Extra-column Effects Research Articles

Considerations for method development and method translation in capillary liquid chromatography: A tutorial

HPLC remains one of the most widely used measurement techniques for chemical analysis. Capillary LC, which utilizes narrow diameter columns operated at lower flow rates than analytical-scale LC, continues to gain adoption based on its reduced mobile phase consumption and increased sensitivity when coupled to MS detection. This tutorial offers practical insights into the most critical aspects of translating analytical-scale separations to the capillary scale. The selection of pumping systems, detectors, and the potential for performance loss due to extra-column effects are examined within the context of separations using columns with inner diameters ≤ 0.3 mm. Column choices within this diameter range are also detailed, both in terms of stationary phase support options and general commercial availability. The impact of these various factors on the effective development/translation of LC methods down to flow rates under 10 µL/min is described to provide readers with a basis for implementing these strategies within their own analytical workflows.

Standardized approach for accurate and reliable model development of ion-exchange chromatography based on parameter-by-parameter method and consideration of extra-column effects.

Developing an accurate and reliable model for chromatographic separation that meets regulatory requirements and ensures consistency in model development remains challenging. In order to address this challenge, a standardized approach was proposed in this study with ion-exchange chromatography (IEC). The approach includes the following steps: liquid flow identification, system and column-specific parameters determination and validation, multi-component system identification, protein amount validation, steric mass action parameters determination and evaluation, and validation of the calibrated model's generalization ability. The parameter-by-parameter (PbP) calibration method and the consideration of extra-column effects were integrated to enhance the accuracy of the developed models. The experiments designed for implementing the PbP method (five gradient experiments for model calibration and one stepwise experiment for model validation) not only streamline the experimental workload but also ensure the extrapolation abilities of the model. The effectiveness of the standardized approach is successfully validated through an application about the IEC separation of industrial antibody variants, and satisfactory results were observed with R2 ≈ 0.9 for the majority of calibration and validation experiments. The standardized approach proposed in this work contributes significantly to improve the accuracy and reliability of the developed IEC models. Models developed using this standardized approach are ready to be applied to a broader range of industrial separation systems, and are likely find further applications in model-assisted decision-making of process development.

Automated Regularized Deconvolution for Eliminating Extra-Column Effects in Fast High-Efficiency Separations.

With the introduction of ultrahigh efficiency columns and fast separations, the need to eliminate peak deformation contributed by the instrument must be effectively solved. Herein, we develop a robust framework to automate deconvolution and minimize its artifacts, such as negative dips, wild noise oscillations, and ringing, by combining regularized deconvolution and Perona-Malik (PM) anisotropic diffusion methods. A asymmetric generalized normal (AGN) function is proposed to model the instrumental response for the first time. With no-column data at various flow rates, the interior point optimization algorithm extracts the parameters describing instrumental distortion. The column-only chromatogram was reconstructed using the Tikhonov regularization technique with minimal instrumental distortion. For illustration, four different chromatography systems are used in fast chiral and achiral separations with 2.1 and 4.6 mm i.d. columns. Ordinary HPLC data can approach highly optimized UHPLC data. Similarly, in fast HPLC-circular dichroism (CD) detection, 8000 plates were gained for a fast chiral separation. Moment analysis of deconvolved peaks confirms correction of the center of mass, variance, skew, and kurtosis. This approach can be easily integrated and used with virtually any separation and detection system to provide enhanced analytical data.

Determining the True Performance of a HPLC Column: A Simulation-Based Experiment

Performing HPLC-based experiments in the undergraduate practical classroom is difficult because of the time required to perform an analysis. This limits the type of practical to one that undertakes a simple quantitative analysis. However, with the use of sophisticated simulators, such as the one employed in this exercise, more complex experiments can be designed. In this exercise, the students use the simulator to evaluate the significance of extra column dead volume on the efficiency of a column. HETP curves are constructed by measuring the column efficiency at a variety of flow rates. In addition, the variance of a band migrating through the system without a column connected is determined, and subsequently, the band variance of the solute migrating through the column is corrected for system variance at each flow rate. By comparing the HETP plots (corrected and uncorrected), the significance of the extra column effects can be determined. In the present study, this amounted to a doubling of the number of theoretical plates at the highest flow rate test.

Fast super/subcritical fluid chromatographic enantioseparations on superficially porous particles bonded with broad selectivity chiral selectors relative to fully porous particles

Superficially porous particles (SPPs) have shown advantages in enantiomeric separations in HPLC by conserving selectivity while providing higher efficiency separations with significantly reduced analysis times. The question arises as to whether the same advantages can be found to the same extent in super/subcritical fluid chromatography. In this work, the low viscosity advantage of carbon dioxide/MeOH mixtures is coupled with high-efficiency 2.7 μm superficially porous particles for enantiomeric separations. Given the fact that the viscosity of the mobile phase is typically ten times lower than liquid mobile phases it is possible to use flow rates as high as 14 mL/min on 5 cm packed columns. Superficially porous particles (SPPs) were grafted with teicoplanin (TeicoShell), a chemically modified macrocyclic glycopeptide (NicoShell), vancomycin (VancoShell), and isopropyl derivatized cyclofructan-6 (LarihcShell-P). One hundred chiral analytes were separated in a very short time frame, as little as 0.2 min (13 s). Even shorter separations can be obtained with advances in SFC instrumentation. The LarihcShell-P is the only chiral crown ether-based selector which showed high selectivity for primary amines. The Teicoshell column offered unique separations for acidic and neutral analytes. The NicoShell and the VancoShell were useful in separating amine (secondary and tertiary) containing pharmaceutical drugs and controlled substances. By chemically modifying a macrocyclic glycopeptide (NicoShell) we report the first enantiomeric separation of nicotine under SFC conditions within 3 min with a resolution of >3. Additionally, van Deemter plots are constructed comparing the fully porous particles and superficially porous particles bonded with the same chiral selectors. In toto the SPP advantages also were found for SFC. However instrumental shortcomings involving extra column effects and pressure limitations need to be addressed by instrument manufacturers to realize the full advantages of SPPs and other smaller particle supports.

Estimation of peak capacity based on peak simulation

Peak capacity (PC) is a key concept in chromatographic analysis, nowadays of great importance for characterising complex separations as a criterion to find the most promising conditions. A theoretical expression for PC estimation can be easily deduced in isocratic elution, provided that the column plate count is assumed constant for all analytes. In gradient elution, the complex dependence of peak width with the gradient program implies that an integral equation has to be solved, which is only possible in a limited number of situations. In 2005, Uwe Neue developed a comprehensive theory for the calculation of PC in gradient elution, which is only valid for certain situations: single linear gradients, absence of delays and extra-column effects, Gaussian peaks and constant plate count. Going beyond these limitations implies resolving algebraic expressions that unfortunately cannot be integrated. In this work, PC is predicted for multiple situations based on peak simulation. The approach is more general and can be applied for situations out of the scope of the Neue outline, such as complex multi-linear gradients, including asymmetrical peaks. The plots of PC versus retention time of the last eluted solute give rise to Pareto fronts, and can be useful for the probabilistic enhancement of peak resolution in situations where complex multi-analyte samples are processed.

Scalability of pre-packed preparative chromatography columns with different diameters and lengths taking into account extra column effects

Small pre-packed columns are commonly used to estimate the optimum run parameters for pilot and production scale. The question arises if the experiments obtained with these columns are scalable, because there are substantial changes in extra column volume when going from a very small scale to a benchtop column. In this study we demonstrate the scalability of pre-packed disposable and non-disposable columns of volumes in the range of 0.2–20 ml packed with various media using superficial velocities in the range of 30–500 cm/h. We found that the relative contribution of extra column band broadening to total band broadening was not only high for columns with small diameters, but also for columns with a larger volume due to their wider diameter. The extra column band broadening can be more than 50% for columns with volumes larger than 10 ml. An increase in column diameter leads to high additional extra column band broadening in the filter, frits, and adapters of the columns. We found a linear relationship between intra column band broadening and column length, which increased stepwise with increases in column diameter. This effect was also corroborated by CFD simulation. The intra column band broadening was the same for columns packed with different media. An empirical engineering equation and the data gained from the extra column effects allowed us to predict the intra, extra, and total column band broadening just from column length, diameter, and flow rate.

Methane Elution from a Helium Flow on Activated Carbon

Methane elution from a helium flow on layers of activated carbon of different length was studied. It was found that the experimental dependences of the corrected retention time on a length of adsorbent layer corresponded to the recently predicted theoretical dependence which considered the extracolumn effects (or an input pulse). It was shown that the experimental dependences of the symmetry coefficient of the breakthrough elution curve on the length of the adsorbent layer were in agreement with the results of numerical experiment.

Low-retention Pre-columns: A Straightforward Approach to Enable Larger Injection Volumes and Reduce Extra-column Effects in HPLC

Background: A common problem in HPLC is the inability to inject a sufficient volume of sample, particularly when the injection solvent (or diluent) is chromatographically strong. Experiments were conducted to evaluate the effectiveness of low-retention pre-columns in allowing larger injection volumes of chromatographically strong solvents. Method: Different combinations of pre-columns and analytical columns were evaluated with respect to their effectiveness in this regard. In addition, the effect of pre-column length, mobile phase conditions, and different injection solvents were evaluated with respect to the maximum injection volume that was possible. Lastly, experiments were conducted to evaluate the additional benefit of placing a mixer upstream of the pre-column. Results: It was demonstrated that significant increases in injection volume were possible by utilizing low-retention pre-columns, which allowed enhanced method sensitivity. Furthermore, it was found that solvents with very limited solubility in the mobile phase, such as ethyl acetate and MTBE, could be used as the sample solvent; and, in fact, the largest injection volumes were possible with these limited-solubility solvents when using the pre-column technique. Conclusion: The general approach was found to be effective in allowing larger injection volumes of chromatographically strong solvents. Furthermore, the focusing in this approach reduces band broadening due to all of the extra-column effects which act upstream of the analytical column. From these observations, it is clear that this technique could significantly reduce some of the difficulties associated with use of smaller diameter columns. Consequently, the use of pre-columns would also enable analytical laboratories to operate in a greener fashion by minimizing the quantity of waste that is generated. Keywords: Band broadening, extra-column effects, focusing, HPLC, Large volume injection, pre-column.

Assessment of intra-particle diffusion in hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography under conditions of identical packing structure

A recently developed stripping protocol to completely remove the stationary phase of reversed-phase liquid chromatography (RPLC) columns and turn them into hydrophilic interaction liquid chromatography (HILIC) columns with identical packing characteristics is used to study the underlying mechanisms of intra-particle diffusion in RPLC and HILIC. The protocol is applied to a column with a large geometrical volume (250×4.6mm, 5μm) to avoid extra-column effects and for compounds with a broad range in retention factors (k” from ∼0.6 to 8). Three types of behavior for the intra-particle diffusion (Dpart/Dm) in RPLC versus HILIC can be distinguished: for nearly unretained compounds (k”<0.6), intra-particle diffusion in HILIC is larger than in RPLC; for compounds with intermediate retention behavior (k”∼0.9–1.2), intra-particle diffusion in HILIC and RPLC are similar; and for well retained compounds (k”>1.8), intra-particle diffusion in RPLC is larger than in HILIC. To explain these observations, diffusion in the stationary phase (γsDs) and in the stagnant mobile phase in the mesopore zone (γmpDm) are deduced from experimentally determined values of the intra-particle diffusion, using models derived from the Effective Medium Theory. It is demonstrated that the larger intra-particle diffusion obtained for slightly retained compounds under HILIC conditions is caused by the higher mesopore diffusion in HILIC (γmp=0.474 for HILIC versus 0.435 for RPLC), while the larger intra-particle diffusion obtained for strongly retained compounds under RPLC conditions can be related to the much higher stationary phase diffusion in RPLC (γsDs/Dm=0.200 for RPLC versus 0.113 for HILIC).

Extensive database of liquid phase diffusion coefficients of some frequently used test molecules in reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography

Diffusion plays an important role in all aspects of band broadening in chromatography. An accurate knowledge of molecular diffusion coefficients in different mobile phases is therefore crucial in fundamental column performance studies. Correlations available in literature, such as the Wilke-Chang equation, can provide good approximations of molecular diffusion under reversed-phase conditions. However, these correlations have been demonstrated to be less accurate for mobile phases containing a large percentage of acetonitrile, as is the case in hydrophilic interaction liquid chromatography.A database of experimentally measured molecular diffusion coefficients of some 45 polar and apolar compounds that are frequently used as test molecules under hydrophilic interaction liquid chromatography and reversed-phase conditions is therefore presented. Special attention is given to diffusion coefficients of polar compounds obtained in large percentages of acetonitrile (>90%). The effect of the buffer concentration (5–10mM ammonium acetate) on the obtained diffusion coefficients is investigated and is demonstrated to mainly influence the molecular diffusion of charged molecules.Diffusion coefficients are measured using the Taylor-Aris method and hence deduced from the peak broadening of a solute when flowing through a long open tube. The validity of the set-up employed for the measurement of the diffusion coefficients is demonstrated by ruling out the occurrence of longitudinal diffusion, secondary flow interactions and extra-column effects, while it is also shown that radial equilibration in the 15m long capillary is effective.

Adsorbent layer efficiency upon methane elution through Cu3(BTC)2 metal-organic material

Methane elution from helium flowing through a sample of Cu3(BTC)2 metal-organic framework material has been studied by gas chromatography. The efficiency of a short adsorbent layer has been calculated in terms of a version of the moments method assuming that there are additive contributions to the variance and retention from both the input pulse (or the extracolumn effects) and the processes of substance movement in the flow of the mobile phase through the permeable porous adsorbent. It has been found that the efficiency or the relative length of the layer of the studied metal-organic material sample, as calculated by the moments method, is considerably lower than the values observed for microporous activated carbon.

General Solution of the Extended Plate Model Including Diffusion, Slow Transfer Kinetics and Extra-Column Effects for Isocratic Chromatographic Elution

The plate model proposed by Martin and Synge has been used for the characterization of columns up-to-date. In this approach, the column is divided into a large number (N) of identical theoretical plates. Mobile phase transference between plates takes place in infinitesimal steps with mixing of the solutions in the adjacent plates during the flow. The plate height is related to the band broadening that occurs in the mixing process due to the microscopic heterogeneities in the mobile phase flow. According to the original Martin and Synge model, solutes reach the equilibrium instantaneously in each theoretical plate, where dispersion is produced by: (i) convection or mixing of the mobile phase reaching a theoretical plate with that existing in that plate; and (ii) the equilibrium of the solute that is partitioned between mobile phase and stationary phase. In this work, a general method is proposed to solve the problem of chromatographic elution by means of an extended plate model assuming slow mass transfer, longitudinal diffusion in both mobile phase and stationary phase, and the extra column dispersion. The final equation was validated by comparing the results with those obtained through the numerical simulation of the solute migration using the finite differential approach. Experimental data were also used to check the validity of the derived equations.

Measurement and Modeling of Extra-Column Effects Due to Injection and Connections in Capillary Liquid Chromatography

As column volumes continue to decrease, extra-column band broadening has become an increasingly important consideration when determining column performance. Combined contributions due to the injector and connecting tubing in a capillary LC system were measured and found to be larger than expected by Taylor-Aris theory. Variance from sigma-type and tau-type broadening was isolated from eluted peaks using the Foley-Dorsey Exponentially Modified Gaussian peak fitting model and confirmed with computational fluid dynamics. It was found that the tau-type contributions were the main cause for the excessive broadening because of poorly-swept volumes at the connection between the injector and tubing. To reduce tau-type contributions (and peak tailing), a timed pinch mode could be used for analyte injection.

Efficiency of short, small-diameter columns for reversed-phase liquid chromatography under practical operating conditions

Prototype small-size (1.0mm I.D., 5cm long) columns for reversed-phase HPLC were evaluated in relation to instrument requirements. The performance of three types of columns, monolithic silica and particulate silica (2μm, totally porous and 2.6μm, core–shell particles) was studied in the presence of considerable or minimal extra-column effects, while the detector contribution to band broadening was minimized by employing a small size UV-detector cell (6- or 90nL). A micro-LC instrument having small system volume (<1μL) provided extra-column band variance of only 0.01–0.02μL2. The three columns generated about 8500 theoretical plates for solutes with retention factor, k>1–3 (depending on the column), in acetonitrile/water mobile phase (65/35=vol/vol) at 0.05mL/min, with the instrument specified above. The column efficiency was lower by up to 30% than that observed with a 2.1mm I.D. commercial column. The small-size columns also provided 8000–8500 theoretical plates for well retained solutes with a commercial ultrahigh-pressure liquid chromatography (UHPLC) instrument when extra-column contributions were minimized. While a significant extra-column effect was observed for early eluting solutes (k<2–4, depending on column) with methanol/water (20/80=vol/vol) as weak-wash solvent, the use of methanol/water=50/50 as wash solvent affected the column efficiency for most analytes. The results suggest that the band compression effect by the weak-wash solvent associated with partial-loop injection may provide a practical means to reducing the extra-column effect for small-size columns, while the use of an instrument with minimum extra-column effect is highly desirable.

Band broadening along gradient reversed phase columns: A potential gain in resolution factor

The advantages of using columns packed with stationary phases having a composition gradient so that retention factors increase toward the outlet and eluting them with an isocratic mobile phase may provide increased peak resolution in liquid chromatography. This approach is discussed from a theoretical viewpoint. The retention factor is assumed to increase linearly along the column. The peak width can be predicted under three different conditions: (1) the possible band compression is neglected (the Giddings model); (2) band compression is taken into account (the Poppe model); and (3) both band compression and extra-column effects are considered in the calculations. The impact of a stationary phase gradient on the resolution of a pair of compound difficult to separate (constant selectivity α=1.05) is illustrated for a 3mm×100mm silica monolithic column of the second generation (porosity ϵt=0.85, plate height H=6μm) operated with a new generation of liquid chromatograph (pre- and post-column volume variances of 10 and 5μL2, respectively). The results show that the resolution factor RS becomes optimum for a specific positive value of the retention gradient along the column. This optimum depends on the retention factor at the column inlet.

Modeling the extra-column volume in a small column setup for bulk gas adsorption

This study aims at highlighting the importance of an accurate characterization of the extra-column volume (ECV) and presents an experimental and computational protocol based on the characterization of the extra-column volume in terms of step-response experiments performed under various flow rates and pressures of 1 bar, 5 bar and 10 bar. The experiments are interpreted by describing the extra-column volume with a compartment model that reflects the geometry of the physical setup and that involves a stagnant zone to account for the non-ideal flow behavior through the piping system. The use of a mathematical model combining the description of the adsorption column and of the ECV can successfully predict experimental CO2–H2 breakthrough profiles performed at different pressures on an activated carbon adsorbent. This work shows how the presence of non-negligible extra-column effects can be accounted for, for the determination of adsorption transport parameters.

Kinetic performance optimisation for liquid chromatography: Principles and practice

This HPLC tutorial focuses on the preparation and use of kinetic plots to characterise the performance in isocratic and gradient LC. This graphical approach allows the selection of columns (i.e. optimum particle size and column length) and LC conditions (operating pressure and temperature) to generate a specific number of plates or peak capacity in the shortest possible analysis time. Instrument aspects including the influence of extra-column effects (maximum allowable system volume) and thermal operating conditions (oven type) on performance are discussed. In addition, the performance characteristics of porous-shell particle-packed columns and monolithic stationary phases are presented and the potential of future column designs is discussed.

Effect of bioparticle size on dispersion and retention in monolithic and perfusive beds

Single-component pulse response studies were used to compare the retention and transport behavior of small molecules, proteins, and a virus on commercially available monolithic and perfusive ion-exchangers. Temporal distortion and extra-column effects were corrected for using a simple algorithm based on the method of moments. It was found that temporal distortion is inversely related to the number of theoretical plates. With increasing bioparticle size, retention increased and the transition from a non-eluting to a non-adsorbing state with increasing ionic strength became more abrupt. Both of these observations are qualitatively explained by calculations of particle–surface electrostatic attractive energy. Calculations also suggest that, for sufficiently large bioparticles, such as viruses or cells, hydrodynamic drag can promote elution. Under non-adsorbing conditions, plate height increased only weakly with flow rate and the skew remained unchanged. With increasing retention, plate height increased dramatically for proteins. Plate height was scaled by permeability rather than bead diameter to enable comparison among different stationary phases.

Instrumental considerations for the effective operation of short, highly efficient fused-core columns. Investigation of performance at high flow rates and elevated temperatures

Fused core or superficially porous columns offer the advantages of higher efficiency compared with totally porous columns of the same particle size, but similar operating pressures. However, their performance may be adversely affected by extra-column effects that become more significant as the column efficiency increases, and as the diameter of the column is reduced. In this study, we show that 10 cm × 0.46 cm fused-core columns can be used on modified conventional instruments (“microbore systems”) without serious loss in performance, and this approach is at present likely to yield superior results compared with use of 0.21 cm columns (of identical efficiency) on current UHPLC instruments that have minimised extra-column volume. Furthermore, the true efficiency of commercial narrow-bore fused-core columns appears to be reduced compared with those of conventional bore, which may be due to packing difficulties for the former type. The fused-core columns in general gave excellent performance, showing no evidence of an upturn in the Knox plots at high flow velocities and elevated temperatures. Careful control of experimental conditions is necessary to ensure accurate data for these plots.