Effects of nonuniform column packing in analytical gel chromatography. I. Monodisperse solutes

The effect of nonuniform column packing on solute profile shapes in analytical gel chromatography has been investigated for monodisperse solutes. This investigation considers the influence of a family of nonuniform partition cross sections on the concentration profiles for small-zone experiments. The nonuniformity causes the dispersive and translational transport coefficients to be functions of position. It is shown that for presently encountered amounts of nonuniformity, the principal effect is in the deviation of peak position from linear dependence on time. There is very little effect on peak shape, when concentrations are expressed in terms of bulk solution values.     

Toward a robust model of packing and scale-up for chromatographic beds. 1. Mechanical compression

The packing of compressible biochromatographic resins at large scale suffers from a poor understanding of how column packing method, resin properties, and column geometry impact column performance. To improve understanding, we develop and evaluate a one-dimensional, continuum mechanics model of column packing by mechanical compression. We show that the model can quantitatively predict the change in bed height, applied stress, and internal axial porosity profile without adjustable parameters when the modulus and wall friction coefficients are determined independently. The model possesses theoretical relationships for wall support and resin rigidity that should enable it to describe the mechanical compression of any biochromatographic resin for any column diameter. Moreover, this framework could provide a path to analogous models for flow packing and dynamic axial compression.     

A radiochemical study of irreversible adsorption of proteins on reversed-phase chromatographic packing materials

A radiochemical study of the irreversible adsorption of proteins on commercial reversed-phase HPLC packing materials is reported. The conditions of study are similar to those used in HPLC separation of protein. The effects of the amount and contact time of two proteins, ovalbumin and cytochrome c, are reported. Additional results include the effect of column pretreatment with protein, silanophilic mobile-phase blocking agent, and type of packing material on the extent of irreversible adsorption. The loss process is shown to be at least biphasic and the mechanisms of loss distinct for different proteins.     

Liquid saturation in concurrent gas-liquid downflow through packed beds

The total and dynamic liquid saturation under concurrent gas-liquid downflow through packed beds were experimentally measured for non-foaming, foaming Newtonian and non-Newtonian liquids. The variables include the column diameter, packing size and shape, flow rate of the phases, and physical properties. Correlations were presented in terms of Lockhart-Martinelli parameter, χ for non-foaming Newtonian and non-Newtonian liquids and in terms of modified Lockhart-Martinelli parameter, χ′ for foaming Newtonian liquids.     

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.     

Rapid quantitative determination of underivatized carbamazepine,phenytoin, phenobarbital and p-hydroxyphenobarbital in biological fluids by packed column gas chromatography

A method is described for measuring, without derivatization, the concentrations of phenobarbital, p-hydroxyphenobarbital, carbamazepine and phenytoin in biological fluids of epileptic patients undergoing long-term therapy. This method uses, at an isothermal temperature, a special column packing (GP-2% SP-2510-DA on 100–120 mesh Supelcoport).The lower limit of detection for all substances analyzed as 1 μg/ml of biological material. The recovery of the compounds is about 95%, the reproducibility of the method is good (coefficient of variation, 4.3%). The mass spectra confirm the identity of substances eluting from the column. There is no interference from other commonly used antiepileptic drugs or endogenous substances.The method has the advantages of high specificity, sensitivity and rapidity and it appears to be suitable for the routine monitoring of blood and urine concentrations in patients receiving multi-drug therapy.     

An HPLC system for the automatic sampling and analysis of time-series kinetic studies

Robotics were used to automate a high performance liquid chromatography (HPLC) system which was designed for the unattended sampling and analysis of time-series kinetic studies. The system allowed for the direct injection of samples containing whole cells and particulates witout plugging the analytical column. Coarse debris was removed from the injected sample by a 0.5 μm inline filter. Finer particulates were removed by a guard column that was layered with three different size packing resins. To demonstrate the system's capabilities, data are presented from an 8 day progress curve showing the anaerobic biodegradation of p-cresol by a sulfate-reducing enrichment. On fitting the data to the Michaelis-Menten equation by nonlinear regression, the apparent kinetic constants K_m and V_max were calculated to be 126 μM and 4.0 μM/h, respectively, for p-cresol metabolism by the enrichment.     

A stereochemical model for phospholipids. II: Conformational analysis and molecular packing of phosphatidylethanolamine (PE)

A partition energy method procedure was applied to select the energetically favoured conformations of phosphatidylethanolamine (PE) as polar constituents of phospholipid molecules. The result indicated a large degree of freedom for the two torsion angles of the ester bond of the phosphate and a gauche, gauche star conformation for the ethane bond.A packing process of the molecule was carried out through a potential energy calculation by considering the conformers selected above, using previously published procedure and conventions. All the arrangements which possess the best packing energy values were characterised by an orientation of the PN dipolar segment parallel to the lattice plain. Rotation of the internal torsion angles and rotation in the eulerian space of the molecule produced differences in the charged groups that interact. An additional minimum was present in the energy packing process of those conformers which have the first torsion angle of the phosphate in a trans conformation. This minimum, which corresponds to an orientation of the molecule orthogonal to the lattice plane, requires a complete neutralisation of the point charges on the system.The results of the calculation underline the importance of changes in the behaviour of the polar group of the phospholipids in the packing process.     

Adsorption of polyhedra of a cytoplasmic-polyhedrosis virus by soil particles

Tests on the quantitative adsorption of polyhedra of a cytoplasmic-polyhedrosis virus of Bombyx mori to soil showed an increase in the adsorption of polyhedra with an increase in the amount of soil, but the number of polyhedra adsorbed per unit weight of soil decreased. The number of polyhedra adsorbed to a fixed quantity of soil was in direct proportion to the polyhedron concentration, and the amount of adsorption increased with acidity but decreased with the addition of reagents which masked the polyvalent cations exposed on the surfaces of soil particles. The polyhedra applied to the top of a soil column were detected microscopically within 4 cm from the surface after the equivalent of 1,120 cm depth of water was passed through the column. The polyhedra occurred on the upper surface of the soil column and on the walls of voids formed by random packing of the soil particles.     

Starch analysis using hydrodynamic chromatography with a mixed-bed particle column

Columns packed with commercial glass beads 5 and 19 μm average size and a mixture of both (0.7 volume fraction of large particles) were used to analyse starch composition by hydrodynamic chromatography (HDC), applying water as mobile phase. To obviate retrogradation, experiments were carried out at column temperatures of 15 and 3 °C and several types of starch were assayed. In what concerns amylopectin and amylose separation, a better resolution and a lower pressure drop were obtained for the mixed binary packing when compared with the packing containing uniform 5 μm glass beads. A more efficient cooling of the mobile phase was also obtained with the mixed packing, which was determinant for improving resolution. For the Hylon VII starch the relative retention times (RRT) were 0.777 and 0.964 for amylopectin and amylose, respectively, while for the Tapioca starch the obtained RRTs were 0.799 and 0.923. Application of unbound glass beads as column packing not only might reduce equipment and running costs in preparative scale separations, but also proved to be useful as a fast and reliable method to monitor the amylose and amylopectin content of starch samples of different sources.     

Two conformations of crystalline adenylate kinase.

Pig muscle adenylate kinase (EC2.7.4.3) can exist in three crystal forms, which are interconvertible. For crystal form A the enzyme structure is known in atomic detail. We report the X-ray diffraction analysis of crystal form B at 4.7 Å resolution and a comparison with the A form. During the transition from A to B the packing arrangement of the molecules changes slightly. Moreover, the individual molecule undergoes an appreciable conformational change: by displacing a chain segment of seven residues and two adjacent α-helices a hydrophobic pocket is opened deep in the cleft near the centre of the molecule. Concomitantly the β-pleated sheet is enlarged by about four hydrogen bonds in the B form. Several lines of evidence indicate that the observed conformational change is an intrinsic property of the molecule and is not induced by crystal packing forces.     

Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion-exchange adsorbents

Although it is commonly believed that a column packing used for chromatofocusing must have an "even" buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong-acid cation-exchange column packing or a strong-base anion-exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak-acid or weak-base functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using chromatofocusing and a strong-acid cation-exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ion-exchange chromatography using a salt gradient for this purpose.     

Toward a robust model of packing and scale-up for chromatographic beds. 2. Flow packing

We developed and evaluated a model for predicting the flow packing of nonrigid chromatographic resins. The model is based on elasticity theory and accounts for resin rigidity and column diameter. When a modulus determined from a standard mechanical compression (consolidation) test is used, the model captures the primary phenomena of the scale-up process. However, moduli determined from flow-packing experiments improve the accuracy of the predictions and show that the apparent rigidity of chromatographic resins is lower for flow packing than for mechanical compression. Using a modulus from flow-packing experiments provided quantitative scale-up predictions of flow packing carried out in columns with diameters between 200 and 450 mm at different locations and by different operators.     

A modified procedure for the large scale preparation of tRNA from E. coli

A procedure for the preparation of about 50 g batches of tRNA from 25 kg E. coli W is described. The method involves phenolic extraction of the cells, batch absorption of the tRNA on DEAE-cellulose, washing the DEAE-cellulose and packing it into a column, elution of the tRNA from the column and precipitation of the tRNA with ethanol. The method is less time and labor consuming than the methods described in the literature and can be carried out with relatively simple equipment.     

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.     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

Influences of nonuniform activity distributions on the apparent maximum reaction rate and apparent Michaelis constant of immobilized enzyme reactions

The influences of nonuniform activity distribution within a porous solid support on the apparent kinetic parameters, V_m^app and K_m^app, of immobilized enzyme reactions following the Michaelis-Menten kinetics were theoretically investigated. As the enzyme is distributed to the neighborhood of the external surface of the support, V_m^app and K_m^app approach their respective intrinsic values over a wide range of substrate concentration. There is a close relationship between the nonuniform distribution and internal diffusional resistance. Changes in these two factors provide similar effects on V_m^app and K_m^app. As long as the immobilized enzyme reaction follows Michaelis-Menten kinetics, the nonuniform activity distribution never makes K_m^app less than its intrinsic value.     

General model of myosin filament structure. 3. Molecular packing arrangements in myosin filaments

Following the original proposals about myosin filament structure put forward as part of a general myosin filament model (Squire, 1971, 1972) it is here shown what the most likely molecular packing arrangements within the backbones of certain myosin filaments would be assuming that the model is correct. That this is so is already indicated by recently published experimental results which have confirmed several predictions of the model (Bullard and Reedy, 1972; Reedy et al., 1972; Tregear and Squire, 1973).The starting point in the analysis of the myosin packing arrangements is the model for the myosin ribbons in vertebrate smooth muscle proposed by Small &; Squire (1972). It is shown that there is only one reasonable type of packing arrangement for the rod portions of the myosin molecules which will account for the known structure of the ribbons and which is consistent with the known properties of myosin molecules. The dominant interactions in this packing scheme are between parallel myosin molecules which are related by axial shifts of 430 Å and 720 Å. In this analysis the myosin rods are treated as uniform rods of electron density and only the general features of two-strand coiled-coil molecules are considered.Since the general myosin filament model is based on the assumption that the structures of different types of myosin filament must be closely related, the packing scheme derived for the myosin ribbons is used to deduce the structures of the main parts (excluding the bare zones) of the myosin filaments in a variety of muscles. It is shown in each case that there is only one packing scheme consistent with all the available data on these filaments and that in each filament type exactly the same interactions between myosin rods are involved. In other words the myosin-myosin interactions involved in filament formation are specific, they involve molecular shifts of either 430 Å or 720 Å, and are virtually identical in all the different myosin filaments which have been considered. Apart from the myosin ribbons, these are the filaments in vertebrate skeletal muscle, insect flight muscle and certain molluscan muscles.In the case of the thick filaments in vertebrate skeletal muscle the form of the myosin packing arrangement in the bare zone is considered and a packing scheme proposed which involves antiparallel overlaps between myosin rods of 1300 Å and 430 Å. It is shown that this scheme readily explains the triangular profiles of the myosin filaments in the bare zone (Pepe, 1967, 1971) and many other observations on the form of these myosin filaments.Finally it is shown that the cores of several different myosin filaments, assuming they contain protein, may consist of different arrangements of one or other of two types of core subfilament.     

A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.     

Comparison of single- and three-stage tower loop reactors

Summary Hansenula polymorpha was cultured for long periods in 254 cm high single and three-stage countercurrent tower loop reactors 20 cm in diameter using ethanol as a substrate in the absence and presence of antifoam agents (Desmophen 3600 and/or soy oil). In the absence of antifoam agents in the three-stage column, much higher volumetric mass transfer coefficients were attained than in the corresponding single-stage column. The cell productivity in the former, however, was only slightly higher than in the single-stage column due to considerable enrichment of the cells in the foam and nonuniform cell concentration distribution in the three-stage column. In the presence of antifoam agents the three-stage column has a higher cell productivity, OTR, k_L a and a lower specific energy requirement with regard to the absorbed oxygen and/or produced cell mass than the single stage column. The reactor performance is especially high if the bubbling layer height is reduced to 20 cm. Soy oil has considerably less foam eliminating property than Desmophen. Since the soy oil is metabolized by the yeast, large amounts are needed to operate these reactors.