Journal of Chromatography A

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Journal of Chromatography A

http://rss.sciencedirect.com/publication/science/5248

Selected papers from the latest issue:

Relative hydrophobicity between the phases and partition of cytochrome-c in glycine ionic liquids aqueous two-phase systems

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Changzeng Wu , Jianji Wang , Zhiyong Li , Jun Jing , Huiyong Wang
In this work, glycine ionic liquids tetramethylammonium glycine ([N1111][Gly]), tetraethylammonium glycine ([N2222][Gly]), tetra-n-butylammonium glycine ([N4444][Gly]), tetra-n-butylphosphonium glycine ([P4444][Gly]) and tetra-n-pentylammonium glycine ([N5555][Gly]) were synthesized and used to prepare aqueous two-phase systems (ATPSs) in the presence of K2HPO4. Binodal curves of such ATPSs and partition coefficients of a series of dinitrophenylated (DNP) amino acids in these ATPSs were determined at 298.15K to understand the effect of cationic structure of the ionic liquids on the phase-forming ability of glycine ionic liquids, relative hydrophobicity between the phases in the ionic liquids ATPSs, and polarity of the ionic liquids-rich phases. With the attempt to correlate the relative hydrophobicity of the phases in the ATPSs with their extraction capability for proteins, partition coefficients of cytochrome-c in the ATPSs were also determined. It was shown that partition coefficients of cytochrome-c were in the range from 2.83 to 20.7 under the studied pH conditions. Then, hydrophobic interactions between cytochrome-c and the ionic liquid are suggested to be the main driving force for the preferential partition of cytochrome-c in the glycine ionic liquid-rich phases of the ATPSs. Result derived from polarity of the ionic liquids-rich phases supports this mechanism.

Continuous protein purification using functionalized magnetic nanoparticles in aqueous micellar two-phase systems

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Ingo Fischer , Chia-Chang Hsu , Markus Gärtner , Christine Müller , Tim W. Overton , Owen R.T. Thomas , Matthias Franzreb
A novel technique for technical-scale continuous purification of proteins is presented. It is based on the combined use of functionalized magnetic nano-particles and an Aqueous Micellar Two-Phase System featuring the non-ionic surfactant, Eumulgin ES, which undergoes temperature induced phase separation at ∼25°C. In the first step, conducted below the transition temperature (i.e. 15°C), the magnetic sorbent particles are added into the single dispersed phase and bind the protein of interest. Next, on raising the temperature to 30°C the protein-laden magnetic particles partition strongly into the micelle-rich top phase of the micellar two-phase system that's formed. The magnetically susceptible top phase is then continuously separated from the micelle-poor phase in a flowthrough tailor-made magnetic extractor featuring a permanent magnet providing an upwardly acting magnetic force. This separation device was shown to be effective for continuous separation of a wide range of differently sized magnetic particle sorbents (i.e. from 2μm diameter to as small as 25nm) from a 10% (w/w) Eumulgin ES system; high separation efficiencies were recorded for the phase-forming surfactant (87 to >98%), and all magnetic sorbent particles tested (95–99.9%). Finally, protein purification by continuous magnetic extraction was demonstrated at 15L scale for the recovery of an antibody fragment, A33 Fab′, from a crude extract of Escherichia coli periplasm. Nearly 70% of the A33 Fab′ initially present in the extract at 15.6% of the total protein content was recovered in a 2-fold concentrated and highly purified (>98%) state. Further, the amounts of magnetic sorbent and phase-forming surfactant lost in the process were very small; thus recycling of both components into subsequent rounds of continuous magnetic extraction is highly feasible.

Hollow fiber liquid-liquid-liquid microextraction combined with high performance liquid chromatography-ultraviolet detection for the determination of various environmental estrogens in environmental and biological samples

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Beibei Chen , Yunlin Huang , Man He , Bin Hu
A new method of hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) combined with high performance liquid chromatography-ultraviolet detection (HPLC-UV) was developed for simultaneous analysis of various environmental estrogens. Taking eight environmental estrogens (17β-estradiol (E2), estrone (E1), estriol (E3), 17α-ethynylestradiol (EE), diethylstilbestrol (DES), dienestrol (DIS), bisphenol-A (BPA) and 4-t-octylphenol (OP)) as the target analytes, the factors affecting HF-LLLME-HPLC-UV were investigated in detail. Under the optimized experimental conditions, the extraction efficiency of HF-LLLME for eight target estrogens was in the range of 13.9–62.8%, and the enrichment factor was 88–376-fold. The limits of detection (LODs) of the proposed method for eight target estrogens of ES, BPA, E2, EE, E1, DES, DIS and OP were 0.11, 0.055, 0.66, 0.55, 0.51, 0.20, 0.11 and 1.46ng/mL, respectively, which are lower than that obtained by some other sample pretreatment methods followed by HPLC-UV analysis. The relative standard deviations of the method (RSDs, c E3, BPA, DIS =5ng/mL, c E1, E2, EE, OP =40ng/mL, c DES =15ng/mL, n =7) are in the range of 5.5–8.4%. The developed method was applied for the determination of environmental estrogens in environmental and biological samples (sediment and fish), and none of the eight target estrogens was detected. The recoveries for the spiking samples with low, medium and high spiking levels were in the range of 73.2–117.5%. The proposed method has been demonstrated to be suitable for simultaneous determination of multiple environmental estrogens with high sensitivity, high enrichment factors, good sample clean-up ability and no derivatization required.

Fast and easy extraction combined with high resolution-mass spectrometry for residue analysis of two anticonvulsants and their transformation products in marine mussels

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): M.J. Martínez Bueno , C. Boillot , H. Fenet , S. Chiron , C. Casellas , E. Gómez
Environmental field studies have shown that carbamazepine (Cbz) is one of the most frequently detected human pharmaceuticals in different aquatic compartments. However, little data is available on the detection of this substance and its transformation products in aquatic organisms. This study was thus mainly carried out to optimize and validate a simple and sensitive analytical methodology for the detection, characterization and quantification of Cbz and oxcarbazepine (Ox), two anticonvulsants, and six of their main transformation products in marine mussels (Mytilus galloprovincialis). A modified QuEChERS extraction method followed by analysis with liquid chromatography coupled to high resolution mass spectrometry (HRMS) was used. The analyses were performed using two-stage fragmentation to reveal the different fragmentation pathways that are highly useful for the identification of isomeric compounds, a common problem when several transformation products are analyzed. The developed analytical method allowed determination of the target analytes in the lower ng/g concentration levels. The mean recovery ranged from 67 to 110%. The relative standard deviation was under 11% in the intra-day and 18% in the inter-day analyses, respectively. Finally, the method was applied to marine mussel samples collected from Mediterranean Sea cultures in southeastern France. Residues of the psychiatric drug Cbz were occasionally found at levels up to 3.5ng/g dw. Lastly, in this study, other non-target compounds, such as caffeine, metoprolol, cotinine and ketoprofen, were identified in the real samples analyzed.

Analysis of beer volatiles by polymeric imidazolium-solid phase microextraction coatings: Synthesis and characterization of polymeric imidazolium ionic liquids

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Jaime González-Álvarez , Domingo Blanco-Gomis , Pilar Arias-Abrodo , Jairo Pello-Palma , Nicolás Ríos-Lombardía , Eduardo Busto , Vicente Gotor-Fernández , María Dolores Gutiérrez-Álvarez
Two polymeric ionic liquids, 3-(but-3″-en-1″-yl)-1-[2′-hydroxycyclohexyl]-1H-imidazol-3-ium bis(trifluoromethanesulfonyl)imide (IL-1) and 1-(2′-hydroxycyclohexyl)-3-(4″-vinylbenzyl)-1H-imidazol-3-ium bis(trifluoromethylsulfonyl)imide (IL-2), have been synthesized by a free radical polymerization reaction and used as coatings for solid-phase microextraction (SPME). These new fibers exhibit good film stability, high thermal stability (270–290°C) and long lifetimes, and are used for the extraction of volatile compounds in lemon beer using gas chromatography separation and flame ionization detection. The scanning electron micrographs of the fiber surface revealed a polymeric ionic liquid (PIL) film, which is distributed homogeneously on the fiber. The developed PIL fiber showed good linearity between 50 and 2000μg/L with regression coefficients in the range of 0.996–0.999. The relative standard deviations (RSD) obtained in the peak area were found to vary between 1% and 12%, which assured that adequate repeatability was achieved. The spiked recoveries for three beer samples ranged from 78.4% to 123.6%. Experimental design has been employed in the optimization of extraction factors and robustness assessment. The polymeric IL-1 butenyl fiber showed a greater efficiency compared to the PDMS-DVB (65μm) and CAR-PDMS (75μm) for the extraction of all of the analytes studied.

A comparison of essential oils obtained from lavandin via different extraction processes: Ultrasound, microwave, turbohydrodistillation, steam and hydrodistillation

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Sandrine Périno-Issartier , Christian Ginies , Giancarlo Cravotto , Farid Chemat
A total of eight extraction techniques ranging from conventional methods (hydrodistillation (HD), steam distillation (SD), turbohydrodistillation (THD)), through innovative techniques (ultrasound assisted extraction (US-SD) and finishing with microwave assisted extraction techniques such as In situ microwave-generated hydrodistillation (ISMH), microwave steam distillation (MSD), microwave hydrodiffusion and gravity (MHG), and microwave steam diffusion (MSDf)) were used to extract essential oil from lavandin flowers and their results were compared. Extraction time, yield, essential oil composition and sensorial analysis were considered as the principal terms of comparison. The essential oils extracted using the more innovative processes were quantitatively (yield) and qualitatively (aromatic profile) similar to those obtained from the conventional techniques. The method which gave the best results was the microwave hydrodiffusion and gravity (MHG) method which gave reduced extraction time (30min against 220min for SD) and gave no differences in essential oil yield and sensorial perception.

Polymeric beads containing Cyanex 923 for actinide uptake from nitric acid medium: Studies with uranium and plutonium

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): R.B. Gujar , D. Shanthana Lakshmi , A. Figoli , P.K. Mohapatra
Conventional phase inversion technique has been successfully applied for the preparation of the solid phase extractant (SPE), Cyanex 923 loaded polymer beads. Two types of polymer beads prepared by blending Polyetherether ketone with card (PEEKWC)/DMF with 5% Cyanex 923 (SPE-I, av bead size: 900μm) and 10% Cyanex 923(SPE-II, av. bead size: 1100μm) were evaluated for the uptake of actinide ions. The polymer beads were characterized by various physical methods such as thermal analysis, surface morphology analysis by SEM, EDAX techniques, etc. The polymer beads were used for the experiments involving the uptake of both U(VI) and Pu(IV) at tracer scale and U(VI) at milli molar concentrations from nitric acid feeds. The actinide ion uptake studies involved kinetics of metal ion sorption, adsorption isotherms, and column studies. The metal sorption capacities for U(VI) at 3M HNO3 were found to be 38.8±1.9mg and 54.5±1.7mg per g of SPE-I and SPE-II, respectively. The sorption isotherm analysis with Langmuir, D–R and Freundlisch isotherms indicated chemisorption monolayer mechanism. Column studies were also carried out using 4.5mL bed volume columns containing about 0.4 and 0.45g of SPE-I and SPE-II, respectively. The breakthrough profiles were obtained for U(VI) and the elution profiles were obtained using 1M Na2CO3 as the eluent.

Isolation of monoclonal antibody from a Chinese hamster ovary supernatant. I: Assessment of different separation concepts

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Wojciech Marek , Renata Muca , Sylwia Woś , Wojciech Piątkowski , Dorota Antos
The performance of different non-affinity purification techniques commonly used for isolating CHO derived monoclonal antibodies has been investigated. Ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), aqueous-two-phase extraction (ATPE) and their integration has been evaluated in terms of yield and purity of the product obtained. The integration of chromatographic techniques comprised two steps, in which the CHO supernatant was directly injected into the IEC column to capture monoclonal IgG1 and then the isolated fraction was processed using the HIC column. To reduce the influence of the feed media on retention of the target protein, the feed mixture was on-column diluted by use of the multi-injection technique. In the coupled process of extraction and chromatography the ATPE operation was used for the pre-purification of the supernatant as well as for buffer exchange. The bottom ATPE phase containing the target protein was further purified on the HIC column without feed dilution. The influence of operating conditions on the effectiveness of different purification processes has been evaluated. The best performance with respect to the product purity was achieved for the coupled process of IEC and HIC. The experimental data acquired were exploited in subsequent investigations for determining underlying kinetic parameters and for the process prediction and optimization.

Isolation of monoclonal antibody from a Chinese hamster ovary supernatant. II: Dynamics of the integrated separation on ion exchange and hydrophobic interaction chromatography media

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Wojciech Marek , Renata Muca , Sylwia Woś , Wojciech Piątkowski , Dorota Antos
Dynamics of the purification process of a CHO derived monoclonal antibody by ion exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and their integration has been investigated. To quantify the adsorption behavior of the target protein (IgG1) and impurities contained in the supernatant, their elution course on IEC and HIC columns has been analyzed versus pH and/or the salt concentration in the mobile phase. A short-cut method has been proposed for mathematical modeling and determining underlying kinetic and thermodynamic parameters. The accuracy of the model predictions has been verified by comparing the simulated and experimental band profiles recorded in both chromatographic processes. After verification, the model was used to optimize operating conditions for the column loading and chromatographic elution in the integrated process IEC/HIC. Two alternative loading techniques based on the upstream and downstream feed dilution were taken into account in the optimization routine. In the first one the feed stream was diluted with the loading buffer prior to the column loading, while in the latter one the feed dilution was realized inside the column using the multiple-injection technique. It was shown that the downstream dilution allowed significant reduction of the contact time between the protein and the loading buffer.

Protein adsorption to poly(ethylenimine)-modified Sepharose FF: I. A critical ionic capacity for drastically enhanced capacity and uptake kinetics

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Lin-Ling Yu , Shi-Peng Tao , Xiao-Yan Dong , Yan Sun
To explore the details of protein uptake to polymer-grafted ion exchangers, Sepharose FF was modified with poly(ethylenimine) (PEI) to prepare anion exchanger of 10 different ionic capacities (ICs, 100–1220mmol/L). Adsorption equilibria and kinetics of bovine serum albumin (BSA) were then studied. It is found that ionic capacity, i.e., the coupling density of PEI, had significant effect on both adsorption capacity (q m ) and effective protein diffusivity (D e ). With increasing ionic capacity, the q m value increased rapidly at IC<260mmol/L and then increased slowly till reaching a plateau at IC=600mmol/L. In the IC range of 100–600mmol/L, however, the D e values kept at a low level (D e /D 0 <0.07); it first decreased from 0.05±0.01 at IC=100mmol/L to 0.01±0.01 at IC=260mmol/L and then increased to 0.06±0.01 at IC=600mmol/L. Thereafter, sharp increases of the q m and D e values [36% (from 201 to 273mg/mL) and 670% (from 0.06±0.01 to 0.49±0.04), respectively] were observed in the narrow range of IC from 600 to 740mmol/L. Finally, at IC>740mmol/L, the q m value decreased significantly while the D e value increased moderately with increasing the IC. The results indicate that PEI chains played an important role in protein adsorption and transport. In brief, there was a critical IC (cIC) or PEI chain density, above which protein adsorption and transport behaviors changed drastically. The cIC was identified to be about 600mmol/L. Estimation of PEI grafting-layer thickness suggests that PEI chains formed an extended three-dimensional grafting-layer at IC>cIC, which provided high flexibility as well as accessibility of the chains for protein binding. Therefore, at IC>cIC, the adjacent PEI chains became close and flexible enough, leading to facilitated transport of adsorbed protein molecules by the interactions of neighboring chains mediated by the bound molecules. It is regarded as “chain delivery” effect. At the same time, improved accessibility of binding sites led the significant increase of binding capacity. The decrease of q m value at IC>740mmol/L is considered due to the decrease of effective porosity. The research has thus provided new insight into protein adsorption and transport in polymer-grafted ion-exchange media.

Protein adsorption to poly(ethylenimine)-modified Sepharose FF: II. Effect of ionic strength

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Lin-Ling Yu , Yan Sun
In Part I of this work, we have studied the effect of ionic capacity (IC) on bovine serum albumin (BSA) adsorption equilibria and kinetics to poly(ethylenimine) (PEI)-grafted Sepharose FF, and found a critical IC (cIC, 600mmol/L), above which both protein capacity and uptake rate increased drastically. In this work, five PEI-Sepharose FF resins of typical ICs reported earlier were selected to explore the effect of ionic strength (IS) on the adsorption equilibria and kinetics of BSA. Commercially available DEAE (IC=160mmol/L) and Q Sepharose FF (IC=269mmol/L) resins were used for comparisons. It is found that at similar ionic capacities, protein adsorption capacities on both the PEI-Sepharose FF resins and the commercial resins decreased with increasing IS, but on the capacity sensitivity to salt concentration, the former was lower than the latter. In addition, the effective diffusivities (D e ) of the former were smaller than the latter in the entire IS range studied. The low IS sensitivity of adsorption capacity of the PEI-Sepharose FF resins could be interpreted by the increase of pore accessibility with increasing IS; the smaller D e values in the PEI-Sepharose FF resins were considered due to the lack of surface diffusion in the PEI-Sepharose FF resins of low PEI densities. For the PEI-Sepharose FF resins of high ICs (520, 740 and 1220mmol/L), both protein capacity and D e values increased first and then decreased with increasing IS. The increasing trend of protein capacity in the low IS range was considered due to the increase of accessible pores for BSA. The rise–fall trend of D e was attributed to the dependencies of the “chain delivery” effect on protein capacity and binding strength, both of which are related to IS. Moreover, the IS sensitivity of the D e for the resins of ICs>cIC (740 and 1220mmol/L) was much higher than those of ICs<cIC, further proving that the “chain delivery” effect in PEI layer did contribute significantly to the overall mass transfer at IC>cIC. Furthermore, the two PEI-Sepharose FF resins of ICs>cIC kept high adsorption capacities and D e values up to 200–300mmol/L NaCl. Therefore, the operating IS ranges for these two PEI-Sepharose FF resins can be much broader than the traditional ion-exchange media.

Enantiomeric separation of isochromene derivatives by high-performance liquid chromatography using cyclodextrin based stationary phases and principal component analysis of the separation data

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Yasith S. Nanayakkara , Ross M. Woods , Zachary S. Breitbach , Sachin Handa , LeGrande M. Slaughter , Daniel W. Armstrong
Isochromene derivatives are very important precursors in the natural products industry. Hence the enantiomeric separations of chiral isochromenes are important in the pharmaceutical industry and for organic asymmetric synthesis. Here we report enantiomeric separations of 21 different chiral isochromene derivatives, which were synthesized using alkynylbenzaldehyde cyclization catalyzed by chiral gold(I) acyclic diaminocarbene complexes. All separations were achieved by high-performance liquid chromatography with cyclodextrin based (Cyclobond) chiral stationary phases. Retention data of 21 chiral compounds and 14 other previously separated isochromene derivatives were analyzed using principal component analysis. The effect of the structure of the substituents on the isochromene ring on enantiomeric resolution as well as the other separation properties was analyzed in detail. Using principal component analysis it can be shown that the structural features that contribute to increased retention are different from those that enhance enantiomeric resolution. In addition, principal component analysis is useful for eliminating redundant factors from consideration when analyzing the effect of various chromatographic parameters. It was found that the chiral recognition mechanism is different for the larger γ-cyclodextrin as compared to the smaller β-cyclodextrin derivatives. Finally this specific system of chiral analytes and cyclodextrin based chiral selectors provides an effective format to examine the application of principal component analysis to enantiomeric separations using basic retention data and structural features.

High through-put and highly sensitive liquid chromatography–tandem mass spectrometry separations of essential amino acids using active flow technology chromatography columns

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): D. Kocic , L. Pereira , D. Foley , T. Edge , J.A. Mosely , H. Ritchie , X.A. Conlan , R.A. Shalliker
A preliminary investigation was undertaken to assess the performance of a new chromatography column technology in applications involving liquid chromatography coupled to mass spectrometry. The new column design allows mobile phase and solute to be extracted from the radial central region of the column, which reduces the solvent load to the mass spectrometer and improves separation efficiency. Effectively the column functions as a ‘wall-less’ column. The advantages of this design is that the analysis through-put can be increased by a factor of five, while at the same time there is a reduction in baseline noise, which results in an increase in the signal to noise response by up to 10-fold in comparison to standard columns with the same internal diameter and approaching 66-fold in comparison to standard columns with the same virtual internal diameter.

A study of bioactive, branched (1→3)-β-d-glucans in dimethylacetamide/LiCl and dimethyl sulphoxide/LiCl using size-exclusion chromatography with multi-angle light scattering detection

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Fen Qin , Mürşide Kes , Bjørn E. Christensen
Water-soluble (1→3)-β-d-glucans with 1,6-linked branches (SBG) isolated from the cell walls of Saccharomyces cerevisiae were studied by size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) detector using dimethylacetamide (DMAc) containing 0.5% (0.12M) LiCl, or dimethyl sulphoxide (DMSO) in the absence and presence of 0.25M LiCl, respectively, as eluents. The aggregating glucan could be dispersed as single chains in both solvents, with chain length distributions in reasonable agreement with results obtained previously with carboxymethylated glucans in aqueous solvent. However, DMAc is preferred over DMSO because of higher sensitivity in MALLS, and also because the latter produces SEC anomalies. SBG dissolves slowly in DMAc/LICl at room temperature, but heating accelerates the process. The rate of depolymerisation of SBG in DMAc/LiCl at high temperatures (70–105°C) was determined as a basis for defining dissolution procedures at elevated temperatures with a minimum of degradation. The result of the investigation is a simple and reliable protocol for preparing unaggregated, fully dissolved and undegraded SBG in DMAc/LiCl, which is well suited as a standard analysis of the molecular weight distribution of SBG-like molecules without chemical derivatization.

Computational fluid dynamic simulation of axial and radial flow membrane chromatography: Mechanisms of non-ideality and validation of the zonal rate model

13 August 2013, 09:40:25

Publication date: 30 August 2013
Source:Journal of Chromatography A, Volume 1305
Author(s): Pranay Ghosh , Kaveh Vahedipour , Min Lin , Jens H. Vogel , Charles Haynes , Eric von Lieres
Membrane chromatography (MC) is increasingly being used as a purification platform for large biomolecules due to higher operational flow rates. The zonal rate model (ZRM) has previously been applied to accurately characterize the hydrodynamic behavior in commercial MC capsules at different configurations and scales. Explorations of capsule size, geometry and operating conditions using the model and experiment were used to identify possible causes of inhomogeneous flow and their contributions to band broadening. In the present study, the hydrodynamics within membrane chromatography capsules are more rigorously investigated by computational fluid dynamics (CFD). The CFD models are defined according to precisely measured capsule geometries in order to avoid the estimation of geometry related model parameters. In addition to validating the assumptions and hypotheses regarding non-ideal flow mechanisms encoded in the ZRM, we show that CFD simulations can be used to mechanistically understand and predict non-binding breakthrough curves without need for estimation of any parameters. When applied to a small-scale axial flow MC capsules, CFD simulations identify non-ideal flows in the distribution (hold-up) volumes upstream and downstream of the membrane stack as the major source of band broadening. For the large-scale radial flow capsule, the CFD model quantitatively predicts breakthrough data using binding parameters independently determined using the small-scale axial flow capsule, identifying structural irregularities within the membrane pleats as an important source of band broadening. The modeling and parameter determination scheme described here therefore facilitates a holistic mechanistic-based method for model based scale-up, obviating the need of performing expensive large-scale experiments under binding conditions. As the CFD model described provides a rich mechanistic analysis of membrane chromatography systems and the ability to explore operational space, but requires detailed knowledge of internal capsule geometries and has much greater computational requirements, it is complementary to the previously described strengths and uses of the ZRM for process analysis and design.