified dye inside the CCC column. 200mg of purified CBB were obtained from 1g of crude CBB in 3h using as little as 150mL of butanol and 70mL of heptane with 200mL of water. The purified CBB gave total satisfaction in testing amine content in polyclonal antibody containing monolith pipettes.

Multiple dual-mode countercurrent chromatography applied to chiral separations using a ( S)-naproxen derivative as chiral selector

Countercurrent chromatography (CCC) is a liquid–liquid chromatographic technique without a solid support. Several alternative elution modes can be applied to take advantage of the special nature of the liquid stationary phase. Among these dual-mode (DM) and multiple dual-mode (MDM) consist of switching alternatively between Reversed and Normal Phase operation during the experiment (once for DM and several times for MDM). In this paper, MDM has been applied to the chiral CCC separations of two racemic mixtures, (±)- N-(3,4- cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide and N-(3,5-dinitrobenzoyl)-(±)-leucine, using ( S)-naproxen N, N-diethylamide as chiral selector (CS). Although the behaviour of the two analytes differed, improved resolution factors were successfully obtained. Results are rationalized on the basis of the distinct partition behaviour of the CS/enantiomer complexes in the biphasic system.

Multiple dual-mode centrifugal partition chromatography as an efficient method for the purification of a mycosporine from a crude methanolic extract of Lichina pygmaea

Centrifugal partition chromatography method was applied to the separation and purification of a crude methanolic extract of a cyanobacterial lichen, Lichina pygmaea. A multiple dual-mode was used to separate two compounds of interest, namely mycosporine-serinol and a glutamic acid derivative. These compounds are described here for the first time in a lichen. Their structures were identified by UV, IR, ESI-MS, 1H NMR, 13C NMR, and 2D NMR.