Measurement of the axial and radial temperature profiles of a chromatographic column. Influence of thermal insulation on column efficiency

The temperatures of the metal wall along a chromatographic column (longitudinal temperature gradients) and of the liquid phase across the outlet section of the column (radial temperature gradients) were measured at different flow rates with the same chromatographic column (250 mm x 4.6 mm). The column was packed with 5 microm C18-bonded silica particles. The measurements were carried out with surface and immersion thermocouples (all junction Type T, +/-0.1 K) that measure the local temperature. The column was either left in a still-air bath (ambient temperature, T(ext) = 295-296 K) or insulated in a packing foam to avoid air convection around its surface. The temperature profiles were measured at several values of the inlet pressure (approximately = 100, 200, 300 and 350 bar) and with two mobile phases, pure methanol and a 2.5:97.5 (v/v, %) methanol:water solution. The experimental results show that the longitudinal temperature gradients never exceeded 8 K for a pressure drop of 350 bars. In the presence of the insulating foam, the longitudinal temperature gradients become quasi-linear and the column temperature increases by +1 and +3 K with a water-rich (heat conductivity approximately = 0.6 W/m/K) and pure methanol (heat conductivity approximately = 0.2 W/m/K), respectively. The radial temperature gradients are maximum with methanol (+1.5 K at 290 bar inlet pressure) and minimum with water (+0.8 K at 290 bar), as predicted by the solution of the heat transfer balance in a chromatographic column. The profile remains parabolic all along the column. Combining the results of these measurements (determination of the boundary conditions on the wall, at column inlet and at column outlet) with calculations using a realistic model of heat dispersion in a porous medium, the temperature inside the column could be assessed for any radial and axial position.     

Modeling of thermal processes in very high pressure liquid chromatography for column immersed in a water bath: Application of the selected models

Currently, chromatographic analyses are carried out by operating columns packed with sub-2 μm particles under very high pressure gradients, up to 1200 bar for 5 cm long columns. This provides the high flow rates that are necessary for the achievement of high column efficiencies and short analysis times. However, operating columns at high flow rates under such high pressure gradients generate a large amount of heat due to the viscous friction of the mobile phase stream that percolates through a low permeability bed. The evacuation of this heat causes the formation of significant or even large axial and radial gradients of all the physico-chemical parameters characterizing the packing material and the mobile phase, eventually resulting in a loss of column efficiency. We previously developed and successfully applied a model combining the heat and the mass balances of a chromatographic column operated under very high pressure gradients (VHPLC). The use of this model requires accurate estimates of the dispersion coefficients at each applied mobile phase velocity. This work reports on a modification of the mass balance model such that only one measurement is now necessary to accurately predict elution peak profiles in a wide range of mobile phase velocities. The conditions under which the simple equilibrium-dispersive (ED) and transport-dispersive (TD) models are applicable in VHPLC are also discussed. This work proves that the new combination of the heat transfer and the ED model discussed in this work enables the calculation of accurate profiles for peaks eluted under extreme conditions, like when the column is thermostated in a water bath.     

Fundamental chromatographic equations designed for columns packed with very fine particles and operated at very high pressures. Applications to the prediction of elution times and the column efficiencies

The wall temperatures of three Acquity-BEH-C18columns (2.1 mm x 50, 100, and 150 mm) and the temperature of the incoming eluent were maintained constant at 289 K, using a circulating water heat exchanger. The retention times and the band broadening of naphtho[2,3-a]pyrene were measured for each column as a function of the flow rate applied. Pure acetonitrile was used as the eluent. The flow rate dependence of neither elution volumes nor bandwidths can be accounted for by classical models of retention and HETP, respectively, since these models assume columns to be isothermal. Because the heat generated by friction of the eluent against the column bed increases with increasing flow rate, the column bed cannot remain isothermal at high flow rates. This heat is evacuated radially and/or longitudinally by convection, conduction, and radiation. Radial and axial temperature gradients are formed, which are maximum and minimum, respectively, when the temperature of the column wall is kept uniform and constant. The retention times that we measured match well with the values predicted based on the temperature distribution along and across the column, which we calculated and on the temperature dependence of the retention for the same column operated isothermally (i.e., at very low flow rate). The rate of band spreading varies along non-isothermal columns, so the HETP can only be defined locally. It is a function of the axial coordinate. A new contribution is needed to account for the radial thermal heterogeneity of the column, hence the radial distribution of the flow velocities, which warps the elution band. A new model, based on the general dispersion theory of Aris, allows a successful prediction of the unusually large bandwidths observed with columns packed with fine particles, operated at high flow rates, hence high inlet pressures.     

Prediction of the influence of the heat generated by viscous friction on the efficiency of chromatography columns

The combination of the heat balance in a chromatographic column percolated by a stream of mobile phase and of the model of band migration under linear conditions along such a column permits the calculation of the axial and radial temperature distributions in the column, of the elution band profiles, and of the column efficiency under different sets of experimental conditions. The calculated results are always consistent with the experimental results published by different groups and often in good quantitative agreement. Minor discrepancies arise from difficulties in deriving precise estimates of the heat transfers from the column due to the massive endfittings of the column and to uncontrolled heat transfer from the column tube to ambient air.     

Hydrophobic interaction chromatography at low salt concentration for the capture of monoclonal antibodies

We evaluated hydrophobic interaction chromatography (HIC) at low salt concentration for the capture of proteins from feed stocks by using monoclonal antibodies as model samples. It was indicated that the HIC at low salt concentration on critical hydrophobicity supports has a potential for capturing hydrophobic monoclonal antibodies directly from large volumes of feed stocks and recovering bound monoclonal antibodies in high yield. On the other hand, the HIC at low salt concentration did not seem so useful for the capture of weakly hydrophobic monoclonal antibodies. The recovery of weakly hydrophobic monoclonal antibodies from columns packed with critical hydrophobicity supports was not quantitative and significantly decreased as the residence time of the monoclonal antibodies in the columns became longer.     

Generalizing a two-conformation model for describing salt and temperature effects on protein retention and stability in hydrophobic interaction chromatography

A two-conformation adsorption model that includes the effects of salt concentration and temperature on both stability and adsorption has been developed to describe the effects of secondary protein unfolding on hydrophobic interaction chromatography (HIC). The model has been applied to a biotech protein and to beta-lactoglobulin on Phenyl Sepharose 6FF low sub HIC media. Thermodynamic property models for adsorption and protein stability with parameters obtained from experimental chromatographic data successfully describe observed chromatographic behavior over ranges of temperature and salt concentration, provide predictions of distribution among different conformers, and give a basis for calculating trends in retention strength and stability with changing conditions, that might prove useful in HIC process development.     

Modeling of thermal processes in high pressure liquid chromatography: I. Low pressure onset of thermal heterogeneity

Heat due to viscous friction is generated in chromatographic columns. When these columns are operated at high flow rates, under a high inlet pressure, this heat causes the formation of significant axial and radial temperature gradients. Consequently, these columns become heterogeneous and several physico-chemical parameters, including the retention factors and the parameters of the mass transfer kinetics of analytes are no longer constant along and across the columns. A robust modeling of the distributions of the physico-chemical parameters allows the analysis of the impact of the heat generated on column performance. We developed a new model of the coupled heat and mass transfers in chromatographic columns, calculated the axial and radial temperature distributions in a column, and derived the distributions of the viscosity and the density of the mobile phase, hence of the axial and radial mobile phase velocities. The coupling of the mass and the heat balances in chromatographic columns was used to model the migration of a compound band under linear conditions. This process yielded the elution band profiles of analytes, hence the column efficiency under two different sets of experimental conditions: (1) the column is operated under natural convection conditions; (2) the column is dipped in a stream of thermostated fluid. The calculated results show that the column efficiency is remarkably lower in the second than in the first case. The inconvenience of maintaining constant the temperature of the column wall (case 2) is that retention factors and mobile phase velocities vary much more significantly across the column than if the column is kept under natural convection conditions (case 1).     

Numerical modeling of elution peak profiles in supercritical fluid chromatography. Part I—Elution of an unretained tracer

When chromatography is carried out with high-density carbon dioxide as the main component of the mobile phase (a method generally known as “supercritical fluid chromatography” or SFC), the required pressure gradient along the column is moderate. However, this mobile phase is highly compressible and, under certain experimental conditions, its density may decrease significantly along the column. Such an expansion absorbs heat, cooling the column, which absorbs heat from the outside. The resulting heat transfer causes the formation of axial and radial gradients of temperature that may become large under certain conditions. Due to these gradients, the mobile phase velocity and most physico-chemical parameters of the system (viscosity, diffusion coefficients, etc.) are no longer constant throughout the column, resulting in a loss of column efficiency, even at low flow rates. At high flow rates and in serious cases, systematic variations of the retention factors and the separation factors with increasing flow rates and important deformations of the elution profiles of all sample components may occur. The model previously used to account satisfactorily for the effects of the viscous friction heating of the mobile phase in HPLC is adapted here to account for the expansion cooling of the mobile phase in SFC and is applied to the modeling of the elution peak profiles of an unretained compound in SFC. The numerical solution of the combined heat and mass balance equations provides temperature and pressure profiles inside the column, and values of the retention time and efficiency for elution of this unretained compound that are in excellent agreement with independent experimental data.     

Protein adsorption on novel acrylamido-based polymeric ion-exchangers III. Salt concentration effects and elution behavior

The effect of salt concentration on the adsorption and desorption of BSA has been determined for a polymeric anion-exchanger based on acrylamido monomers. The material investigated possesses a high adsorption capacity at low salt concentration and the bound protein can be recovered quantitatively at high salt concentrations. The effects of salt on adsorption and desorption rates were evaluated from batch and shallow-bed experiments, and a model was developed to describe the data quantitatively. The adsorption capacity decreases as the salt concentration is increased, but both adsorption and desorption rates increase at higher salt concentrations. The predictability of the behavior of columns packed with this material was examined by comparing model predictions and experimental results obtained in laboratory columns. In general, a good agreement was obtained between predicted and experimental breakthrough and elution profiles, especially in shorter columns. Thus, the model allows a prediction of the effects of column length, mobile phase flow-rate, protein feed concentration, and salt concentration on dynamic capacity, productivity, and on the concentration of product fractions.     

Measurements and particle resolved modelling of the thermo- and fluid dynamics of a packed bed

The objective of this study is to investigate experimentally and numerically into heat-up, drying and pyrolysis of a packed bed consisting of large single particles. The novelty of the current approach is that the numerical model contrary to continuum mechanic approaches considers a packed bed as an ensemble of a finite number of particles, which may have different material properties or sizes. The heat-up, drying and pyrolysis process of each particle is described sufficiently accurate by a set of one-dimensional and transient differential conservation equations for mass and energy. Applying this model to all particles, including interactions between them, of a packed bed forms the entire backed bed process as a sum of individual particle processes. The arrangement of particles within a bed defines a void space between the particles. The flow through the void space of a packed bed is modelled as a flow through a porous media taking into account interaction between the solid and the gaseous phase by heat and mass transfer. Experiments for drying and pyrolysis of a packed bed were carried out for validation in a temperature range of T=120–530 °C. The temperatures and the mass loss due to drying and pyrolysis were recorded during the experiments. The measured mass loss of the packed bed due to drying were well predicted by the constant evaporation temperature model of the particles and thus, indicating, that the drying process is transport limited by heat transfer for large wood particles in a temperature range of T=120–530 °C. A comparison between experiments and predictions of pyrolysis yielded reasonable agreement for temperatures above T=300 °C. For temperatures of T≈200 °C the deviations were not acceptable. However, the results show, that a particle resolved approach is well suited to describe packed bed processes.     

Modeling of thermal processes in high pressure liquid chromatography: II. Thermal heterogeneity at very high pressures

Advanced instruments for liquid chromatography enables the operation of columns packed with sub-2 μm particles at the very high inlet pressures, up to 1000 bar, that are necessary to achieve the high column efficiency and the short analysis times that can be provided by the use of these columns. However, operating rather short columns at high mobile phase velocities, under high pressure gradients causes the production of a large amount of heat due to the viscous friction of the eluent percolating through the column bed. The evacuation of this heat causes the formation of significant axial and radial temperature gradients. Due to these thermal gradients, the retention factors of analytes and the mobile phase velocity are no longer constant throughout the column. The consequence of this heat production is a loss of column efficiency. We previously developed a model combining the heat and mass balance of the column, the equations of flow through porous media, and a linear isotherm model of the analyte. This model was solved and validated for conventional columns operated under moderate pressures. We report here on the results obtained when this model is applied to columns packed with very fine particles, operated under very high pressures. These results prove that our model accounts well for all the experimental results. The same column that elutes symmetrical, nearly Gaussian peaks at low flow rates, under relatively low pressure drops, provides strongly deformed, unsymmetrical peaks when operated at high flow rates, under high pressures, and under different thermal environments. The loss in column efficiency is particularly important when the column wall is kept at constant temperature, by immersing the column in a water bath.     

Multiple-injection technique for isolating a target protein from multicomponent mixtures

An integrated chromatographic process comprising ion exchange (IEC) and hydrophobic interaction chromatography (HIC) for isolating a target protein form multicomponent mixtures has been analyzed. The model mixture contained immunoglobulin G that was the key product of the separation process, cytochrome C and ovalbumin. The adsorption characteristics and the mass transport kinetics of the model proteins have been determined along with their dependencies on the operating variables such as pH, temperature and the salt concentration for IEC as well as HIC media. Limitations of the process efficiency resulting from kinetic effects, solubility constraints and the necessity of the mobile phase exchange between chromatographic steps have been discussed. To improve the performance of the integrated process the multiple-injection technique has been suggested. This technique consisted in loading feed mixtures dissolved in a good solvent onto the column by several small-volume injections under conditions of strong protein adsorption. It allowed diminishing interactions between the sample-solvent and protein and elimination of undesired effects such as band splitting and band broadening. For the process design and optimization a dynamic model has been used accounting for thermodynamics and kinetics of the process. The optimization results indicated superiority of the multiple-injection technique over standard isocratic injections in terms of the process yield and productivity.     

pH shifting effect on anionic dye removal by surfactant-modified sugar beet pulp: Modeling of column studies

pH shifting effect on the adsorption of anionic RBB dye was tested by using untreated and CTAB-treated SBP as adsorbent in both batch and continuous systems. Characterization of the sorbents revealed the effects of surface modification. Enhanced binding sites and more porous surface structure resulted in improved adsorption capability. Flow rate and initial RBB concentration effects were tested in packed bed column. Optimum pH value of the adsorption, which was determined as 2.0 in the batch studies with untreated SBP, shifted to 8.0 with 20 g/L CTAB treated SBP. Experimental data in column studies showed the decreasing capacity with increasing flow rate and enhanced performance with increasing inlet RBB concentration for both sorbents. Maximum capacities of the columns were found as 36.9 and 2.6 mg/g with dried SBP at pH 2.0 and 8.0, respectively, at a maximum inlet RBB concentration of 500 mg/L and a minimum flow rate of 0.8 mL/min. The highest capacity value at pH 8.0 was found as 140.0 mg/g under the same operating conditions, which reveals positive effect of the treatment on adsorptive performance. Langmuir isotherm was found to be most convenient model for the all equilibrium cases in the column. Moreover, Thomas model accurately predicted the breakthrough curves of each system. This is the first study reporting the modeling data of an anionic dye adsorption in a packed bed column by using modified SBP.     

Sorptive elimination of fluoride from contaminated groundwater in a fixed bed column: A kinetic model validation based study

The current investigation involves a continuous adsorption experiment in a packed bed column for the sorptive elucidation of fluoride from contaminated groundwater using an activated soil-clay mixture. Through the combination of naturally accessible laterite soil with silica enriched clay (3:1 ratio), a low-cost Al–Si heterogeneous material has been developed. Following detailed characterization, the developed materials were employed in a long-time column process to achieve a high degree of fluoride separation from real-world groundwater. In a packed bed column investigation, the effect of bed height, initial fluoride concentration, and flow rate on the breakthrough properties of the adsorption system were investigated. By using a non-linear regression equation, three model kinetics, such as the Thomas Model, Adams-Bohart Model, and Yoon-Nelson Model, were fitted to validate the column-based experimental data, by analysing the breakthrough curves profiles, and distinct kinetic parameters. The Bed Depth Service Time Analysis (BDST) model was tested to express the effect of bed height on breakthrough curves, as well as to predict the time for breakthrough, and material depletion under optimal conditions. The Thomas and Yoon-Nelson models were identified to be the most appropriate ones for describing the entire breakthrough curve, whereas the Adams-Bohart model was only utilised to predict the first half of the dynamic process. With correlation coefficients (R²) 0.96, the experimental results were well suited to Thomas, Yoon-Nelson, and Adams-Bohart models. Finally, regeneration assessment was carried out where even after four cycles of operation, regenerated adsorbent showed a rejection efficacy of 78% to fluoride that proves the viability of the material and methodology.     

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

A novel dual‐retention mechanism mixed‐mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high‐salt‐concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low‐salt‐concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion‐exchange chromatography and HIC columns. The results indicated that the novel dual‐retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a ‘2D column’. In addition, the mixed retention mechanism of proteins on this ‘2D column’ was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual‐retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this ‘2D column’, a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of the thermal heterogeneity of the column on chromatographic results

The influence of the thermal heterogeneity of HPLC columns on retention data was investigated. The retention factor of the retained compound phenol was measured at 24 increasing values of the flow rate, from 0.025 to 4.9 mL/min, on six different packing materials prepared with the same batch of silica particles (5 microm diameter, 90 A pore size). One column was packed with the neat silica particles, another with the silica endcapped with trimethylchlorosilane (TMS)(C(1), 3.92 micromol/m(2)), and the other four with silica first derivatized with octadecyl-dimethyl-chlorosilane (C(18), 0.42, 1.01, 2.03, and 3.15 micromol/m(2)), and second endcapped with TMS. Four different sources of heat contributing to raise the column temperature were considered: (1) the heat supplied by the hot high-pressure pump chamber to the solvent; (2) the adiabatic (dS=0) compression of the solvent in the high-pressure pump; (3) the isenthalpic (dH=0) decompression of the solvent during its migration along the porous chromatographic bed; and (4) the heat released by the friction of the solvent percolating through the column bed. The main contributions appear to be the heat supplied to the solvent by the HP pump and the friction heat. The average column temperature (ACT) was indirectly derived from the measurements of the first moment, mu(1), of phenol peak, of the column pressure drop, DeltaP, and of the retention factors of the phenol peak apices as a function of the flow rate applied. If the column is placed in a still-air bath at 298 K (and its temperature is not externally controlled), a longitudinal temperature gradient is established along the column and the average column temperature is about 6 K higher when this column is operated at 4.9 mL/min than when the flow rate is only 0.025 mL/min. If the column is placed in a heated air bath (temperature controlled at 316 or 338 K), the ACT changes by less than 3 K over the whole flow rate range applied.     

Chromatography of human prothrombin from Nitschmann fraction III on DEAE Sepharose Fast Flow using axial and radial flow column

An axial column (3 x 2.6 cm) and a radial flow column (3.5 x 5 cm) packed with DEAE Sepharose Fast Flow media was evaluated for the separation of human prothrombin from Nitschmann fraction III. Under radial flow conditions, a sample flow rate up to 14 mL/min (approximately 18 bed vols/h) was achieved. Breakthrough capacity was determined and both columns had almost the same breakthrough capacity per mL media, indicating that the sample loading was independent of radial column geometry.     

Joule heating in packed capillaries used in capillary electrochromatography

Effective heat dissipation is critical for reproducible and efficient separations in electrically driven separation systems. Flow rate, retention kinetics, and analyte diffusion rates are some of the characteristics that are affected by variation in the temperature of the mobile phase inside the column. In this study, we examine the issue of Joule heating in packed capillary columns used in capillary electrochromatography (CEC). As almost all commonly used CEC packings are poor thermal conductors, it is assumed that the packing particles do not conduct heat and heat transfer is solely through the mobile phase flowing through the system. The electrical conductivity of various mobile phases was measured at different temperatures by a conductivity meter and the temperature coefficient for each mobile phase was calculated. This was followed by measurement of the electrical current at several applied voltages to calculate the conductivity of the solution within the column as a function of the applied voltage. An overall increase in the conductivity is attributed to Joule heating within the column, while a constant conductivity means good heat dissipation. A plot of conductivity versus applied voltage was used as the indicator of poor heat dissipation. Using theories that have been proposed earlier for modeling of Joule heating effects in capillary electrophoresis (CE), we estimated the temperature within CEC columns. Under mobile and stationary phase conditions typically used in CEC, heat dissipation was found to be not always efficient. Elevated temperatures within the columns in excess of 23 degrees C above ambient temperature were calculated for packed columns, and about 35 degrees C for an open column, under a given set of conditions. The results agree with recently published experimental findings with nuclear magnetic resonance (NMR) thermometry, and Raman spectroscopic measurements.     

Peak tailing and column radial heterogeneity in linear chromatography

The correlation between the radial heterogeneity of a column and the tailing of the elution profiles of chromatographic peaks was studied using a numerical method. A parabolic distribution of the linear flow velocity of the mobile phase and of the column efficiency in the radial direction were assumed. Moment analysis showed that peak tailing takes place under such experimental conditions and that it increases with increasing range of radial variations of the flow velocity and the column efficiency. It was also found that the higher the column efficiency, the larger the effect of a given degree of radial heterogeneity on the extent of peak tailing. Peak tailing behavior of columns having different efficiencies could be correlated with each other by an equation. Some characteristic features of tailing peaks were analyzed in connection with the column radial heterogeneity.     

氨基己酸为配基的新型弱阳离子交换/疏水双功能色谱固定相对蛋白质的分离纯化

以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水（WCX/HIC）双功能混合模式色谱固定相。该固定相配基具有一定的疏水性且含有羧基,在高盐浓度下表现为HIC的性质,可作为HIC固定相使用;在低盐浓度条件下表现为离子交换的性质,可作为WCX固定相使用。分别考察了该介质在WCX和HIC两种模式下对标准蛋白质的分离性能,并与商品柱进行比较。结果表明,所合成的WCX/HIC双功能固定相在WCX和HIC两种模式下对蛋白质均有较高的分离度和选择性,且分离能力与商品柱相当,两种模式下标准蛋白质的质量和活性回收率均大于93%,表明该柱具有“一柱二用”的功能,适于生物大分子的分离纯化。基于此双功能色谱柱构建的在线单柱二维液相色谱（2DLC-1C）可在60 min内实现8种蛋白质的快速分离。在70 min内完成了对蛋清中溶菌酶的二维纯化,纯度可达到98.3%。该技术中一根色谱柱可当作两根色谱柱使用,对蛋白质组学研究和重组蛋白药物的生产具有重要的应用价值。