Multiphase flow modeling in centrifugal partition chromatography

Correlating physical properties of aqueous-organic solvent systems and stationary phase retention in a centrifugal partition chromatograph in descending mode

Centrifugal partition chromatography (CPC) is an emerging separation technology in pharmaceutical and natural product purifications, reaching recently industrial-scale solutions. Because both the stationary and mobile phases are liquids in CPC, the solvent consumption of such separation processes is considered significant. Thus, automation is highly required in the preparation and recycling of the solvent mixtures for economical (i.e., continuous) CPC operation. Here, we report a feasible solution for this industrial issue, including the concept, algorithm, and instrumentation of solvent system handling. For majority of ternary solvent systems used in CPC separations, the linear correlation between the density and composition of the phases was recognized and utilized during the continuous solvent recycling. Hence, an efficient density-based composition adjustment algorithm was established in a mixer-settler unit using Coriolis flow meters for precise density monitoring of the upper and lower phases of the biphasic liquid system (BLS). In addition, a complete cascade consisting of a buffer, waste, a recycler (evaporator), and mixer-settler units was designed around the industrial-scale CPC device. The proof of concept was demonstrated by sequential industrial-scale CPC separations of a binary model mixture in the n-hexane/methanol/water (5/4/1, v/v/v) solvent system, followed by the purification of a crude steroid active pharmaceutical ingredient performed in the methyl isobutyl ketone/acetone/water (2/2/1, v/v/v) solvent system. The reproducibility of chromatographic performance and the productivity of the mixer-settler and evaporator units were satisfactory, enabling a robust and continuous operation of the developed separation process.

A water-free solvent system containing an L-menthol-based deep eutectic solvent for centrifugal partition chromatography applications

Countercurrent chromatography, based on liquid-liquid partitioning, has many technological variants. One of them is centrifugal partition chromatography, introduced by Wataru Murayama and Kanichi Nunogaki in 1982. This technique, like other countercurrent chromatography techniques, is based on the phenomenon of liquid-liquid partitioning between two immiscible liquid phases that stay at equilibrium. But the significant difference between this technique and others is the retention mechanism of stationary phase. In the case of centrifugal partition chromatography, this mechanism is based on hydrostatic force, formed by the centrifugal field in the rotor in one-axis centrifuge. Sometimes that allows more control of stationary phase, for example, when aqueous two-phase and other difficult solvent systems are used. However, the efficiency of the separation in centrifugal partition chromatography is also affected by a variety of parameters dependent on the sample properties in the solvent system, physical properties of the solvent system, parameters of the instrument, and the method. This article includes also recent ideas for improvements to the technique and broadening its application (e.g., (multiple) dual-mode or elution-extrusion procedure, pH-zone-refining centrifugal partition chromatography, ion-exchange centrifugal partition chromatography, online and offline coupling of centrifugal partition chromatography).

Influence of temperature on the separation performance in solid support-free liquid-liquid chromatography

Obtaining an ideal solvent system for target compounds is still an obstacle to the wide application of high-speed counter-current chromatography (HSCCC). The partition coefficient and retention of the stationary phase are two key parameters for solvent system selection. The retention of the stationary phase of the solvent system is roughly judged by settling time using a test tube, which is subjective and inaccurate. In this study, we demonstrated that high-resolution separation of HSCCC is tightly connected with the retention of the stationary phase. Notably, unlike the in vitro test of settling time, we investigated the retention of the stationary phase of classical biphasic solvent systems by a TBE300C HSCCC apparatus. Our results revealed that settling time is not always inversely proportional to the retention of the stationary phase. The n-hexane–ethylacetate–methanol–water solvent systems showed the highest correlation coefficient of settling time and retention of the stationary phase (r = −0.91, n = 16). N-heptane–n-butanol–acetonitrile–water solvent system showed the lowest correlation coefficient (r = −0.26, n = 7). These results may be helpful for HSCCC solvent system selection and accelerate the application of this technique.

Mass transfer studies in shallow bubble column reactors

Cellulose was chemically modified with hydrophobic dodecanoyl groups followed by 3,5-dimethylphenylcarbamoyl substituents forming mixed ester/carbamate derivatives in order to improve the solubility in lipophilic solvents compared to the corresponding homosubstituted cellulose tris(3,5-dimethylphenylcarbamate). Two mixed derivatives of different degree of substitution were prepared, tested as chiral selectors (CSs) in counter-current chromatography (CCC) and compared to the homo-substituted derivative. Alternatively, cellulose tris(3,5-dichlorophenylcarbamate), was synthesised and tested with the same purpose. The racemic drugs pindolol and warfarin were used as test compounds. Biphasic organic/aqueous solvent systems, composed of methyl isobutyl ketone, t-butyl methyl ether or ethyl acetate and aqueous ammonium acetate or sodium phosphate buffer, were used. Centrifugal partition chromatography, a variety of CCC, in the classical elution mode and the pH-zone-refining displacement mode was applied. The enantioseparation of pindolol and warfarin was achieved in the latter conditions. The presence of dodecanoyl chains on the CS increased solubility in the organic solvents used. The selectivity of the mixed dodecanoyl/3,5-dimethylphenylcarbamoyl cellulose derivative with low degree of dodecanoyl groups (degree of substitution 0.3/degree of substitution 2.6, respectively) was similar to that of the homo-substituted derivative. Furthermore, the loading capacity for pindolol was increased by a factor of three compared to cellulose tris(3,5-dimethylphenylcarbamate). Nevertheless, the increasing degree of substitution with dodecanoyl groups on the CS, although improved solubility in the stationary phase, contributed negatively to the enantioselectivity, where warfarin was more affected than pindolol.

A high-performance thin-layer chromatographic methodology was developed and validated for the isolation and quantitative determination of oleuropein in two extracts of Olea europaea leaves. OLE_A was a crude acetone extract, while OLE_AA was its defatted residue. Initially, high-performance thin-layer chromatography was employed for the purification process of oleuropein with fast centrifugal partition chromatography, replacing high-performance liquid-chromatography, in the stage of the determination of the distribution coefficient and the retention volume. A densitometric method was developed for the determination of the distribution coefficients, KC = CS/CM. The total concentrations of the target compound in the stationary phase (CS) and in the mobile phase (CM) were calculated by the area measured in the high-performance thin-layer chromatogram. The estimated Kc was also used for the calculation of the retention volume, VR, with a chromatographic retention equation. The obtained data were successfully applied for the purification of oleuropein and the experimental results confirmed the theoretical predictions, indicating that high-performance thin-layer chromatography could be an important counterpart in the phytochemical study of natural products. The isolated oleuropein (purity > 95%) was subsequently used for the estimation of its content in each extract with a simple, sensitive and accurate high-performance thin-layer chromatography method. The best fit calibration curve from 1.0 µg/track to 6.0 µg/track of oleuropein was polynomial and the quantification was achieved by UV detection at λ 240 nm. The method was validated giving rise to an efficient and high-throughput procedure, with the relative standard deviation % of repeatability and intermediate precision not exceeding 4.9% and accuracy between 92% and 98% (recovery rates). Moreover, the method was validated for robustness, limit of quantitation, and limit of detection. The amount of oleuropein for OLE_A, OLE_AA, and an aqueous extract of olive leaves was estimated to be 35.5% ± 2.7, 51.5% ± 1.4, and 12.5% ± 0.12, respectively. Statistical analysis proved that the method is repeatable and selective, and can be effectively applied for the estimation of oleuropein in olive leaves' extracts, and could potentially replace high-performance liquid chromatography methodologies developed so far. Thus, the phytochemical investigation of oleuropein could be based on high-performance thin-layer chromatography coupled with separation processes, such as fast centrifugal partition chromatography, showing efficacy and credibility.

Centrifugal partition chromatography (CPC) was successfully used for isolation of three lignan compounds such as phyllanthin, hypophyllanthin, and niranthin from Phyllanthus amarus by using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (2:1:2:1, v/v/v/v) (Arizona R solvent system) in ascending mode. In CPC method, the 250 mL CPC column was filled with the lower stationary phase at a flow rate of 30 mL/min with a maintaining rotation speed at 500 rpm. Then, the upper mobile phase was pumped into the column at a flow rate of 8 mL/min and the rotation speed was set to 1600 rpm. From 0.10 (g) of the crude lignan extract, phyllanthin (1, 25.3 mg, 92.8% purity), hypophyllanthin (2, 7.4 mg, 88.9% purity), and niranthin (3, 14.8 mg, 94.5% purity) were obtained by a single CPC run. The isolated substances were identified by ESI-MS and comparison of retention times with standards.

Effect of NAPL film stability on the dissolution of residual wetting NAPL in porous media: A pore-scale modeling study

In a WFGD spray column, spraying droplets have dual duties of optimizing flow uniformity and providing interfacial area for mass transfer but have contradiction in selection of diameter. In this paper, a synergistic effect between smaller and bigger droplets is proposed to improve the performance of the column, which is to enlarge the interfacial area and optimize the flow uniformity in different layers, and it is used to create a novel design scheme. Through the study of the flow uniformity in the spray column, the detail of this design scheme is proposed as followed: The minimum flow rate and proper droplet diameter should be selected in the bottom layer to achieve the flow uniformity, and smaller droplets can be used in the rest layer to enlarge the interfacial area as much as possible. Compared with the traditional scheme, the liquid hold‐up has increased 16%–30%, and the interfacial area has increased 32%–56% in novel scheme. In practice, this novel design scheme can provide reference and guidance for large scale spray columns.

Study question Can ooplasm streaming (OS) velocity anticipate the fertilization outcome before the time for pronuclei appearance (tPNa) and infer the ultrastructural cytoskeleton profile of failed-to-fertilize oocytes? Summary answer Changes in OS-velocity post-ICSI identify normally and failed-to-fertilize (FF) oocytes before tPNa, and reveal an association between specific OS-velocities and different fertilization failure patterns. What is known already The ooplasm is a highly dynamic and fluidic milieu, which enables an efficient redistribution of its components in preparation for fertilization and the subsequent ultrastructural reorganization. Particle Image Velocimetry (PIV) has been employed to study the OS behaviour during oocyte activation and changes in velocity and directionality have been associated with the calcium oscillations triggered by a fertilizing sperm. These observations suggest that the ooplasm undergoes meaningful dynamic shifts during the process of fertilization. The question remains how OS swings in the event of failed fertilization, and whether PIV analysis could anticipate this outcome and shed light on the interpretation. Study design, size, duration This retrospective analysis includes 101 FF-oocytes from 22 patients experiencing ICSI failures (mean fertilization rate 31,4±13,5%). FF-oocytes underwent immunodetection to assess meiotic spindle, sperm nucleus and flagellum configurations. FF-oocytes were grouped based on the sperm nucleus configuration: Intact head, Decondensed nucleus, and Premature chromosome condensation (PCC). The absence or the presence of the second polar body (PB) was annotated revisiting time-lapse videos. PIV analysis was applied to FF-oocytes (Study group) and diploid/euploid zygotes (Control group). Participants/materials, setting, methods For immunocytochemistry (ICC) studies we used Hoechst (DNA), alpha-tubulin (maternal spindle) and acetylated-alpha-tubulin (sperm flagellum) monoclonal antibodies. Microinjected MII-oocytes were cultured under hypoxia (Geri; Genea-Biomedx, AUS) using G-TL (Vitrolife, SWE). PIV analysis (v3.08PIVlab, MATLAB) determined the average OS-velocity (µm/s) per hour post-ICSI (hpi). Further, we calculated the velocity change rate (%) from 1hpi to 8hpi, to compare amongst grouped FF-oocytes and the controls. Main results and the role of chance A total of 87 FF-oocytes (86,1%) were informative after ICC. Informative FF-oocytes were grouped following their sperm nucleus profiles: Intact-head (n = 9; 1stPB 11,1%; 2ndPB 88,9%); Decondensed-nucleus (n = 13; 1stPB 61,5%; 2ndPB 38,5%); PCC (n = 53; 1stPB 39,6%; 2ndPB 60,4%); Other patterns (n = 12). This last group (other patterns) and non-stationary time-lapse images (n = 21) were excluded from PIV analysis. Control-oocytes (n = 14) had an OS-velocity (1hpi) of 9,8±0,7 µm/s, declining by 48% at 8hpi. Intact-Head FF-oocytes (n = 7) showed an OS-velocity (1hpi) of 9,8±0,8 µm/s, with a 16% decrease at 8hpi. Decondensed-nucleus FF-oocytes (n = 7) had an initial OS-velocity of 10,6±0,9 µm/s, declining by 7% at 8hpi. PCC FF-oocytes (n = 40) showed an OS-velocity at 1hpi of 8,8±1,0 µm/s, decreasing by 25% at 8hpi. The results reveal a decline in the OS velocity over time, which is significantly slower in FF-oocytes than in controls (p-value <0,05). The velocity change rate showed significant differences from 4hpi between Intact-Head and Decondensed-Nucleus FF-oocytes and the controls, and from 6hpi in PCC FF-oocytes. These data suggest an association between OS velocities and specific ultrastructural cytoskeleton profiles. No differences were observed in PIV when sub-analysing PCC FF-oocytes by the 2ndPB extrusion. Low sample sizes of Head-Intact and Decondensed-nucleus FF-oocytes restricted similar subgroupings. Limitations, reasons for caution This study is a cohort analysis based on a limited sample size; therefore, the results should be interpreted with caution. Prospective studies are required to validate the present observations. PIV analysis only applies to oocytes that remain focused and stationary within the well during incubation. Wider implications of the findings To the best of our knowledge, this is the first study to establish a correlation between failed-to-fertilize immunodetection data, time-lapse imaging, and PIV analysis. Our findings suggest that PIV analysis could serve as a non-invasive method to infer the underlying causes of fertilization failure after ICSI. Trial registration number No

A new prototype, large‐scale countercurrent chromatograph was constructed. It was equipped with a three‐layer spiral column made from a continuous piece of convoluted Teflon tubing (8.5 mm I.D. and 9.7 m in length), which was accommodated in a spirally carved foam plastic holder. Two different types of two‐phase solvent systems were used to evaluate stationary phase retention and mixing effect between stationary and mobile phases, i.e., chloroform/water (1:1, v/v) for separation of caffeine and theophylline; and 1‐butanol/acetic acid/water (4:1:5, v/v/v) for separation of dipeptides, valyl‐tyrosine (val‐tyr), and tryptophyl‐tyrosine (trp‐tyr). A set of different elution modes (inside–outside and head–tail), and rotational speed (0–400 rpm) was assessed for stationary phase retention and separation efficiency. The elution mode of inside to outside of the spiral for the lower mobile phase or the opposite direction for the upper mobile phase, is rated the best for stationary phase retention due to observed spiral effect for liquid movement. Different operational parameters, such as rpm and flow rate of the mobile phase were evaluated for separation efficiency and speed. The obtained data revealed that the spiral effect and Archimedean screw force were two important contributing factors for yielding the enhanced speed and higher partition efficiency, compared to conventional low‐speed rotary countercurrent chromatography.

Cross-axis synchronous flow-through coil planet centrifuge (type XLL) : I. Design of the apparatus and studies on retention of stationary phase