Behavior of packing materials in axially compressed chromatographic columns

The behavior of a packing material (Luna C18 from Phenomenex, Torrance, CA, USA) was studied during the consolidation of a column bed under axial compression stress. The kinetics of this consolidation, the permeability and efficiency of the columns obtained, and the reproducibility of these column properties were measured under different conditions. The consolidation process and the column properties are considerably affected by the friction between the packing material in the bed and the column wall. Clear evidence of this wall effect was demonstrated. The apparent permeability of columns consolidated under the same axial stress increases with increasing column length. The apparent modulus of elasticity of the beds increases with increasing column length. The shear resistance between the packed bed and the column wall was measured for columns of different lengths. It increases rapidly with increasing bed length. The column efficiency for thiourea (unretained) and phenyloctane (retention factor, k' approximately 1) was much poorer after recompression than after the first compression. It depended little on the compression stress. The effect of the column length was small.     

Use of ultrasound to monitor the packing of large-scale columns,the monitoring of media compression and the passage of molecules,such as monoclonal antibodies,through the column bed during chromatography

The novel use of ultrasound as a detector in pilot- and production-scale chromatography is described. The difficulties in packing production scale chromatography columns using an integral packing valve are reviewed. Results are presented from the packing of 400- and 600-mm diameter columns with various medias. From these results it is proposed that when packing large columns using a packing valve, for a given medium and column size, there is an ideal rate and pattern (or control "corridor") by which the bed builds in order to give optimum performance. Ultrasound was shown to be able to monitor the building of such a column bed as the medium was pumped into the column. It was found that the ultrasound detector was sensitive to bed compression, mobile phase composition and components such as acetone, albumin, casein and monoclonal antibodies while on the chromatography bed. This enabled the visualisation, by ultrasound, of these components as they were chromatographed through the column.     

制备型液相色谱动态轴向压缩柱技术和应用

综述了动态轴向压缩技术装填液相色谱柱的原理、装置及装置设计的发展状况,对动态轴向压缩装填过程和柱性能的影响因素如柱长、填料和调浆溶剂的性质、压缩压力进行了分析,讨论了柱内密度分布不均、柱壁效应两种现象和解决的办法,评价了这种技术的优缺点,介绍了其应用、发展现状与前景。引用文献50篇。     

Axial and radial diffusion coefficients in a liquid chromatography column and bed heterogeneity

The axial and transverse diffusion coefficients of a band of iodine in a chromatographic column were measured optically as a function of time. It was found that the axial diffusion coefficient remains constant even when the edges of the sample band get close to the wall. By contrast, the radial diffusion coefficient decreases progressively with increasing time when the edges of the sample band leave the core region and begin to diffuse inside the wall region. The local axial and transverse diffusion coefficients of the band decrease from the column center toward the wall. Hence, the increase in local height equivalent to a theoretical plate observed in the region close to the wall must be explained by increasing mass transfer resistances and degree of heterogeneity of the bed.     

工业制备色谱在中药分离制备中的应用

概述了工业制备色谱柱型结构、填料、色谱技术特性的研究进展,对目前普遍使用的工业制备色谱的工作原理作了较为全面的评述,总结了动态轴向压缩色谱和模拟移动床色谱技术在中药分离制备中的应用和发展。     

The reverse auto-compression system: a new type of column for preparative liquid chromatography

Axial compression of the stationary phase is an easy technique for packing preparative liquid chromatography columns. However, the compression piston often needs an important external device to move the external jack. A new type of column is described which uses an auto-compression system to move the piston and compress the packing material. The system is fitted with a new annular injector which is known to reduce the peak tailing. The results of the tests demonstrate the ease of use of this column, its efficiency and stability.     

Are axial and radial flow chromatography different?

Radial flow chromatography can be a solution for scaling up a packed bed chromatographic process to larger processing volumes. In this study we compared axial and radial flow affinity chromatography both experimentally and theoretically. We used an axial flow column and a miniaturized radial flow column with a ratio of 1.8 between outer and inner surface area, both with a bed height of 5 cm. The columns were packed with affinity resin to adsorb BSA. The average velocity in the columns was set equal. No difference in performance between the two columns could be observed. To gain more insight into the design of a radial flow column, the velocity profile and resin distribution in the radial flow column were calculated. Using mathematical models we found that the breakthrough performance of radial flow chromatography is very similar to axial flow when the ratio between outer and inner radius of the radial flow column is around 2. When this ratio is increased, differences become more apparent, but remain small. However, the ratio does have a significant influence on the velocity profile inside the resin bed, which directly influences the pressure drop and potentially resin compression, especially at higher values for this ratio. The choice between axial and radial flow will be based on cost price, footprint and packing characteristics. For small-scale processes, axial flow chromatography is probably the best choice, for resin volumes of at least several tens of litres, radial flow chromatography may be preferable.     

Variations of particle size and bed voidage distributions in expanded bed during transient operation processes

Changes in bed expansion are frequently encountered during an expanded bed adsorption, such as during the initial bed expansion, feed loading and washing processes. We have here studied the changes of local particle size distribution and bed voidage of an expanded bed in the initial bed expansion process as well as those during the changes in mobile phase viscosity, which imitated feed loading and column washing processes. Using a glass column modified with three side sampling ports and Streamline AC as the solid phase, experimental measurements on a series of operation moments during the transient processes were carried out by sampling the particles from within the column at different axial positions. In the initial bed expansion process, the gradual formation of an axial classification from a settled bed to a stable expanded bed was first displayed. By changing the mobile phase from water to 10% (w/w) glycerol solution or vice versa, the variations in both the particle size distribution and bed voidage corresponding to the increase or decrease of the bed height caused by the changes of the mobile phase viscosity were examined as well. The transient changes of the local particle size distribution and bed voidage first occurred in the bed bottom and then progressed from bottom to top along the axial direction. However, the changes of bed voidage at different axial positions were not unidirectional. That is, by changing the mobile phase to the high-viscosity glycerol solution, a constant increase of the bed voidage was observed in the bed bottom, while a distinct decrease of the bed voidage before its increase was involved at the middle and top positions. This is ascribed to the compression effect caused by the upward movement of the lower part particles.     

十二烷基键合氧化锆填充电色谱柱的研究

以十二烷基键合氧化锆(C12-ZrO2)作为固定相,制备了填充毛细管电色谱(CEC)柱,较为系统地研究了流动相条件对电渗流的影响、填充CEC柱的稳定性、碱性与中性化合物的保留与流动相pH值和有机溶剂含量的关系。C12-ZrO2固定相填充CEC柱在pH3～11．7范围内具有极好的稳定性;利用磷酸盐与氧化锆表面之间较强的相互作用,能够有效解决传统硅胶键合烷基固定相在有机溶剂含量低的流动相条件下不稳定的问题;同时吸附磷酸盐的固定相表面使得在更宽的流动相pH值范围内CEC柱有足够的电渗流,进一步拓宽CEC的应用领域。     

Phenomenological study of the bed--wall friction in axially compressed packed chromatographic columns

The properties of column beds prepared with slurries of Kromasil C8 in 12 different solvents, using the same axial compression skid, were investigated. The extent of the consolidation of the column beds, their permeabilities, and the friction shear stress of these beds against the column wall were determined, as well as the column efficiencies (for an unretained tracer). The results of this study illustrate the influence of the wall effect on the consolidation. The permeability of columns consolidated under a constant compression stress was found to increase with increasing bed length. The bed-wall friction shear stress increases rapidly with increasing bed length and varies widely with the nature of the solvent used. No correlation was found between this shear stress and any physico-chemical property of the solvent. The best efficiency was observed for a column consolidated from a slurry in ethanol.     

Hydrodynamic impact of particle shape in slurry packed liquid chromatography columns

We report on a series of flow velocity and efficiency profiles, which were measured across the cross section of preparative chromatographic columns packed with different stationary phase materials using computed tomography. It is shown that this non-invasive technique is very useful for visualization of the inner part of a packed column and measurement of the spatial resolved column packing properties. For evaluation of the influence of the particle shape on the velocity distribution and column performance, irregular and spherical reversed phases were studied in detail. The results showed a decreasing velocity towards the column wall most certainly due to a lower permeability. This effect was much less pronounced in the case of spherical particles, indicating a more homogenous packing structure. The influence of the column packing pressure, as a possible measure for improvement of the packing homogeneity was also studied. It was shown that under the same packing conditions spherical particles always lead to a more homogeneous packing. The overall results of this work contribute to the origin of the fact that spherical material is superior to irregular one from the hydrodynamic point of view.     

Characterization of the properties of stationary phases for liquid chromatography in aqueous mobile phases using aromatic sulphonic acids as the test compounds

We investigated the effects of the concentration of naphthalene sulphonic acids (NSAs) as anionic test compounds in the injected sample and of the salt additives to the mobile phase on ion-exclusion. The retention behaviour of NSAs sensitively reflects even minor changes in the ionic and hydrophobic interactions and can be useful for predicting the effects of the stationary phases in reversed-phase chromatography of polar and ionic compounds, both small ones and biopolymers, e.g., oligonucleotides. We studied chromatographic properties of several stationary phases intended for separations in aqueous mobile phases: a C18 column end-capped with polar hydrophilic groups, a densely bonded C8 column doubly end-capped with short alkyl groups, a short alkyl stationary phase designed to keep full pore accessibility in highly-aqueous mobile phases and a Bidentate column with “bridged” C18 groups attached to the silica hydride support. The chemistry and pore structure of various types of column packing materials and of the salt additives to the mobile phase affect the proportion of the pore volume non-accessible to anions due to ion-exclusion and consequently the peak asymmetry and hydrophobic selectivity in reversed-phase chromatography of organic acids. We also addressed the problems connected with the determination of column hold-up volume in aqueous mobile phases. The accessibility of the stationary phase for anionic compounds in contact with the sample zone is affected by ion-exclusion due to repulsive interactions with the negatively charged surface in the pores of the stationary phase. The accessible part of the stationary phase increases and consequently the migration velocity along the column decreases with increasing concentration of the sample in the zone moving along the column. Because of a limited access to the stationary phase, its capacity can be easily overloaded. The combination of the column overload and ion-exclusion effects may result in fronting or tailing peak asymmetry. To explain this behaviour, we proposed a modified Langmuir model, respecting the variation of the column capacity due to the effects of sample concentration on ion-exclusion.     

Performance and lifetime of slurry packed capillary columns for high performance liquid chromatography

Fused silica capillary columns of the internal diameter of 320 μm were packed with the Nucleosil C18 stationary phase of 5 μm using the slurry packing method. The time of the bed compaction phase, packing pressure, and the use of ultrasound varied to study their influence on the column performance. Van Deemter curves were measured and separation impedance values were calculated in order to assess both separation efficiency and kinetic performance of the columns. Selected columns were tested again after nine months to evaluate the stability of their beds. Separation efficiencies of all columns were similar, but a major difference, caused by the use of ultrasound, was observed in the bed stability. Columns sonicated for 25 minutes during the bed compaction phase exhibited unchanged performance in the course of several months, while the performance of non-sonicated columns decreased.     

Identification of the factors that influence the reproducibility of chromatographic retention data

Principal component analysis was used to identify the parameters that influence the column-to-column and batch-to-batch reproducibility of retention times and retention factors measured on Symmetry C18, Kromasil C18, Luna C18 (2) and Vydac RP C18, all reversed-phase silica columns. We devised a procedure that allows the determination of the differences in column volume and packing density between two columns, provided that these columns are packed with identical stationary phases (i.e., phases that originate from the same batch). Principal component analysis of the retention times confirmed that the column-to-column variations of the column volume and the total porosity of the bed are the factors that influence the reproducibility of the retention times, the column volume being the major factor. For the fluctuations of the retention factors, the column phase ratios (or the bed porosities) and some specific, secondary retention mechanisms are responsible. All the C18 columns investigated proved to behave in a very similar fashion. Two principal components were always sufficient to characterize the variations of either the retention times or the retention factors.     

Size exclusion chromatography of polypeptides on Bio-Gel P2 in the dynamic axial compression mode

The effect of dynamic axial compression (DAC) upon the structure of a bed packed with semirigid Bio-Gel P2 and its chromatographic parameters was studied in the separation of peptides of various mass. In the studied range of DAC pressure, 0–5 bar, a decrease in the retention times of the studied substances and the standard deviation of their peaks is observed. In spite of a reduced packed bed height and an increase in the linear velocity of the eluent, the height equivalent to the theoretical plate (HETP) does not rise. The resolution of the separated substances increases throughout the studied range of DAC pressure with the Bio-Gel P2 exclusion limit extended. According to the results obtained, DAC provides more efficient optimisation of the bed parameters in columns packed with Bio-Gel P2 than in columns packed with Sephadex G-25 due to the greater rigidity of the former packing.     

Inherent peak compression of charged analytes in electrochromatography

This work resolves peak compression of charged analytes in CEC with strong cation‐exchange stationary phase particles. By combining electrochromatographic peak shape analysis with the results of numerical simulations and confocal laser scanning microscopy in the packed capillaries, we identify electrical field‐induced concentration polarization as the key physical phenomenon responsible for the inherent existence of local electrical field gradients on the scale of an individual support particle. Consequently, positive and negative field gradients exist between and inside the particles along the whole packing. Their intensity depends on the particles cation‐selectivity (governed by the particles volume charge density and the mobile phase ionic strength) and the applied field strength. The interplay of these local field gradients with the analytes retention (intraparticle adsorption) determines whether fronting, tailing, or spiked analyte peaks are observed, and it provides a mechanism by which strongly retained analytes can be eluted over long distances with little zone dispersion. Our analysis explains the “anomalous” peak compression effects with strong cation‐exchange particles, which have been reported more than a decade ago (Smith, N. W., Evans, M. B., Chromatographia 1995, 41, 197–203) and since then remained largely unresolved.     

A new split-flow injector for preparative liquid chromatography columns. Annular injection system

The packing of large-diameter columns for liquid chromatography is still difficult and numerous publications have reported results from tests which prove the packing is heterogeneous. The slurry is more compact in the wall region and this reduces the flow of the mobile phase, leading to distortion of the sample zone in the column and generation of peak tailing. A new type of injection system for the head of the column has been developed which divides the flow of the solvent from the pump into two parts. One, without sample, is directed to a crown injector, close to the wall. By adjusting the ratio of this flow to that of the bulk flow it is possible to increase the speed of the mobile phase in this part of the stationary phase and reduce distortion of the sample zone. The other part of the solvent carries the sample to the stationary phase through a distributor. The results demonstrate the benefits of this annular injection system, which include increased efficiency and improved column stability.     

Conformational changes of poly(N-isopropylacrylamide) chains at air/water interface: effects of temperature, compression rate, and packing density

This study concerns the effects of temperature, compression rate, and packing density on conformational properties of polystyrene-block-poly(N-isopropylacryamide) (PS-b-PNIPAM) diblock copolymer monolayers at the air/water interface using the Langmuir balance technique. The dependency of surface pressure changes on temperature and compression rate is strongly influenced by the conformations of PNIPAM chains at the interface, which can be adjusted by varying the packing density. Specifically, when loops or tails are formed at the interface, PNIPAM chains display thermosensitive properties due to hydration/dehydration and obvious dependence on compression rate. When PNIPAM chains take train conformation at the air/water interface, however, the surface pressure changes are nearly independent of temperature and compression rate because almost all segments of the PNIPAM chains are adsorbed at the interface and the nonpolar isopropyl groups are preferentially oriented toward the air. Our work reveals that one could manipulate stimuli-responsive properties of PNIPAM chains at the interface simply by adjusting the conformations of PNIPAM chains.     

Enantiomer separation by strong anion-exchange capillary electrochromatography with dynamically modified sulfated beta-cyclodextrin

A novel mode of capillary electrochromatography (CEC) based on a dynamically modified stationary phase was presented for chiral separation. The capillary column was packed with strong anion-exchange (SAX) stationary phase packing; the sulfated beta-cyclodextrin (S-CD), which was added to the mobile phase, was dynamically adsorbed to the packing surface. Separation of enantiomers was achieved by their different abilities to form an inclusion complex with the adsorbed S-CD. The enantiomers of tryptophan, praziquantel, atropine, metoprolol, and verapamil were successfully separated in this system with a column efficiency of 36000-412000 plates/m. The resolution value obtained for atropine was as high as 11.23. The superiority of CEC with a dynamically modified stationary phase over that with a physically adsorbed stationary phase was demonstrated. The influence of ionic strength, S-CD concentration, and methanol content on separation was also studied.     

Packings and stationary phases in preparative column liquid chromatography

Although the theoretical treatment of chromatographic processes on a preparative scale provides guidelines to the extent to which packing and stationary phase properties affect the target quantities such as sample input, throughput and resolution times sample input, a series of additional criteria were established to judge the quality of a packing in preparative column liquid chromatography. These include bed stability and flow resistance, chemical resistance and purity, solute accessibility, mass and biological recovery, fouling, regeneration and cost. Applying these criteria, the relative importance of physical and chemical structure parameters of packings and stationary phases was assessed. Commercial packings with mean particle diameters dp greater than 20 micron were listed for adsorption, size exclusion, ion-exchange and affinity chromatography. An analysis of the characteristic features of phase systems showed that adsorption media offer a high selectivity combined with adequate loadability, whereas ion exchangers and affinity media were best suited for biospecific solutes, particularly biopolymers, which can be attributed to their high selectivity and loadability.