Enzyme immobilized in a packed-bed reactor: kinetic parameters and mass transfer effects.

To describe axial dispersion, particle film mass transfer, intraparticle diffusion, and the chemical reaction of the substrate for enzymes immobilized in porous particles in packed columns, we have developed mathematical models for first- and zero-order limits of Michaelis-Menten kinetics. Steady-state solutions were derived for both long and short column boundary conditions and for plug flow. Theory was compared to experiments by hydrolysis of sucrose catalyzed by invertase bound to porous glass particles. Steady-state conversions were measured for a range of flow rates. Pulse response experiments with inert packing were used to determine values of bed void fraction and particle porosity.     

Solute exclusion from cellulose in packed columns: process modeling and analysis

In this investigation, process modeling and analysis were used to explore the behavior of solute exclusion from cellulose in packed columns. The study focused on modeling the effects of dispersion, mass transport, and pore diffusion. Three mathematical models were used to predict the behavior of the columns: an equilibrium model, a mass transfer model, and a combined mass transfer and pore diffusion model. Computer implementations of these models were tested against experimental conditions where cellulose particle size and solution velocity were used to either amplify or minimize dispersion or skewness in the elution curves. For small cellulose particles (200-300 mesh), all three models accurately predicted the shape of the elution curve and the particle porosity. For larger particles (45-60 mesh), the mass transfer model and the combined mass and pore diffusion model best represented the behavior of the column. At high solution velocities (0.63 cm(3) min(-1)) and large particles, only the combined mass transfer and pore diffusion model accurately represent the column behavior. Sensitivity analysis revealed that the mass transfer coefficient had little effect on the elution curves for the range of values (10(-6)-10(-3) cm s(-1)) calculated from the experimental data. The combined mass transfer and pore diffusion model presented in this article can be used to design solute exclusion measurement experiments for the larger cellulose particles found in a commercial cellulose-to-ethanol plant.     

Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the forensic analysis of eleven frequently used cyclic antidepressant drugs (ADSs) (amitriptyline, amoxapine, clomipramine, desipramine, dosulepine, doxepin, imipramine, maprotiline, melitracen, mianserine and nortriptyline) using a recently developed reversed-phase column with 2 μm particles for the analysis of biological samples. The separation was carried out using two different C₈ reversed-phase columns (column 1: 100 mm × 4.6 mm I.D., particle size 2 μm, TSK gel Super-Octyl; column 2: 100 mm × 4.6 mm I.D., particle size 5 μm, Hypersil MOS-C₈) for comparison. The mobile phase was composed of methanol-20 mM KH₂PO₄ (pH 7) (60:40, v/v) and the flow-rate was 0.6 ml/min for both columns. The absorbance of the eluent was monitored at 254 nm. When the eleven drugs were determined, the sensitivity with the 2 μm particles was about five times greater than with the 5 μm particles. Retention times on column 1 were shorter than those on column 2. These results show that the new ODS column packing with a particle size of 2 μm gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.     

Influence of added packing on the volumetric efficiency in a Karr column

Dispersed phase holdup and volumetric mass transfer coefficient were measured in a reciprocating plate column with high porosity packing in interplate spaces of the column and the performance was compared with that of the column without packing. The data and its analysis show substantial increase in the dispersed phase holdup and in the interfacial area to give an enhancement of 4 to 5 times in the volumetric efficiency of the column with the added packing.     

Modeling of immobilized cell columns for bioconversion and wastewater treatment

Immobilized cells are widely used in bioconversions to produce biological products as well as in wastewater treatment such as solvent removal from wastewater streams. In this work, a rate model is proposed to simulate this kind of process in an axial-flow fixed-bed column packed with porous particles containing immobilized cells. The transient model considered various mass transfer mechanisms including axial dispersion, interfacial film mass transfer, and intraparticle diffusion. Cell death in the immobilized cell system was also considered. Effects of various parameters such as kinetic constants and mass transfer parameters were studied. Operational situations such as feed fluctuation flow rate increase and two columns in series were also investigated. The model can be used to study the behavior and characteristics of immobilized cell columns in order to perform scale-up predictions of effluent profiles and for the purpose of process optimization.     

Size-exclusion chromatography of large molecules from coal liquids,petroleum residues,soots, biomass tars and humic substances

Size-exclusion chromatography (SEC) using 1-methyl-2-pyrrolidinone (NMP) as eluent has been calibrated using various standard polymers and model compounds and applied to the analysis of extracts of coal, petroleum and kerogens, to petroleum vacuum residues, soots, biomass tars and humic substances. Three separate columns of different molecular mass (MM) ranges were used, with detection by UV absorption; an evaporative light scattering detector was used for samples with no UV absorption. Fractionation was useful to separate signal from the less abundant high-mass material, which was normally masked by the strong signal from the more abundant low-mass material in the absence of fractionation. Fractionation methods used to isolate high-mass materials before SEC analysis included planar chromatography, column chromatography and solvent solubility. The apparently large molecules were concentrated into the fractions not soluble in common solvents and were relatively immobile in planar chromatography. All samples and fractions contained some material excluded from the column porosity. Evidence from other techniques suggests that the excluded material is of different structures from that of the resolved material rather than consisting of aggregates of small molecules. We speculate that the excluded material may elute early because the structures of this material are three-dimensional rather than planar or near planar.     

Improved packing of preparative biochromatography columns by mechanical vibration

The bioprocessing industry relies on packed-bed column chromatography as its primary separation process to attain the required high product purities and fulfill the strict requirements from regulatory bodies. Conventional column packing methods rely on flow packing and/or mechanical compression. In this work, the application of ultrasound and mechanical vibration during packing was studied with respect to packing density and homogeneity. We investigated two widely used biochromatography media, incompressible ceramic hydroxyapatite, and compressible polymethacrylate-based particles, packed in a laboratory-scale column with an inner diameter of 50 mm. It was shown that ultrasonic irradiation led to reduced particle segregation during sedimentation of a homogenized slurry of polymethacrylate particles. However, the application of ultrasound did not lead to an improved microstructure of already packed columns due to the low volumetric energy input (~152 W/L) caused by high acoustic reflection losses. In contrast, the application of pneumatic mechanical vibration led to considerable improvements. Flow-decoupled axial linear vibration was most suitable at a volumetric force output of ~1,190 N/L. In the case of the ceramic hydroxyapatite particles, a 13% further decrease of the packing height was achieved and the reduced height equivalent to a theoretical plate (rHETP) was decreased by 44%. For the polymethacrylate particles, a 18% further packing consolidation was achieved and the rHETP was reduced by 25%. Hence, it was shown that applying mechanical vibration resulted in more efficiently packed columns. The application of vibration furthermore is potentially suitable for in situ elimination of flow channels near the column wall.     

Optimal packing characteristics of rolled, continuous stationary-phase columns

Rolled, continuous stationary phases were constructed by tightly rolling and inserting a whole textile fabric into a chromatography column. This work reports the column performance, in terms of plate height, void fraction, and resolution, of 10 cellulose-based fabrics. The relation between fabric structural properties of yarn diameter, fabric count, fabric compressibility, and column performance are quantitated. General requirements, including reproducibility of packing, for choosing fabrics to make a good SEC column are identified. This research showed that the packed columns have an optimal mass of fabric that minimizes plate height and maximizes resolution, in a manner that is consistent with chromatography theory. Mass of material packed is then an important column parameter to consider when optimizing columns for the rapid desalting of proteins. Proteins were completely separated from salt and glucose in less than 8 min at a pressure drop less than 500 psi on the rolled, continuous stationary-phase columns. These results, together with stability and reproducibility, suggest potential industrial applications for cellulose-based rolled, continuous stationary-phase columns where speed is a key parameter in the production process.     

Modeling and simulation of liquid–solid circulating fluidized bed ion exchange system for continuous protein recovery

Liquid–solid circulating fluidized bed (LSCFB) is an integrated two‐column (downcomer and riser) system which can accommodate two separate processes (adsorption and desorption) in the same unit with continuous circulation of the solid particles between the two columns. In this study, a mathematical model based on the assumption of homogeneous fluidization was developed considering hydrodynamics, adsorption‐desorption kinetics and liquid–solid mass transfer. The simulation results showed good agreement with the available experimental results for continuous protein recovery. A parametric sensitivity study was performed to better understand the influence of different operating parameters on the BSA adsorption and desorption capacity of the system. The model developed can easily be extended to other applications of LSCFB. Biotechnol. Bioeng. 2009; 104: 111–126 © 2009 Wiley Periodicals, Inc.     

Prediction of reversible IgG1 aggregation occurring in a size exclusion chromatography column is enabled through a model based approach

One important aspect of antibody separation being studied today is aggregation, as this not only leads to a loss in yield, but aggregates can also be hazardous if injected into the body. The aim of this study was to determine whether the methodology applied in the previous study could be used to predict the aggregation of a different batch of IgG1, and to model the aggregation occurring in a SEC column. Aggregation was found to be reversible. The equilibrium parameter was found to be 272 M^‐1 and the reaction kinetic parameter 1.33 × 10^‐5 s^‐1, both within the 95% confidence interval of the results obtained in the previous work. The effective diffusivities were estimated to be 1.45 × 10^‐13 and 1.90 10^‐14 m²/s for the monomers and dimers, respectively. Good agreement was found between the new model and the chromatograms obtained in the SEC experiments. The model was also able to predict the decrease of dimers due to the dilution and separation in the SEC column during long retention times.     

Roles of interstitial fraction and load conditions on the dynamic binding capacity of proteins on capillary‐channeled polymer fiber columns

Capillary‐channeled polymer (C‐CP) fibers are used as a stationary phase for ion‐exchange chromatography of proteins. Collinear packing of the fibers permits operation at high linear velocities (U_o > 100 mm s^?1) and low backpressure (<2,000 psi) on analytical‐scale columns. Rapid solvent transport is matched with very efficient solute mass transfer as fibers are virtually non‐porous with respect to the size of the target protein molecules. Lack of porosity of course limits the equilibrium binding capacity of stationary phases. Breakthrough curves and frontal analysis are used to better understand trade‐offs between the kinetic and thermodynamic properties as C‐CP fibers are applied in preparative situations. Fiber columns packed to different interstitial fraction values affect both the total fiber surface area (e.g., equilibrium binding capacity [EBC]) and the permittivity to flow and mass transport characteristics (e.g., dynamic binding capacity [DBC]). The EBC of the nylon 6 C‐CP fibers was found to be 1.30 mg g^?1, with isotherms that were best matched by a Moreau model, showing linearity up to solute concentrations of ～0.4 mg mL^?1. Isotherms generated under flow conditions were equally well approximated using Langmuir, Freundlich, and Moreau isotherm models. Fairly linear responses were seen up to the maximum load concentration of 1.2 mg mL^?1. Counterintuitively, dynamic studies revealed that conditions of high column porosity yielded a DBC that is ～70% higher than the EBC. These findings point to potential advantages in terms downstream processing applications, where protein throughput and yield are critical metrics. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:97–109, 2015     

Intraparticle diffusion alters the dynamic response of immobilized cell/enzyme columns

The effect of intra-particle tracer diffusion on the transient response of a fixed-bed column containing porous Ca-alginate beads is investigated experimentally. Dynamic response experiments are carried out using acetone solutions as tracer, whose concentration in the effluent stream is monitored on-line using a UV analyzer. Comparison of the response of such columns for two particle sizes (radii of 2 and 1?mm) to that of otherwise identical beds filled with solid glass particles of equal size reveals a substantial difference, mainly characterized by a trailing of the effluent concentration-vs-time curves at longer times. This cannot be explained by assuming a change in the effective porosity of the system, nor a change in the hydrodynamic dispersion. Following independent experimental determination of the pertinent physical parameters, a model based on the tanks-in-series concept [1,?2] is implemented on a personal computer and its predictions are compared to the experimental data. The essential assumption of the model is that tracer convection in the bulk liquid is substantially faster than intra-particle diffusion and, as a result, a bead is always surrounded by fluid of uniform (albeit time-dependent) concentration. In the absence of external mass transfer resistances and assuming that the porous nature of the Ca-alginate beads does not substantially alter the hydrodynamic dispersion in the column, the agreement between model and experiment is remarkably good. This leads to the suggestion that the observed response can be explained as the combined result of (fast) tracer convection in the bulk liquid and (slower) tracer diffusion from the bulk liquid to the beads.     

Identification of fatty acids by GC-MS using polar siloxane liquid phases

A practical method is described for the unambiguous characterization of all fatty acids in natural mixtures from a single injection into a GC-MS instrument. The fatty acids are separated as the methyl esters on a polar siloxane column which yields resolutions similar to those commonly obtained on polyester columns, but has higher thermal stability. Any leakage of the polar siloxane into the mass spectrometer is further reduced by placing a small amount of a thermally stable methyl siloxane packing at the outlet end of the column which serves as a trap. The glc peaks emerging from such columns yield characteristic mass spectra, including molecular ions, which along with the retention data are adequate for the accurate identification of both saturated and unsaturated fatty acids and dimethyl acetals.     

Biosorption Processes for Natural and Waste Water Treatment – Part II: Experimental Studies and Theoretical Model of a Biosorption Fixed Bed

A model has been developed for fixed‐bed biosorption performance, i.e. combined action of adsorption of organic water contaminants and their biological destruction in a column. The model contains an adsorption isotherm of the Freundlich type, adsorption kinetics by an overall film mass transfer (Glueckauf equation), maximum bacterial growth,and biological aerobic destruction (Monod model) of the organics by exoenzymes. Bacteria can not penetrate into the pores of the adsorbent. The model was tested using the system aqueous solution of aniline/Pseudomonas putida/Polysorb 40/100. Breakthrough curves in shorter columns have been measured and a velocity‐dependent steady‐state exit concentration was achieved. These curves could be simulated with sufficient accuracy on the basis of isotherm data, mass transfer coefficients and values of biological growth and destruction activity estimated from independent measurements.     

High-performance size-exclusion chromatography of hydrolyzed plant proteins

A high-performance size-exclusion chromatographic (SEC) procedure has been developed to determine the molecular size distribution of several sources of hydrolyzed plant proteins including soy, potato, cotton seed, corn gluten, wheat bran, and wheat germ. The SEC packing consisted of a glycerylpropylsilyl layer covalently bonded to 100-Å pore-size silica particles (10 μm). A number of mobile phases were evaluated in an attempt to reduce adsorption between hydrolyzed components and packing material. Systems containing either sodium dodecyl sulfate or methanol showed the best performances; however, adsorption could not totally be eliminated. The combination of size-exclusion and retention mechanisms of separation was valuable in establishing differences among samples. Based on dialysis of hydrolyzed soy protein, it appeared that most of the adsorbed material was of low molecular weight. Column recovery studies indicated complete elution of samples.     

Influence of Bed‐Sediment Features on the Interstitial Habitat Available for Macroinvertebrates in 15 French Streams

The interstitial space of bed‐sediments is a major component of the stream system as it influences both physical and biological processes and patterns, such as exchanges between surface water and groundwater or the assemblage of invertebrates. To investigate the influence of grain‐size distribution and vertical bed‐packing on the interstitial habitat, we used 99 frozen cores collected from 15 French streams. On our data, overall invertebrate density, density of Chironomidae and taxon richness were more strongly related to effective porosity than to other physical variables describing bed‐sediments. We built two models to quantify the influence of grain‐size distribution and vertical bed‐packing on effective porosity. The effective porosity decreased with increasing grain‐size heterogeneity and was strongly related to grain‐size distribution. These relationships differed with vertical bed‐packing, but were consistent across three geologic types of sediments that varied in particle shape and particle texture. These general relationships suggest that effective porosity could be helpful to better understand the distribution patterns of invertebrates in bed‐sediments.     

Utilisation of controlled pore topology for the separation of bioparticles in a mixed-glass beads column

To study the flow of shaped particles in porous media, elution of spherical and rod-like micro-organisms was performed through beds of spherical glass beads. A 0.04 cm/s constant flow rate was used with 5 microm yeast suspensions, 1 microm latex micro-spheres and rod-like bacilli Lactobacillus bulgaricus 6 microm long and 0.5 microm in diameter. Yeast cells' diameter is close to the bacilli length and micro-spheres have the same diameter as bacilli. All particle types have similar density. To make the different packing beds, 1.125 mm coarse beads and 0.1115 mm fine beads were used. Experiments were carried out using a column loaded with the binary packing (volume fraction of coarse particles in the mixture 0.7) or a monosize packing with the same amount of coarse or fine particles as used in the binary packing. Analysis of experimental results was based on two models: pure exclusion effect and hydrodynamic separation model [hydrodynamic chromatography (HDC)]. Results for spheres show that the classic HDC model fits to the experimental data whenever the ratio of particle size to the pathway bend scale is high ( approximately 1/100, micro-spheres). However, if this ratio increases and becomes approximately 1/20, the HDC model needs to be corrected due to the effect of channel wall curvature on exclusion. This led to a modified HDC equation of the form R=B/(1+2lambda-2.8lambda(2)), where R is the retention, lambda is the aspect ratio and constant B>or=1. Bacillus separation follows an exclusion mechanism, since pore topology is important in the separation of shaped particles when the aspect ratio approaches lambda=0.1. In the case of a binary packing bed, rod-like particles display a different behaviour than the one exhibited by the spherical particles of the same scale as bacilli, either in length or in diameter. This may be explained by the interaction between rod-like bacilli and the bed's pore topology. A generalised exclusion model for particles was proposed to be R=A/(1-lambda)(z), where A is the coefficient proportional to the tortuosity and the parameter z=1, 2 or 3 depends mainly on pore shape. Controlled pore topology opens interesting applications for bio-separation (in porous micro-fluidic devices, deep bed filtration) and might be especially important for macromolecules and micro-organisms separation with different shapes.     

Superficially Porous Particle Based Hydroxypropyl‐β‐cyclodextrin Stationary Phase for High‐Efficiency Enantiomeric Separations

A superficially porous particle (SPP)‐based hydroxypropyl‐β‐cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)‐based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP‐based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation. Chirality 27:788–794, 2015. © 2015 Wiley Periodicals, Inc.     

Fractionation and characterization of a Polysaccharide from the sclerotia of Pleurotus tuber-regium by preparative size-exclusion chromatography

A kind of regenerated cellulose gel (RCG) particles were treated by toluene to obtain particles with smaller mean pore size, then was mixed with the cellulose gel with pore size of 370 and 525 nm. A preparative size-exclusion chromatography (SEC) column (700 x 20 mm) packed with three gel particles was used to fractionate water-soluble polysaccharide (WEP) extracted from the sclerotia of Pleurotus tuber-regium by aqueous solution. The exclusion limit and fractionation range of the stationary phase of the preparative SEC were molecular mass 8 x 10(5) and 5 x 10(3) to 8 x 10(5), respectively. The calibration curve of the preparative SEC was represented as: log M=13.96-0.53 Ve. The WEP sample (weight-average molecular mass M(w)=2.2 x 10(4), polydispersity=2.4) was divided into three fractions with M(w) ranging from 1.4 x 10(4) to 3.4 x 10(4) by the preparative SEC column, and the fractions were characterized by gas chromatography GC, SEC combined with laser light scattering (SEC-LLS) and viscometry. The unfractionated WEP exhibited triple peaks due to different molecular mass, but each fraction exhibited single peak with the polydispersity of 1.1-1.8 in the SEC patterns. The results indicated that the preparative SEC was efficient for fractionation of polysaccharides having low molecular weight and for determination of their molecular mass.     

Simulation of the dynamic packing behavior of preparative chromatography columns via discrete particle modeling

Preparative packed‐bed chromatography using polymer‐based, compressible, porous resins is a powerful method for purification of macromolecular bioproducts. During operation, a complex, hysteretic, thus, history‐dependent packed bed behavior is often observed but theoretical understanding of the causes is limited. Therefore, a rigorous modeling approach of the chromatography column on the particle scale has been made which takes into account interparticle micromechanics and fluid–particle interactions for the first time. A three‐dimensional deterministic model was created by applying Computational Fluid Dynamics (CFD) coupled with the Discrete Element Method (DEM). The column packing behavior during either flow or mechanical compression was investigated in‐silico and in laboratory experiments. A pronounced axial compression–relaxation profile was identified that differed for both compression strategies. Void spaces were clearly visible in the packed bed after compression. It was assumed that the observed bed inhomogeneity was because of a force‐chain network at the particle scale. The simulation satisfactorily reproduced the measured behavior regarding packing compression as well as pressure‐flow dependency. Furthermore, the particle Young's modulus and particle–wall friction as well as interparticle friction were identified as crucial parameters affecting packing dynamics. It was concluded that compaction of the chromatographic bed is rather because of particle rearrangement than particle deformation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:363–371, 2016