Mobile Phase Viscosity Research Articles

Comprehensive analysis of the effective and intra-particle diffusion of weakly retained compounds in silica hydrophilic interaction liquid chromatography columns

A detailed analysis of intra-particle volumes and layer thicknesses and their effect on the diffusion of solutes in hydrophilic interaction liquid chromatography (HILIC) was made. Pycnometric measurements and the retention volume of deuterated mobile phase constituents (water and acetonitrile) were used to estimate the void volume inside the column, including not only the volume of the mobile phase but also part of the enriched water solvent acting as the stationary phase in HILIC. The mobile phase (hold-up) volume accessible to non-retained components was estimated using a homologous series approach. The joint analysis of the different approaches indicated the formation of enriched water layers on the hydrophobic silica mesopore walls with a thickness varying significantly with mobile phase composition. The maximal thickness of the enriched water layers, which corresponded to the minimum void volume accessible to unretained solutes, marked a transition in the retention behavior of the studied analytes. Discrepancies between deuterated solvent measurements and pycnometry were explained by the existence of an irreplaceable water layer adsorbed on the silica surface. Regarding the diffusion behavior in HILIC, peak parking experiments were used to interpret the influence of the acetonitrile content on the effective diffusion coefficient Deff. A systematic decrease in Deff and molecular diffusion Dm was observed with decreasing acetonitrile concentration, primarily attributed to variations in mobile phase viscosity. Notably, Deff/Dm remained nearly unaffected by variations in mobile phase composition. Finally, the effective medium theory was used to make a comprehensive analysis of Dpart/Dm to study the contribution to band broadening when the solute resides in the mesopores. The obtained data unveiled a curvature with a minimum corresponding to conditions of maximum water-layer thickness and retention. For the weakly retained compounds (k’ < 0.5) the Dpart/Dm-values were found to be relatively high (order of 0.35-0.5), which directly reflects the high γsDs/Dm-values that were observed (order 0.35-7).

Diffusion coefficients of an extensive set of pharmaceutical compounds in supercritical fluid chromatography over a wide range of mobile phase compositions

Diffusion data are essential for adequate analysis of the kinetic separation performance of any chromatographic system. Unfortunately, for Supercritical Fluid Chromatography (SFC), very little data is available of the diffusion coefficients in mobile phases typically used in contemporary methods, i.e. with a non-negligible amount of polar modifier such as methanol. In this work, a relative simple method which only requires minor modifications to a standard commercially available SFC instrument is used to determine the diffusion coefficient of an extensive set of pharmaceutical compounds in the range of 10–50 vol% of modifier (methanol) in CO2. By using a traditional SFC column, the solute is first separated from the sample solvent plug, before entering a long capillary, where the band broadening can be linked to its diffusion coefficient using the Taylor-Aris equation. By using two UV-detectors, before and after the capillary, the effect of the dispersion in the column can be eliminated and the true volumetric flow rate determined. It was found that in the investigated range of conditions, the change in mobile phase viscosity in a first approximation allows to predict the variation in diffusion coefficient. Chemical structure and more particularly functional groups can however have a significant effect on the diffusion coefficient.

The effect of water on the large-scale supercritical fluid chromatography purification of two factor XIa active pharmaceutical ingredients

BMS-962212, a parenteral Factor XIa inhibitor, was scaled-up for toxicity studies. Two steps of supercritical fluid chromatography (SFC) were developed for the chiral resolution of the penultimate and achiral purification of final active pharmaceutical ingredient (API), BMS-962212. A robust SFC process using Chiralcel OD-H with methanol-acetonitrile as modifier in CO2 was established to achieve a stable and uninterrupted operation with reduced mobile phase viscosity and system pressure drop. More than 230 g of the racemic penultimate was chirally resolved to reach >99% chiral purity, ready for final tert-butyl ester deprotection to provide the API. There were a significant number of impurities in BMS-962212 generated from the final step that needed to be removed. In contrast to conventional SFC conditions, an SFC method exploiting water and ammonia as additives in both the mobile phase and sample solution was developed to accomplish purification and desalting (i.e. removing TFA) of the zwitterionic API in one step. Water as an additive eliminated salt precipitation and improved the resolution while ammonia contributed to the desalting, details of which will be discussed in this article. A throughput of 2 g/h was achieved, and >80 g of the crude API was purified. The same strategy was applied to another Factor XIa API (compound A) and its penultimate.

Hydrodynamics and dynamic capacity of cryogels produced with different monomer compositions

ABSTRACTCryogels have received great attention in the area of bioseparation due to their characteristic of very large and interconnected pores. These materials allow rapid isolation of various biomolecules and permit that chromatographic processes are carried out at high flow rates. Nevertheless, studies regarding the effect of monomer concentration on the hydrodynamics and dynamic capacity of cryogels are scarce. In this study, supermacroporous polyacrylamide cryogels with different monomer and crosslinker concentrations were produced by cryopolymerization and activated with tris(hydroxymethyl)aminomethane. Porosity and permeability were determined and it was observed that these parameters changed with different monomer concentrations, indicating changes in the structure of the cryogels. Residence time distribution experiments were conducted at different flow rates and different mobile phase viscosities. As a function of composition of the cryogels, the axial dispersion increased and the flow pattern deviated considerably from the ideal plug flow model. For cryogels with lower crosslinker content, the effect of the mobile phase viscosity was more evident. The monomer composition changed the dispersion number and height equivalent to one theoretical plate, resulting in an impact on the adsorption capacity of the cryogel. Consequently, proper monomer and crosslinker concentrations may produce cryogels with suitable performance. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48507.

Effect of temperature on adsorption and chromatography of polystyrene macromolecules

Study of the effect of temperature on the parameters of adsorption and liquid chromatography of&#x0D; molecules and macromolecules constantly attracts the attention of specialists. The potential benefits of ele- vated temperature column, particularly enhanced kinetic and transport properties, which are based on the reduction of mobile phase viscosity and increase the diffusion of analyte at high temperature, began to be actively used for the rapid analysis of molecules and macromolecules by chromatographic methods in recent years. It is now recognized that temperature is an important tool to optimize chromatographic parameters, such as retention, selectivity and efficiency, particularly for macromolecules&#x0D; The effect of temperature on adsorption and liquid chromatography of polystyrene macromolecules on silicas has been investigated by static and .chromatographic methods. The sign and value of the tempera- ture adsorption coefficient depend on many factors, namely, the chemical nature of the solvent, the chemical structure of macromolecules and adsorbent surface chemistry. Thus temperature elevation in the chromato- graphic system alters the macromolecular coil dimensions and total energy of elution of solvent molecules from the adsorption space of the porous adsorbents. Keeping these factors in view we can find the sign of the temperature adsorption coefficient. Diffusion coefficients increase in proportion to temperature, thus effi- ciency is usually enhanced, too, as peaks become more narrow. Prospects for optimization of the chromato- graphic process and, above all, the efficiency of separation of components of mixtures, include the effect of temperature on the adsorption of the components and their retention in the chromatographic column.

Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals

A new stationary phase based on wide-pore hybrid silica bonded with amide ligand has been used to explore the utility of HILIC for the analytical characterization of protein biopharmaceuticals. Various, highly-relevant samples were tested, including different insulins, interferon α-2b and trastuzumab. This work shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs, using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA. In terms of elution order and selectivity, these HILIC separations have proven to be highly orthogonal to RPLC, while the kinetic performance remains comparable. In the case of characterizing trastuzumab, HILIC was uniquely able to resolve several important glycoforms at the middle-up level of analysis (fragments of 25–100kDa). Such a separation of glycoforms has been elusive by other separation mechanisms, such as RPLC and IEX. Besides showing orthogonality to RPLC and improved separations of glycoforms, HILIC offers several additional benefits for biopharmaceutical characterization: i) an inherent compatibility with MS, ii) a reduced requirement for very high mobile phase temperatures that are otherwise needed in RPLC to limit undesirably strong adsorption to the surface of the stationary phase, and iii) the possibility to couple several columns in series to improve resolving power, thanks to comparatively low mobile phase viscosity.

Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A “green” technique

A “green” hydrophilic interaction liquid chromatography (HILIC) technique for separating the components of mixtures with a broad range of polarities is illustrated using enhanced-fluidity liquid mobile phases. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquid CO2 to conventional liquid mobile phases. Decreased mobile phase viscosity and increased analyte diffusivity results when a liquefied gas is dissolved in common liquid mobile phases. The impact of CO2 addition to a methanol:water (MeOH:H2O) mobile phase was studied to optimize HILIC gradient conditions. For the first time a fast separation of 16 ribonucleic acid (RNA) nucleosides/nucleotides was achieved (16min) with greater than 1.3 resolution for all analyte pairs. By using a gradient, the analysis time was reduced by over 100% compared to similar separations conducted under isocratic conditions. The optimal separation using MeOH:H2O:CO2 mobile phases was compared to MeOH:H2O and acetonitrile:water (ACN:H2O) mobile phases. Based on chromatographic performance parameters (efficiency, resolution and speed of analysis) and an assessment of the environmental impact of the mobile phase mixtures, MeOH:H2O:CO2 mixtures are preferred over ACN:H2O or MeOH:H2O mobile phases for the separation of mixtures of RNA nucleosides and nucleotides.

Evaluation and characterization of axial distribution in expanded bed: II. Liquid mixing and local effective axial dispersion

Expanded bed adsorption (EBA) is a promising technology to capture proteins directly from unclarified feedstock. In order to better understand liquid mixing along the bed height in expanded beds, an in-bed sampling method was used to measure residence time distribution at different bed heights. A 2cm diameter nozzle column was tested with agarose raw beads (3% crosslinked agarose containing tungsten carbide). Two settled bed heights (11.5 and 23.1cm) with different expansion factors (1.4–2.6) were investigated and the number of theoretical plates (N), the height equivalent of theoretical plate (HETP) and the local effective axial dispersion coefficient (Dax) were calculated for each bed height-defined zone. The effects of expansion factor, settled bed height and mobile phase were evaluated. The results showed that N increased with the increase of expansion factors, but Dax was unaffected under fixed bed heights. Dax and HETP were found similar as a function of relative bed height for two settled bed heights tested. Higher mobile phase viscosity resulted in stronger axial dispersion. In addition, the local effective Dax under the expansion factor near 2.0 had a different profile which showed minimum values at 0.6–0.8 relative bed height, and the potential mechanism was discussed. These results would be useful for the characterization of axial dispersion and modeling protein adsorption in expanded beds under varying operation conditions.

Relative importance of column and adsorption parameters on the productivity in preparative liquid chromatography II: Investigation of separation systems with competitive Langmuir adsorption isotherms

In this study we investigated how the maximum productivity for commonly used, realistic separation system with a competitive Langmuir adsorption isotherm is affected by changes in column length, packing particle size, mobile phase viscosity, maximum allowed column pressure, column efficiency, sample concentration/solubility, selectivity, monolayer saturation capacity and retention factor of the first eluting compound. The study was performed by generating 1000 random separation systems whose optimal injection volume was determined, i.e., the injection volume that gives the largest achievable productivity. The relative changes in largest achievable productivity when one of the parameters above changes was then studied for each system and the productivity changes for all systems were presented as distributions. We found that it is almost always beneficial to use shorter columns with high pressure drops over the column and that the selectivity should be greater than 2. However, the sample concentration and column efficiency have very limited effect on the maximum productivity. The effect of packing particle size depends on the flow rate limiting factor. If the pumps maximum flow rate is the limiting factor use smaller packing, but if the pressure of the system is the limiting factor use larger packing up to about 40μm.

Hydrophilic interaction chromatography and its recent applications in environmental analysis

Hydrophilic interaction chromatography (HILIC) has been used as a supplement method of reversed-phase liquid chromatography (RPLC) for the separation of the polar and hydrophilic compounds, and has been received more and more attention in recent years. One reason is that the separation problems of strongly polar compounds are rising in various fields. Another is that there are some obvious advantages of HILIC compared with RPLC in the separation of polar compounds, such as low viscosity of mobile phase, good permeability of the column, high sensitivity when coupled to mass spectrometry and low back pressure. In this review, the development history, characteristics and retention mechanism of HILIC are summarized. The main attention in this review is on the applications of HILIC in environmental analysis. In addition, the advantages and disadvantages of HILIC and RPLC in the separation of pollutants are discussed, and the development trends of HILIC applications in environmental analysis are proposed.

Quantification aspects of constant pressure (ultra) high pressure liquid chromatography using mass-sensitive detectors with a nebulizing interface

The present contribution investigates the quantitation aspects of mass-sensitive detectors with nebulizing interface (ESI-MSD, ELSD, CAD) in the constant pressure gradient elution mode. In this operation mode, the pressure is controlled and maintained at a set value and the liquid flow rate will vary according to the inverse mobile phase viscosity. As the pressure is continuously kept at the allowable maximum during the entire gradient run, the average liquid flow rate is higher compared to that in the conventional constant flow rate operation mode, thus shortening the analysis time. The following three mass-sensitive detectors were investigated: mass spectrometry detector (MS), evaporative light scattering detector (ELSD) and charged aerosol detector (CAD) and a wide variety of samples (phenones, polyaromatic hydrocarbons, wine, cocoa butter) has been considered. It was found that the nebulizing efficiency of the LC-interfaces of the three detectors under consideration changes with the increasing liquid flow rate. For the MS, the increasing flow rate leads to a lower peak area whereas for the ELSD the peak area increases compared to the constant flow rate mode. The peak area obtained with a CAD is rather insensitive to the liquid flow rate. The reproducibility of the peak area remains similar in both modes, although variation in system permeability compromises the ‘long-term’ reproducibility. This problem can however be overcome by running a flow rate program with an optimized flow rate and composition profile obtained from the constant pressure mode. In this case, the quantification remains reproducibile, despite any occuring variations of the system permeability. Furthermore, the same fragmentation pattern (MS) has been found in the constant pressure mode compared to the customary constant flow rate mode.

Imprinted polymers for chiral resolution of (±)-ephedrine, 4: Packed column supercritical fluid chromatography using molecularly imprinted chiral stationary phases

(−)-Ephedrine-molecularly imprinted polymers (MIPs) have been successfully used as stationary phases in supercritical fluid chromatography for the separation of (±)-ephedrine enantiomers. This approach combines the simple preparation and predictable elution order of MIP stationary phases with the superior mobile phase diffusivity and low viscosity of supercritical fluid mobile phases. The optimised mobile phase comprised supercritical carbon dioxide with a modifier consisting of MeOH/isopropylamine/H2O 93:5:2 (v/v/v). In many cases, better resolution separations were observed compared to when liquid mobile phases were used, and better separations achieved at high sample loads, although interestingly the MIPs which work best in SFC are different from the MIPs that work best in HPLC with an amine modifier. The MIP stationary phases were stable under the conditions employed and the chromatography was reproducible. This work opens the door to exploiting MIP stationary phases in preparative SFC.

Kinetic optimisation of the reversed phase liquid chromatographic separation of proanthocyanidins on sub-2 μm and superficially porous phases

Phenolic compounds, and proanthocyanidins in particular, are important natural molecules which are of significant importance due to their sensory and biological activities. The analysis of proanthocyanidins in natural products is very challenging due to their complex nature. In this study, the kinetic performance of a range of recently developed C18 columns, including sub-2μm fully porous and 2.6μm superficially porous particle-packed columns, was evaluated for improved proanthocyanidin analysis. The kinetic plot method was employed to compare the ultimate performance limits of each column in terms of efficiency and speed for different maximum pressures and temperatures using representative proanthocyanidins comprising a range of molecular weights and functionalities as test analytes. By combining plate height data with relevant parameters such as column permeability and mobile phase viscosity, plots of practically attainable efficiencies as a function of analysis time for specific experimental configurations were obtained and performance limits for all investigated supports could accurately be compared. Both fully- and superficially porous particles provided significant speed and/or efficiency gains compared to conventional 5μm particle packed columns. Analyte properties, particle size and packing quality as well as analysis temperature were all found to have a significant influence on the performance of the presently investigated chromatographic supports. For smaller compounds, higher optimal linear velocities and better performance in the low-efficiency range were observed, while the lower diffusion coefficients of larger procyanidins resulted in lower optimal linear velocities and better performance in the high-efficiency regime. Faster analyses become possible at higher temperatures due to decreased eluent viscosity and faster mass transfer, which was especially beneficial for larger compounds and resulted in dramatic improvement in efficiency. A possible explanation of the abnormal behaviour of oligomeric procyanidins is presented. Our findings indicate that new column formats, when used under optimal conditions, significantly improve the speed and/or efficiency of reversed phase liquid chromatographic analyses of proanthocyanidins.

Optimization and validation of a fast ultrahigh‐pressure liquid chromatographic method for simultaneous determination of selected sulphonamides in water samples using a fully porous sub‐2 μm column at elevated temperature

High temperature in HPLC is considered a valuable tool helping to overcome the increase in the column backpressure when using small packing particles such as sub-2 μm, as it allows reduction in the mobile-phase viscosity. In this study, a fast analytical method based on HPLC-UV was developed using a sub-2 μm column at elevated temperature for the simultaneous determination of nine sulphonamides. Owing to the lower viscosity of the mobile phase, the separation could be achieved in 3 min at 60°C for all analytes. The effect of temperature, the organic modifier percentage and the flow rate on the retention time was studied. The method developed was used for the determination of selected sulphonamides in surface and wastewater samples. Sample preparation was carried out by solid-phase extraction on Oasis HLB cartridges. The method developed was validated based on the linearity, precision, accuracy, detection and quantification limits. The recovery ranged from 70.6 to 96 % with standard deviations not higher than 4.7%, except for sulphanilamide. Limits of detection ranged from 1 to 10 μg/L after optimization of all analytical steps. This method has the highest performance in terms of analytical speed compared with other published HPLC-UV methods for the determination of sulphonamides in water.

The mobile phase motion in ascending Micellar thin-layer chromatography with normal-phase plates

The physical chemical characteristics (surface tension and viscosity) of micellar mobile phases based on the cationic surfactant cetylpiridinium chloride and additives of alcohols (ethanol, 1-propanol, 1-butanol, 1-pentanol) have been obtained in this work. The effect of mobile phase properties on the motion of mobile phase in the ascending mode of thin-layer chromatography (TLC) was investigated. The applicability of a quadratic relationship between the time of mobile phase motion and the distance from the solvent entry position of mobile phase and front of the mobile phase was validated for micellar TLC. It was observed that the retardation factors of hydrophilic test compounds change significantly only at low contents of the modifier in micellar mobile phase.

Comparison of the quantitative performance of constant pressure versus constant flow rate gradient elution separations using concentration-sensitive detectors

This contribution discusses the difference in chromatographic performance when switching from the customary employed constant flow rate gradient elution mode to the recently re-introduced constant pressure gradient elution mode. In this mode, the inlet pressure is maintained at a set value even when the mobile phase viscosity becomes lower than the maximum mobile phase viscosity encountered during the gradient program. This leads to a higher average flow rate compared to the constant flow rate mode and results in a shorter analysis time. When both modes carry out the same mobile phase gradient program in volumetric units, normally identical selectivities are obtained. However, small deviations in selectivity are found due to the differences in pressure and viscous heating effects. These selectivity differences are of the same type as those observed when switching from HPLC to UHPLC and are inevitable when speeding up the analysis by applying a higher pressure. It was also found that, when using concentration-sensitive detectors, the constant pressure elution mode leads to identical peak areas as the constant flow rate mode. Also the linearity is maintained. In addition, the repeatability of the peak area and retention time remains the same when switching between both elution modes.

High-Efficiency Liquid Chromatography Using Sub-2 μm Columns at Elevated Temperature for the Analysis of Sulfonamides in Wastewater

Efficiency in HPLC can be enhanced by increasing the column length and/or decreasing the particle size. The use of high temperature in HPLC has emerged as a valuable tool to overcome the increase in column backpressure when using small packing particles, as it allows for reduction in mobile phase viscosity. In this study, high plate count was obtained by coupling sub-2 μm columns at elevated temperature to reduce the viscosity of the mobile phase, thus reducing the column backpressure. At 80 °C, up to three columns of 15 cm × 4.6 mm I.D. packed with 1.8 μm particles could be coupled generating ~84,000 theoretical plates for the last eluting compound. The number of theoretical plates was increased on average by a factor of ~3.6 when three columns were coupled at 80 °C compared with one column at 30 °C. The relationships between separation efficiency and column length were examined using Van Deemter plots constructed at 30 °C and 80 °C for different column lengths. The advantages of using coupled columns in combination with elevated temperature for the environmental analysis were illustrated using test mixtures comprised of eight sulfonamides separated on one column at 30 °C and three coupled columns at 80 °C by isocratic elution. Sample clean up was carried out by employing solid-phase extraction (SPE) using Oasis HLB cartridges. The method developed was validated based on parameters such as linearity, precision, accuracy, detection and quantification limits. Recoveries generally ranged from 71.7 to 99% (with the exception of sulfanilamide), with standard deviations not higher than 4.7%. The detection limits of the method ranged from 0.6–2 μg L−1, while limits of quantification were in the range 2–6.7 μg L−1 with UV detection.

Differential magnetic catch and release: experimental parameters for controlled separation of magnetic nanoparticles

Differential magnetic catch and release (DMCR) has been used as a method for the purification and separation of magnetic nanoparticles. DMCR separates nanoparticles in the mobile phase by magnetic trapping of magnetic nanoparticles against the wall of an open tubular capillary wrapped between two narrowly spaced electromagnetic poles. Using Au and CoFe(2)O(4) nanoparticles as model systems, the loading capacity of the 250 μm diameter capillary is determined to be ∼130 μg, and is scalable to higher quantities with larger bore capillary. Peak resolution in DMCR is externally controlled by selection of the release time (R(t)) at which the magnetic flux density is removed, however, longer capture times are shown to reduce the capture yield. In addition, the magnetic nanoparticle capture yields are observed to depend on the nanoparticle diameter, mobile phase viscosity and velocity, and applied magnetic flux. Using these optimized parameters, three samples of CoFe(2)O(4) nanoparticles whose diameters are different by less than 10 nm are separated with excellent resolution and capture yield, demonstrating the capability of DMCR for separation and purification of magnetic nanoparticles.

Use of kinetic plots for the optimization of the separation time in ultra‐high‐pressure LC

The kinetic-plot approach, in which experimental t(0) and N-values are extrapolated to the performance at maximum system pressure by increasing the column length, was validated by coupling 250×3 mm columns packed with 3 μm particles. The extra-column volume introduced by coupling columns could be neglected with respect to the peak volumes. Plate numbers of up to 132,000 were experimentally achieved by coupling four columns. The maximum deviation between the experimental and predicted plate numbers was 7% for two coupled columns, and decreasing to 0.1% for four coupled columns. Kinetic plots were used to find the conditions to separate a critical pair, with a preset value for the effective plate number, in the shortest possible time. For high-efficiency separations yielding 100,000 effective plates, the optimum critical-pair retention factor was around 4.5. Kinetic plots are presented to find the optimal column length to obtain the fastest possible 100,000 effective-plate separation, taking into account the effect of mobile-phase viscosity on column pressure, and consequently the optimum column length.

Direct recovery of recombinant nucleocapsid protein of Nipah virus from unclarified Escherichia coli homogenate using hydrophobic interaction expanded bed adsorption chromatography

A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPas. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.