Extra-column Effects Research Articles

Approaches to characterise chromatographic column performance based on global parameters accounting for peak broadening and skewness

Peak broadening and skewness are fundamental parameters in chromatography, since they affect the resolution capability of a chromatographic column. A common practice to characterise chromatographic columns is to estimate the efficiency and asymmetry factor for the peaks of one or more solutes eluted at selected experimental conditions. This has the drawback that the extra-column contributions to the peak variance and skewness make the peak shape parameters depend on the retention time. We propose and discuss here the use of several approaches that allow the estimation of global parameters (non-dependent on the retention time) to describe the column performance. The global parameters arise from different linear relationships that can be established between the peak variance, standard deviation, or half-widths with the retention time. Some of them describe exclusively the column contribution to the peak broadening, whereas others consider the extra-column effects also. The estimation of peak skewness was also possible for the approaches based on the half-widths. The proposed approaches were applied to the characterisation of different columns (Spherisorb, Zorbax SB, Zorbax Eclipse, Kromasil, Chromolith, X-Terra and Inertsil), using the chromatographic data obtained for several diuretics and basic drugs (β-blockers).

Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-μm porous particles

The effects of extra-column band spreading, LC system operating pressure, and separation temperature were investigated for sub-2-μm particle columns using both a conventional HPLC system as well as a UPLC ® system. The contributions of both volume- and time-based extra-column effects were analyzed in detail. In addition, the performance difference between columns containing 2.5 and 1.7-μm particles (same stationary phase) was studied. The performance of these columns was compared using a conventional HPLC system and a low dead volume UPLC system capable of routine operation up to 1000 bar. The system contribution to band spreading and the pressure limitations of the conventional HPLC system were found to be the main difficulties that prevented acceptable performance of the sub-2-μm particle columns. Finally, an increase in operating temperature needs to be accompanied by an increase in flow rate to prevent a loss of separation performance. Thus, at a fixed column length, an increase in temperature is not a substitute for the need for very high operating pressures.

Correction procedures for extra-column effects in dynamic column breakthrough experiments

Dynamic column breakthrough experiments, routinely used to complement adsorption and diffusion studies at the particle scale, constitute an important step in the development and verification of dynamic models for simulation of adsorption processes. Various parts of the experimental setup contribute to the retention time and band broadening of the experimental breakthrough curve. However, the effect of the extra-column contributions have to be properly accounted for in order to compare the experimental results with theoretical calculations. A common practice is to measure a blank response under the same flow rate, pressure and temperature conditions as the actual experiment by simply bypassing the adsorption column with a tube (or a connector) of negligible volume. This blank response is then subtracted point-by-point from the composite response (i.e., including the adsorption column) to account for extra-column contributions. The underlying assumption here is that blank and column responses are linearly additive, both in terms of mean residence time and band broadening. It is shown that this method of correction can, under certain operating conditions, lead to erroneous results. An alternative procedure based on linear regression is introduced and the improvements achieved by this method are illustrated using simulation examples.

Dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns

Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.

Effective diffusivities and pore-transport characteristics of washcoated ceramic monolith for automotive catalytic converter

Using the chromatographic technique (carrier-gas: N 2; tracer-gases: He, Ar) the effective diffusivity in single-pellet string columns (SPSC) packed with porous slab particles was studied. Dispersion due to extra-column effects was eliminated via convolution of column responses for two lengths. The measurements were done for cordierite particles and for cordierite coated with alumina-based washcoat. The effective diffusion coefficients for two tracer–carrier pairs were evaluated by fitting column response (chromatographic) peaks in time-domain. Mean transport parameters were evaluated for both types of porous particles. The obtained mean transport pore radii are in reasonable agreement with pore-size distribution from mercury porosimetry.

A study of interlaboratory influence on column evaluation

A liquid chromatography method for the characterization of base deactivated columns was investigated in a collaborative study involving six laboratories. This work was carried out on two chromatographic supports (Xterra RP 18 and Symmetry Shield). Different cooling systems, namely water bath and air oven, were tested and it was shown that column thermoregulation did not significantly influence chromatographic data. In order to control the mobile phase composition, the latter was prepared by weight rather than volume. Thanks to the injection of a set of selected neutral compounds, extra-column effects were evaluated in each of the participating laboratories. The results showed that chromatographic supports tested in different laboratories and following the same test protocol could be effectively compared.

Axial dispersion in single pellet-string columns packed with unusually shaped porous pellets

Abstract Axial dispersion in the single pellet-string arrangement of a chromatographic column (SPSC) packed with 15 types of pellets with various shapes was studied. Non-porous cylindrical pellets and non-spherical porous pellets with pores blocked by a liquid (Porofil) were used. For different tracer- and carrier-gases (carrier-gases: N2, H2; tracer-gases: H2, He, N2, Ar) comparable dependencies of Bodenstein numbers, Bo, versus the product of Reynolds and Schmidt numbers, Re Sc, were obtained. Tracer dispersion due to extra-column effects was eliminated via convolution of column responses for two column lengths. Reproducibility of axial dispersion determination was verified by repeated column repacking. An interpolation equation for different pellet shapes was proposed. The pellet shape is characterized through pellet sphericity, φ, and the pellet/column diameter ratio, dp/dc. The effectiveness of axial dispersion correlations was confirmed for sphericity 0.427–0.89 and the pellet/column diameter ratios 0.35–0.87.

How to utilize the true performance of monolithic silica columns

Ways of utilizing the true separation efficiency of monolithic silica (MS) columns were studied. The true performance of MS columns, both regular-sized (rod-type clad with PEEK resin, 4.6 mm ID, 10 cm) and capillary sized (in 100 or 200 microm ID fused silica capillary, 25-140 cm) was evaluated by calculating the contribution of extra-column effects. HETP values of 7-9 microm were observed for solutes having retention factors (kvalues) of up to 4 for rod columns and up to 15 for a capillary column. The high permeability of MS columns allowed the use of long columns, with several connected together in the case of rod columns. Narrow-bore connectors gave good results. Peak variance caused by a column connector ranges from 50 to 70% of that caused by one rod-type column for up to three connectors or four columns in 80% methanol, but the addition of a 4th or 5th connector to add a 5th and 6th column, respectively, caused a much greater increase in peak variance, especially for long-retained solutes, which is greater than the variance caused by one rod column. Rod columns seem to show slightly lower efficiency at a pressure higher than 10 MPa or so. The use of acetonitrile-water as a mobile phase better preserved the ability of individual rod columns to generate up to 100,000 theoretical plates with 14 columns connected. Methods for eliminating extra-column effects in micro-HPLC were also studied. Split injection and on-column detection resulted in optimum performance. A long MS capillary measuring 140 cm produced 160,000 theoretical plates. The column efficiency of a capillary column was not affected by the pressure, showing advantages over the rod columns that exhibited peak broadening caused by connectors and pressure.

Mass transfer characteristics of plasmids in monoliths.

The hydrodynamic properties and pore-structure of monoliths based on functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) were characterised by pulse response experiments using different probes representing a wide range of molecular mass. On a small scale, band spreading was found to be caused to the extent of more than 90% by extra-column effects. These monoliths have large channel diameters, providing a suitable chromatography adsorbent for processing of large molecules. Dynamic and static binding capacity for plasmid DNA was investigated. For our model plasmid, consisting of 4.9 kbp, a capacity of 7 mg/mL was observed in comparison to 0.3 mg/mL for a conventional medium designed for protein separation. When plasmids were loaded on the monolith a gradual increase in pressure drop was observed. The channels filled up and the cross-sectional area available for liquid flow decreased. Therefore, a higher pressure drop was observed during elution. This is caused by (i) shrinking of the channels as effect of the high salt concentration, (ii) high viscosity of the mobile phase due to high concentration of plasmids, and (iii) an increase of the hydrodynamic radius of the plasmid with salt concentration from 45 nm at 150 mM to 70 nm at 2 M NaCl, as measured by dynamic light scattering. These types of monoliths are considered to be the preferred adsorbents for plasmid separation.

Axial dispersion in single pellet-string columns with non-porous packing

Using the chromatographic technique (carrier-gas: nitrogen; tracer-gases: H 2, He, Ar, C 2H 6) the axial dispersion in single pellet-string columns (SPSC) packed with non-porous spherical and cylindrical particles was studied. Dispersion due to extra column effects was eliminated via convolution of column responses for two column lengths. For spherical pellets the pellet/column diameter ratios ( d p / d c ) were in the range 0.57–0.79; for cylindrical pellets d p / d c =0.52–0.69 with pellet height/diameter ratios H/ D=0.78–1.5. With different tracer-gases and the same packing and column sizes analogous dependencies of Bodenstein numbers, Bo (Peclet number, Pe, with pellet size, d p , as characteristic dimension), versus the product of Reynolds and Schmidt numbers, ReSc, were obtained. For spherical column packing the Bo– ReSc curves depend also on the d p / d c ratio; for cylindrical pellets the sphericity (which depends on H/ D ratio) plays an additional role. Empirical interpolation equations for both shapes of pellets were set up. The obtained information on axial dispersion significantly increases the confidence of pore-diffusion characteristic when the chromatographic method with SPSC packed with porous pellets is applied.

Evaluation of packed capillary liquid chromatography columns and comparison with conventional-size columns

Apart from extracolumn effects peak dispersion in liquid chromatographic columns is caused by the column inlet, the packed bed, and the column outlet. A strategy applicable for independent evaluation of the individual sources of column band broadening was developed on the basis of the linear extrapolation method (LEM). This method was applied to compare the performance of packed capillary LC columns from various commercial suppliers with conventional-size columns. The columns differed widely in their performance with respect to peak shapes and widths for standard substances. The capillary columns were found well packed, but in some cases overall performance would benefit from improving the design of the area between the packed bed and the connecting capillaries, containing frits as well as dead volumes.

Modeling of separations by closed-loop steady-state recycling chromatography of a racemic pharmaceutical intermediate

Closed-loop steady-state recycling (SSR) is a cyclic, one-column process that is similar to simulated moving bed (SMB) chromatography in several respects. Both processes are cyclic. In both SMB and SSR, fractions are collected from the leading and trailing portions of the circulating chromatographic profile, and fresh sample is injected into the interior of the profile. However, SMB is a continuous process whereas SSR is a discontinuous, repetitive process. This paper presents a model for the closed-loop SSR process and its experimental validation in a case of practical importance. For this last purpose, we used the closed-loop SSR separation of the enantiomers of a racemic pharmaceutical intermediate. The experimental determination of the competitive adsorption equilibria was performed by frontal analysis in a system composed of a chiral HPLC column as the stationary phase and pure acetonitrile as the mobile phase. All the adsorption data were well correlated by the Langmuir model. The Langmuir model was used to calculate overloaded band profiles corresponding to the separation of racemic mixtures at both analytical and preparative scales. Theoretical band profiles were calculated using the equilibrium–dispersive model. With proper corrections for the contributions of the sources of extra-column band broadening, the model properly predicts the experimental band profiles obtained in the closed-loop SSR setup and demonstrates that a cyclic steady-state develops after the completion of a finite number of cycles. The results also show that the extra-column effects must be accounted for in order to model the closed-loop SSR process accurately.

Amperometric Detection of Nonelectroactive Cations in Electrolyte-Free Flow Systems at Zeolite Modified Electrodes

An electrochemical detector was designed and constructed, based upon a thin-layer cell with a working electrode prepared from carbon paste modified with copper- (or silver-) exchanged zeolite particles. This electrode was then evaluated as a sensor for nonelectroactive cations in an electrolyte-free flow injection system and as an amperometric detector for suppressed ion chromatography. In the absence of supporting electrolyte, a higher signal-to-background ratio enhanced by about two order of magnitude the response obtained in electrolytic medium. Peak currents resulted from an important faradic component which was due to the electrochemical activity of the mediator-containing zeolites, while the capacitive counterpart was much lower. When applied to the analysis of alkali, alkaline earth metal and ammonium ions, in combination with a suppressed ion chromatography system, the zeolite-modified detector gave rise to reproducible amperometric responses. This resulted in chromatograms comparable to those obtained simultaneously by using a conductivity detector in series, and any extra-column effects were never observed. The most sensitive responses were achieved when using zeolites displaying the largest pore size, the highest ion exchange capacity, and the most mobile mediator, to ensure the highest exchange rates for the analytes within the zeolite framework.

Ultrafast DNA analysis by capillary electrophoresis/laser-induced fluorescence detection.

The limits of ultrafast DNA analysis by CE were determined by investigating the influence of the effective capillary length and the electric field strength on the analysis time for a given peak resolution (10 bp). In accordance with theory, the use of a fast ramp power supply for narrow plug electrokinetic injection was found to be essential to minimize the extra column effects on peak dispersion. Two major column dispersion factors, longitudinal diffusion and thermal dispersion, were determined experimentally, as well as the influence of the electric field strength on the electrophoretic mobilities and diffusion coefficients of DNA. It was found that higher field strengths can be applied with lower thermal dispersion than predicted by classical CE models. This was attributed to the faster mass transport in the radial direction due to field-induced DNA orientation. Short capillaries (approximately 3-7 cm effective length) and moderate to high electric field strengths (approximately 600-800 V/cm) were used to perform a series of fast DNA separations. The dsDNA fragment standards phiX174/HaeIII and pBR322/HaeIII were separated within 30 s. The possibility for fast mutation detection was demonstrated using constant denaturant capillary electrophoresis (CDCE) for the analysis of a single base mutation in mitochondrial DNA in 72 s. The potential for fast DNA sequencing was illustrated by separating 300 ssDNA fragments within 180 s.

Determination of effective diffusivities and transport parameters of porous solids in the Single-Pellet-String-Column

The chromatographic technique which employs SPSC and takes into account the extra-column effects of the measuring system can provide consistent transport characteristics of porous solids. These characteristics are independent of the kind of gases which are transported through pores, as well of temperature and pressure. Dispersion of the tracer band in the interparticle spaces of the SPSC increases at higher carrier gas velocities when the non-porous column packing is replaced by a porous one. This points to some additional mechanism of band spreading caused by the presence of interface gas-porous solid.

High-resolution pyrolysis–gas chromatography with a movable reaction zone

A new device was constructed for pyrolysis–gas chromatography and it was laboratory tested. The device enables the thermal degradation of polymers inside a capillary pre-column and transfer of the reaction zone into a column oven. The pyrolysis procedure described protects the thermally sensitive compounds prior to pyrolysis, prevents the process of irreversible condensation of high-boiling pyrolysis products during the chromatographic process and eliminates extracolumn effects on the peak broadening.

Prediction of the preparative chromatography performance with a very small column

Practical considerations lead to the use of small columns in preparative chromatography. In these applications extra column effects may overwhelm all broadening effects. The influence of the extra column effects on the band broadening and migration is demonstrated by simulation. The model assumes linearity of extra column effects. Consequently, these effects could be described by the convolution of the initial applied band width, an exponential decay and Gaussian broadening. An attempt has been made to discriminate between the column, pre- and postcolumn broadening.

Peak broadening in capillary zone electrophoresis.

A review on peak broadening in capillary zone electrophoresis in free solutions is given which covers a selection of the literature published on this topic over the period mainly between 1992 and the beginning of 1997 (consisting of 71 publications). The contributions to peak dispersion from extracolumn effects (e.g. due to the finite length of the injection zone, or the aperture of the detector), from longitudinal diffusion, Joule heating, electromigration dispersion (concentration overload), a different path length of the solute ions, wall adsorption, laminar flow and the (longitudinally) homogeneous or nonhomogeneous electroosmotic flow are described. The latter may also occur when a longitudinally nonhomogeneous radial electric field is applied. Peak dispersion is depicted either by the plate-height model, or the concentration of the solute as a function of space and time is calculated either analytically or numerically by solving the equation of continuity with appropriate initial and boundary conditions and possibly completed by equations governing further quantities.

Flow characteristics of a versatile wall‐jet or radial‐flow thin‐layer large‐volume cell for electrochemical detection in flow‐through analytical systems

AbstractFlow characteristics of a versatile large‐volume flow‐through electrochemical cell, which features exchangeable disk, ring‐disk or microdisk working electrodes, are presented. The cell operates in a radial‐flow thin‐layer, wall‐jet or wall‐tube mode, under amperometric or voltammetric conditions, being therefore suitable for flow analytical techniques, such as high performance liquid chromatography (HPLC) and flow injection analysis (FIA). The operation mode is selected by using the inlet capillary either of a flat or conical nozzle and by suitably adjusting the nozzle‐to‐electrode distance. The cell is equipped with a micrometer screw which allows for adjusting of this distance to be as small as 10 ± 5 μm. The inlet solution stream is centered axially against the working electrode surface. Under radial‐flow thin‐layer conditions, flow‐through detection at a ring‐disk electrode benefits from shielding and collecting properties which are enhanced as compared to those for the rotating ring‐disk electrode conditions. The HPLC extracolumn effects due to detection were determined by the peak variance analysis of a mixture of regio isomers of nitrobenzene derivatives. Despite the large (35mL) geometric cell volume, the effective detection volume of the cell is of the microliter order. Therefore, the cell is suitable for detection both in the analytical and microbore‐column HPLC. Due to the large geometric cell volume, solution composition can be different, under wall‐jet conditions, for detection than that for HPLC separation. Hence, in situ postcolumn modification of the mobile phase or analyte is feasible.

Factors Affecting Zone Variance in a Capillary Flow Injection System

Dye dispersion studies are reported to characterize zone variance in a capillary flow injection system. Variance sources are compared with similar sources in a conventional flow injection system. The inside diameters of the reactors in the capillary and conventional systems were 64 μm and 0.5 mm, respectively. In both systems, reactor length, detector flow cell volume, and injection volume are the major contributors to the total zone variance. The individual contributions of each variance source were determined. Results show that, in a single-line capillary system, the majority of the peak variance is due to extracolumn effects. However, in the conventional system, the reactor was found to be the major contributor to the overall zone variance. Implications of these findings to the development of improved capillary flow injection systems are discussed.