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Choosing a liquid chromatography (LC) column for a particular application can be a surprisingly challenging task. On one hand, column manufacturers give us many options to choose from, including particle types, pore sizes, particle sizes, and different lengths and diameters. On the other hand, we usually don't have time to experimentally evaluate many combinations of these parameters, and sometimes we end up picking something similar to the columns that are already in the drawer. The "kinetic plot" is a powerful graphical tool that can help leverage the best available theory to help us understand how different combinations of parameters (that is, particle size and length) will perform in terms of the time needed to get to a particular column efficiency (and thus resolution), and therefore make well-informed decisions when choosing columns. [ABSTRACT FROM AUTHOR]

Choosing a liquid chromatography (LC) column for a particular application can be a surprisingly challenging task. On the one hand, column manufacturers provide us with many options to choose from, including particle types, pore sizes, particle sizes, and different lengths and diameters. On the other hand, we usually do not have time to experimentally evaluate many combinations of these parameters, and sometimes we end up picking something similar to the columns that are already available. The "kinetic plot" is a powerful graphical tool that can help leverage the best available theory to help us understand how different combinations of parameters (that is, particle size, length, among others) will perform in regard to the time needed to get to a particular column efficiency (and thus resolution), and therefore make well-informed decisions when choosing columns. [ABSTRACT FROM AUTHOR]

Choosing a liquid chromatography (LC) column for a particular application can be a surprisingly challenging task. On the one hand, column manufacturers give us many options to choose from, including particle types, pore sizes, particle sizes, and different lengths and diameters. On the other hand, we usually do not have time to experimentally evaluate many combinations of these parameters, and sometimes we end up picking something similar to the columns that are already in the drawer. The "kinetic plot" is a powerful graphical tool that can help leverage the best available theory to help us understand how different combinations of parameters (such as particle size and length) will perform in relation to the time needed to get to a particular column efficiency (and thus resolution), and therefore make well-informed decisions when choosing columns. [ABSTRACT FROM AUTHOR]

Dispersion (broadening, or spreading) of analyte zones (peaks) outside of chromatography columns can seriously erode the resolution that good columns provide. In this instalment, we focus on the contributions of dispersion in connecting tubing and detectors to the total level of extracolumn dispersion in a liquid chromatography (LC) system. [ABSTRACT FROM AUTHOR]

Dispersion (broadening, or spreading) of analyte zones (peaks) outside of chromatography columns can seriously erode the resolution provided by good columns. In this instalment, we focus on the contribution of the sample injection step to the total level of extracolumn dispersion in a liquid chromatography (LC) system. This contribution has both a physical component (that is impacted by injector design and configuration and method variables) and a chemical component (that depends on the difference between the mobile phase and the sample solvent compositions). [ABSTRACT FROM AUTHOR]

Dispersion (the broadening or spreading) of analyte zones (peaks) outside of chromatography columns can seriously erode the resolution provided by good columns. Understanding the relationship between the level of dispersion associated with a given instrument relative to the intrinsic efficiency of a given column can help avoid disappointing results, especially when switching to new column technologies. In this instalment, we focus on basic concepts in extracolumn dispersion as a prelude to future instalments that will focus on dispersion in specific elements of high performance liquid chromatography (HPLC) systems. [ABSTRACT FROM AUTHOR]

By combining separation efficiency data as a function of flow rate with the column permeability, the kinetic plot method allows to determine the limits of separation power (time vs. efficiency) of different chromatographic techniques and methods. The technique can be applied for all different types of chromatography (liquid, gas, or supercritical fluid), for different types of column morphologies (packed beds, monoliths, open tubular, micromachined columns), for pressure and electro‐driven separations and in both isocratic and gradient elution mode. The present contribution gives an overview of the methods and calculations required to correctly determine these kinetic performance limits and their underlying limitations. [ABSTRACT FROM AUTHOR]

This instalment is the first of a series of four white papers on high performance liquid chromatography (HPLC) modules (pump, autosampler, UV detector, and chromatography data system) to be published in 2019. This instalment provides an overview for analytical-scale HPLC pumps, including their requirements, modern designs, operating principles, trends, and best practices for trouble-free operation. [ABSTRACT FROM AUTHOR]

We report on a detailed study of the injection contribution to band broadening in contemporary UHPLC-instruments, using either flow-through needle or fixed loop injection (full loop). Using on-tubing fluorescence measurements at the outlet of the injector valve, very localized and undisturbed measurements were obtained. Varying both the flow rate and the injected volume allowed to split the injection variance (σ V 2 ,inj ) in a volumetric component (related to the amount injected) and a hydrodynamic component (related to the flow rate). For the flow-through needle injector and for the small injection volumes (<2 μL) typically used in UHPLC, it was found that the volumetric contribution (i.e. the part of σ V 2 ,inj , that increases with increasing injection volume) is given by a value of σ V 2 ,inj,vol  = 0.8 to 1·V inj 2 rather than by the value of 0.125 to 0.2·V inj 2 that is normally assumed in literature. For the hydrodynamic contribution to σ V 2 ,inj, (i..e, the part which remains present even for very small injection volumes), a clear increase in dispersion with flow rate is found, reaching a plateau around 0.8ml/min of 0.6 μL² or 1.2 μL² for the 75 μm and 120 μm needle seat capillaries respectively. The difference between both shows the clear advantage of using a low dispersion 75 μm injection needle seat capillary. For a loop-type injector operated in a full-loop mode, the increase in peak variance with the injection volume is much less pronounced, leading to a total injector variance given by σ V 2 ,inj  = 0.34 μL² + 0.12·V inj 2 over the entire range of investigated injection volumes of 1.1 μL up to 4.5 μL when using 120 μm or narrower ID loops. This expression was nearly completely independent of the flow rate. For larger ID sample loops, a clear increase of peak variance with flow rate at fixed injection volume was observed (σ V 2 ,inj increases with 20% for a 170 μm ID loop and with 70% for a 220 μm ID loop from 0.3 to 1 ml/min). [ABSTRACT FROM AUTHOR]

The article focuses on the development in the liquid chromatography instruments with the introduction of fully porous particles rather than the old columns. It discusses how the ultra high-performance liquid chromatography (UHPLC) should be improved with separation quality and operating pressure, and analyze the separation performance while applying kinetic plot methodology.

The article offers information on a study on the comparison of the separation speed of gas chromatography (GC), liquid chromatography (LC), and supercritical fluid chromatography (SFC). Topics discussed include using the kinetic plot method neglecting differences in viscous heating or extra-column band broadening; obtaining fixed length kinetic plot curves; and role of thermodynamic resistance factor in LC.

• Dispersion caused by sudden capillary diameter change has been computed using CFD. • The extra dispersion scales with the square of the flow rate and the Peclet number. • A minor effect of viscosity on this relation starts to develop when Reynolds > 1. • The extra dispersion is very small compared to dispersion in rest of the capillaries. We report on a Computational Fluid Dynamics (CFD) study of the extra dispersion caused by the change in diameter when coupling two pieces of capillary tubing with different diameter. In this first investigation into the problem, the focus is on the typical flow rates (0.25≤F≤2μL/min) and diameters (d≤40μm) used in nano-LC, considering both the case of either a doubling or halving of the diameter. The CFD simulations allow to study the problem from a fundamental point of view, i.e., under otherwise perfect conditions (perfect alignment, zero dead-volume). Flow rates, capillary diameters, diffusion coefficients and liquid viscosities have been varied over a range relevant for nano-LC (Reynolds-numbers Re ≤ 1), with also an excursion made towards high-temperature nano-LC conditions (Re ≥ 10 and more). The extra dispersion caused by the change in diameter has been quantified via a volumetric variance σ2 conn , defined in such a way that the overall dispersion across the entire capillary system can be easily reconstructed from the known analytical solutions in the individual segments. When the two capillaries are longer than their diffusion entry length, covering most of the practical cases, σ2 conn converges to a limiting value σ2 conn,∞ which varies to a close approximation with the square of flow rate. Under the investigated nano-LC conditions, the σ2 conn,∞ -values are surprisingly small (e.g., on the order of 0.01 to 0.15 nL2 in a 20 to 40μm connection) compared to the dispersion occurring in the remainder of the capillaries. [ABSTRACT FROM AUTHOR]

The present study provides experimental evidence for the fact that the peak deconvolution method can be applied to accurately measure the column-only dispersion of the current generation of high speed and high efficiency columns. Unlike the conventional variance difference method, it furthermore preserves any prevailing asymmetry of the column-only peak. This has been demonstrated by testing the same column on three different system configurations, with different extra-column volumes, and showing that, after deconvolution, the resulting column-only peaks coincide very well and produce very similar column-only plate height values (typical relative standard deviation comprising all runs on three different system configurations is 2–2.5%). Extensively studying a large set of theoretically produced peaks (with exactly known variance and asymmetry), it could be shown that the main criterion for the validity of the deconvolution method is that the variance of the system‐only peak is minimum 1.5 times smaller than the variance of the column + system peak. The need to add a radial mixer unit to accurately assess the system-only contributions has been demonstrated as well. To illustrate its use and merits, the deconvolution method has been used to establish so-called multiple van Deemter curves, wherein plate height curves relating to different peak width definitions are shown in the same plot. These plots can give new insights in the intrinsic asymmetry of the column-only dispersion. [ABSTRACT FROM AUTHOR]

We report on the results of an experimental and theoretical study of the effect of the extra-column band broadening (ECBB) on the performance of narrow-bore columns filled with the smallest particles that are currently commercially available. Emphasis is on the difference between the effect of ECBB under gradient and isocratic conditions, as well as on the ability to model and predict the ECBB effects using well-established band broadening expressions available from the theory of chromatography. The fine details and assumptions that need to be taken into account when using these expressions are discussed. The experiments showed that, the steeper the gradient, the more pronounced the extra-column band broadening losses become. Whereas the pre-column band broadening can in both isocratic and gradient elution be avoided by playing on the possibilities to focus the analytes on top of the column (e.g. by using the POISe injection method when running isocratic separations), the post-column extra-column band broadening is inescapable in both cases. Inducing extra-column band broadening by changing the inner diameter of the post-column tubing from 65 to 250 μm, we found that all peaks in the chromatogram are strongly affected (around a factor of 1.9 increase in relative peak width) when running steep gradients, while usually only the first eluting peak was affected in the isocratic mode or when running shallow gradients (factor 1.6–1.8 increase in relative peak width for the first eluting analyte). [ABSTRACT FROM AUTHOR]

Due to the developing insights in the theory of chromatography, column manufacturers of any kind (industrial, academic) nowadays have a broad array of experimental column testing tools at their disposal. The present tutorial aims at helping the novice in the field getting an overview of these tools and provides a fixed procedure to carry out the subsequent steps in the column quality analysis (guided via an Excel template file). After some brief introduction about the main equations, the reader is taken step by step through the theories underlying the measurement methods for the different column and performance parameters. In the final section, the reader is taken through the different items in the Excel template. [ABSTRACT FROM AUTHOR]

Separation performance in chromatography has been extensively studied since the dawn of the technique. Although the basic principles of band broadening and the resulting separation performance in isocratic elution are in general well known and understood, this is much less the case for gradient separations. In this tutorial, first the basic principles, concepts and parameters that determine separation performance, peak width and variance and analysis time in isocratic separations are reviewed. This is subsequently used to discuss the parameters that affect peak width in gradient elution, together with the concepts of plate count and plate height in this elution mode. In addition, the effect of peak compression in gradient elution is elaborated. Finally, the effect of extra-column dispersion on separation performance in gradient elution is discussed, and an overview of how these contributions can be experimentally evaluated is given. [Display omitted] • Basic principles of band broadening in isocratic elution are reviewed. • Parameters affecting observed and true efficiency in gradient elution are elaborated. • The effect of peak compression on peak width in gradient elution is explained. • Extra-column band broadening contributions to performance are discussed. [ABSTRACT FROM AUTHOR]

The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 μm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying t G / t 0 = 5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient ( t G / t 0 = 10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion. [ABSTRACT FROM AUTHOR]

Different automatic peak integration methods have been reviewed and compared for their ability to accurately determine the variance of the very narrow and very fast eluting peaks encountered when measuring the instrument band broadening of today's low dispersion liquid chromatography instruments. Using fully maximized injection concentrations to work at the highest possible signal-to-noise ratio's (SNR), the best results were obtained with the so-called variance profile analysis method. This is an extension (supplemented with a user-independent read-out algorithm) of a recently proposed method which calculates the peak variance value for any possible value of the peak end time, providing a curve containing all the possible variance values and theoretically levelling off to the (best possible estimate of the) true variance. Despite the use of maximal injection concentrations (leading to SNRs over 10,000), the peak variance errors were of the order of some 10–20%, mostly depending on the peak tail characteristics. The accuracy could however be significantly increased (to an error level below 0.5–2%) by averaging over 10–15 subsequent measurements, or by first adding the peak profiles of 10–15 subsequent runs and then analyzing this summed peak. There also appears to be an optimal detector intermediate frequency, with the higher frequencies suffering from their poorer signal-to-noise-ratio and with the smaller detector frequencies suffering from a limited number of data points. When the SNR drops below 1000, an accurate determination of the true variance of extra-column peaks of modern instruments no longer seems to be possible. [ABSTRACT FROM AUTHOR]

After the great commercial success of sub-3µm superficially porous particles, vendors are now also starting to commercialize 5µm superficially porous particles, as an alternative to their fully porous counterparts which are routinely used in pharmaceutical analysis. In this study, the performance of 5µm superficially porous particles was compared to that of fully porous 5µm particles in terms of efficiency, separation performance and loadability on a conventional HPLC instrument. Van Deemter and kinetic plots were first used to evaluate the efficiency and performance of both particle types using alkylphenones as a test mixture. The van Deemter and kinetic plots showed that the superficially porous particles provide a superior kinetic performance compared to the fully porous particles over the entire relevant range of separation conditions, when both support types were evaluated at the same operating pressure. The same observations were made both for isocratic and gradient analysis. The superior performance was further demonstrated for the separation of a pharmaceutical compound (griseofulvin) and its impurities, where a gain in analysis time of around 2 could be obtained using the superficially porous particles. Finally, both particle types were evaluated in terms of loadability by plotting the resolution of the active pharmaceutical ingredient and its closest impurity as a function of the signal-to-noise ratio obtained for the smallest impurity. It was demonstrated that the superficially porous particles show better separation performance for griseofulvin and its impurities without significantly compromising sensitivity due to loadability issues in comparison with their fully porous counterparts. Moreover these columns can be used on conventional equipment without modifications to obtain a significant improvement in analysis time. [ABSTRACT FROM AUTHOR]

Highlights: [•] G2 silica monoliths have smaller characteristic sizes compared to G1. [•] G2 monoliths have improved peak shapes, higher efficiencies but higher flow resistances. [•] The skeleton of G2 monoliths resembles a single chain of spherical globules. [•] G2 monoliths have better kinetic performances than G1 monoliths for N ≤45,000. [•] G2 monoliths can still not compete with state-of-the-art porous particle columns. [ABSTRACT FROM AUTHOR]

In this contribution, we review the recent literature relating to the measurement and modelling of all diffusion-dominated processes contributing to the efficiency of a chromatographic column. In first instance, this involves the measurement and modelling of the overall effective diffusion coefficient D eff (determining the so-called B-term band broadening). The latter manifests itself most clearly during a so-called peak parking experiment. Using effective medium theory modelling, the measured D eff -value can subsequently be decomposed into its constituent contributions, of which the intra-particle or the mesoporous zone and the surface diffusion coefficient are the most important ones. As an accurate estimation of the diffusion processes also allows computing the C-term plate height contribution terms, the review ends with some recent insights obtained when using the established B- and C-term contributions to compute the degree of eddy-dispersion in contemporary packed bed columns. [Display omitted] • Recent literature on measurement and modelling of diffusion in chromatography reviewed. • The effective medium theory modelling is explained and compared with simulations. • Measurements of microscopic diffusion parameters is discussed. • The effect of the diffusion parameters on the eddy-dispersion is considered. [ABSTRACT FROM AUTHOR]

In this work the isopycnic method to construct kinetic plots for SFC was used to investigate the performance limits of an SFC system when using sub-2µm fully porous particles and sub-3µm superficially porous (core–shell) particles. This isopycnic kinetic plot method for SFC was developed and tested earlier for SFC separations on native silica with pure CO2 as mobile phase. In the current work, octadecyl based reversed phase columns were used in combination with a mobile phase that contains 10% methanol as modifier in order to study the applicability of the described methodology to assess the kinetic performance limits of experimental setups in which SFC is used and will, according to all probability, be evolving. SFC and HPLC van Deemter and kinetic plots are constructed for columns packed with fully porous particles with various diameters and for a column packed with core–shell particles. The influence of the experimental kinetic performance limits of the particle diameter and morphology in SFC is shown to be the same as in HPLC. Additionally, kinetic plot predictions were constructed for separations on 1µm and 0.5µm particles using the data measured on the 5µm, 3.5µm and 1.8µm fully porous particles. By doing this the potential applicability of 1µm particles on the contemporary SFC and HPLC systems was demonstrated together with the irrelevance of the use of 0.5µm particles in SFC. [ABSTRACT FROM AUTHOR]

Abstract: Supercritical fluid chromatography (SFC) is attributed many advantages over high performance liquid chromatography (HPLC). Next to the fact that SFC is greener than HPLC, which is especially important for preparative separations, SFC is claimed to be able to deliver faster separations at higher efficiencies (N) than HPLC. This is due to the higher diffusitivity of analytes in supercritical fluids compared to liquids (higher optimum mobile phase velocity) and to the lower viscosity of the mobile phases in SFC compared to HPLC, which results in smaller pressure drops allowing the use of longer columns and/or columns packed with smaller particles at higher velocities. In order to quantify this claimed kinetic performance advantage, it is essential to construct unbiased kinetic plots to make the comparison between HPLC and SFC. The high compressibility of the mobile phase in SFC however makes this problematic. A variable column length (L) kinetic plot method is therefore developed in this work. Because the pressure history in the column is kept constant for every data point in this method, this way of working definitely delivers exact values for the kinetic performance limits in SFC. It is shown that the traditional way of measuring the performance as a function of flow rate (fixed back pressure and column length) cannot deliver the same correct results as this variable L method. However, the isopycnic way of working on a fixed column length has also been proven to be a good alternative for the expensive and time consuming variable L method. Finally, isopycnic kinetic plots are used to compare SFC and HPLC performance in a quantitative way. [Copyright &y& Elsevier]

Abstract: The maximal gain in efficiency that can be expected from the use of the segmented column end fittings that were recently introduced to alleviate the effect of transcolumn packing density gradients has been quantified and generalized using numerical computations of the band broadening process. It was found that, for an unretained compound in a column with a parabolic packing density gradient, the use of a segmented inlet or a segmented outlet allows to eliminate about 60–100% of the plate height contribution (H tc) originating from a parabolic transcolumn velocity gradient in a d c =4.6mm column. In a d c =2.1mm column, these percentages change from 10 to 100%. Using a combined segmented in- and outlet, H tc can be reduced by about 90–100% (d c =4.6mm column) or 20–100% (d c =2.1mm column). The strong variation of these gain percentages is due to fact that they depend very strongly on the column length and the flow rate. Dimensionless graphs have been established that allow to directly quantify the effect for each specific case. It was also found that, in agreement with one''s physical intuition, trans-column velocity profiles that are more flat in the central region benefit more from the concept than sharp, parabolic-like profiles. The gain margins furthermore tend to become smaller with increasing retention and increasing diffusion coefficient. [Copyright &y& Elsevier]

Abstract: The kinetic-performance limits of a capillary silica C18 monolithic column and packed capillary columns with fully-porous 3μm and fused-core 2.7μm silica C18 particles (all 5cm long) were determined in gradient-elution mode for the separation of peptides. To establish a kinetic plot in gradient-elution mode, the gradient time to column dead time ratio (t G /t 0) was maintained constant when applying different flow rates. The normalized gradient approach was validated by dimensionless chromatograms, obtained at different flow rates and gradient times by plotting them as a function of the retention factor. The separation performance of the different column types was visualized via kinetic plots depicting the gradient time required to achieve a certain peak capacity when operating at a maximum system pressure of 350bar. The gradient steepness (applying t G /t 0 =10, 20, and 40) did not significantly affect the gradient performance limits for low (<250) peak-capacity separations. For high peak-capacity separations the peak capacity per unit time increases when increasing the t G /t 0 ratio. The C-term contribution of the porous 3μm and fused-core 2.7μm was comparable yielding the same gradient kinetic-performance limits for fast separations at a column temperature of 60°C. The capillary silica monolithic column showed the lowest contribution in mass transfer and permeability was higher than the packed columns. Hence, the silica monolith showed the best kinetic performance for both fast and high peak-capacity gradient separations. [Copyright &y& Elsevier]

Abstract: The present contribution reviews the foundations of the kinetic-plot method for the direct comparison of the kinetic performance of different chromatographic support and operating modes. The method directly uses experimental data collected for a specific sample and operating condition of one''s interest, and is applicable both under isocratic- and gradient-elution conditions. Experimental proof is provided for the strong relation between the kinetic performance of a given support under isocratic and gradient conditions: a material offering superior kinetic performances under isocratic conditions will remain superior under gradient conditions and vice versa provided the comparison occurs under unbiased conditions. In addition, a review is made of the recent literature using the kinetic-plot method to compare and assess the kinetic performance of high performance HPLC columns and their operation mode. [Copyright &y& Elsevier]

Abstract: The development of a method for the separation of standard compounds of the 15 main phenolics found in rooibos tea is presented. The separation of these compounds in a single HPLC analysis is particularly challenging due to the similarity of rooibos phenolics. As a result, multiple methods are often required to analyze all major phenolics in rooibos tea samples. The method development process is significantly enhanced in this study by using the recently introduced automated column coupler in combination with the variable column length strategy. This strategy consists of performing the initial scouting runs, wherein the best separation conditions are determined, on a short column and subsequently fine-tuning the separation on longer columns to benefit from their higher separation performance. It is demonstrated that the method development process can further be expedited by operating each column length at the maximum pressure, in this case 1000bar. Although this holds in general, it is even more the case for the presently considered sample, since the selectivity of the sample is more pressure- than temperature-dependent. Applying the optimized method to unfermented and fermented aqueous rooibos tea extracts in combination with Q-TOF mass spectrometry, some 30 phenolic compounds are tentatively identified. [Copyright &y& Elsevier]

The throughput of a laboratory can be optimized by selecting the optimum particle diameter and column length for a specific stationary phase. This selection can be made using a step-by-step method based on the recently established kinetic plot method for gradient separations. An example involving the separation of environmental pollutants demonstrates how this approach worked in practice. [ABSTRACT FROM AUTHOR]

Abstract: An experimental study was performed to investigate the effects of column parameters and gradient conditions on the separation of intact proteins using styrene-based monolithic columns. The effect of flow rate on peak width was investigated at constant gradient steepness by normalizing the gradient time for the column hold-up time. When operating the column at a temperature of 60°C a small C-term effect was observed in a flow rate range of 1–4μL/min. However, the C-term effect on peak width is not as strong as the decrease in peak width due to increasing flow rate. The peak capacity increased according to the square root of the column length. Decreasing the macropore size of the polymer monolith while maintaining the column length constant, resulted in an increase in peak capacity. A trade-off between peak capacity and total analysis time was made for 50, 100, and 250mm long monolithic columns and a microparticulate column packed with 5μm porous silica particles while operating at a flow rate of 2μL/min. The peak capacity per unit time of the 50mm long monolithic column with small pore size was superior when the total analysis time is below 120min, yielding a maximum peak capacity of 380. For more demanding separations the 250mm long monolith provided the highest peak capacity in the shortest possible time frame. [Copyright &y& Elsevier]

The use of CO 2 -based mobile phases for separations, more generally referred to as supercritical fluid chromatography (SFC), allows to perform highly efficient and fast separations due to its low viscosity and high diffusivity of the solutes in the mobile phase. Nevertheless, the instrumental capabilities of current state-of-the art instrumentation in SFC is still not on the same level as that of ultra-high performance liquid chromatography. Not only is the maximum operating pressure lower, also the extra-column fluidic path is not fully optimized to exploit the possibilities of high efficiency columns packed with small particles. The effect of the strong solvent injection inherent to SFC hinders the optimal implementation of short small particle columns. The current limits in separation power in SFC are discussed and the requirements for future generation instruments are reviewed. An overview is presented of innovations in column technology, separation conditions and optimization of the instrumental design. • The low viscosity of CO 2 -based mobile phases allows high kinetic performance. • To allow the use of high modifier content, higher operating pressures are required. • Improvements in extra-column dispersion needed to enable the use of narrow ID columns. • New chiral stationary phases allow very fast (<1min) enantioseparations. • Injection solvent effects deteriorate 1D and 2D performance of SFC. [ABSTRACT FROM AUTHOR]

Abstract: To fulfil the increasing demand for faster and more complex separations, modern HPLC separations are performed at ever higher pressures and temperatures. Under these operating conditions, it is no longer possible to safely assume the mobile phase fluid properties to be invariable of the governing pressures and temperatures, without this resulting in significantly deficient results. A detailed insight in the influence of pressure and temperature on the physico-chemical properties of the most commonly used liquid mobile phases: water–methanol and water–acetonitrile mixtures, therefore becomes very timely. Viscosity, isothermal compressibility and density were measured for pressures up to 1000bar and temperatures up to 100°C for the entire range of water–methanol and water–acetonitrile mixtures. The paper reports on two different viscosity values: apparent and real viscosities. The apparent viscosities represent the apparent flow resistance under high pressure referred to by the flow rates measured at atmospheric pressure. They are of great practical use, because the flow rates at atmospheric pressure are commonly stable and more easily measurable in a chromatographic setup. The real viscosities are those complying with the physical definition of viscosity and they are important from a fundamental point of view. By measuring the isothermal compressibility, the actual volumetric flow rates at elevated pressures and temperatures can be calculated. The viscosities corresponding to these flow rates are the real viscosities of the solvent under the given elevated pressure and temperature. The measurements agree very well with existing literature data, which mainly focus on pure water, methanol and acetonitrile and are only available for a limited range of temperatures and pressures. As a consequence, the physico-chemical properties reported on in this paper provide a significant extension to the range of data available, hereby providing useful data to practical as well as theoretical chromatographers investigating the limits of modern day HPLC. [Copyright &y& Elsevier]

It is shown that all Cm and Cs expressions ever established in the literature are intrinsically fully identical and can be derived from a simple parallel plug flow model. Analytically solving this model yields Cm- and Cs-term expressions that exactly correspond to the expressions obtained via either the method of moments (general rate model) or via Giddings' nonequilibrium theory. This holds for open-tubular as well as packed and monolithic columns. From this equivalence, a set of convenient "translation" expressions could be established, enabling a swift transition between the different Cm- and Cs-term expressions that currently coexist in literature. The link with the parallel plug flow model provides a good physical insight into the assumptions underlying the general rate model (e.g., a plug flow in the flow-through pores) and in the physical meaning of the different parameters involved in it. The parallel plug flow model also allows us to illustrate the difference between the zone and the phase retention factor and between the intraparticle diffusion coefficient and the effective diffusion coefficient used in the general rate model. These differences are subtle, but as they can have a large impact, they constitute important potential sources of confusion currently obscuring the modeling of HPLC columns. [ABSTRACT FROM AUTHOR]

Abstract: Using a combination of both analytical and numerical techniques, approximate analytical expressions have been established for the transient and long time limit band broadening, originating from the presence of a thin disturbed sidewall layer in liquid chromatography columns, including packed, monolithic as well as microfabricated columns. The established expressions can be used to compare the importance of a thin disturbed sidewall layer with that of other radial heterogeneity effects (such as transcolumn packing density variations due to the relief of packing stresses). The expressions are independent of the actual velocity profile inside the layer as long as the disturbed sidewall layer occupies less than 2.5% of the column width. [Copyright &y& Elsevier]

• A new in-column methacrylate-based polymerization procedure was developed for imaging the internal morphology of bare silica HPLC columns • Polymerization was performed in a commercial and in a home-made silica column • The obtained silica/polymeric rod can be mechanically cut allowing cross section SEM imaging • The developed procedure allows mapping of the local external porosity in packed column • The procedure could be of assistance to steer the optimization of packing processes of HPLC columns. To allow an enhanced understanding of the order in packed HPLC columns, in this work a methodology for immobilizing native polar silica particles is developed based on the polymerization of a methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) as a cross-linker in the interstitial pores of HPLC columns. Subsequent mechanical cutting then allows scanning electron microscopy (SEM) based imagery of cross-sections of the packed bed. In this way, the packing efficiency of home-made and commercial HPLC columns with 4.6 mm inner diameter and 150 mm length comprising the same packing material of 5 µm silica particles are compared. The methodology is developed for native silica used in e.g. hydrophilic interaction liquid chromatography (HILIC) and in normal phase LC. In order to confirm the feasibility of the developed methodology, the conventional methods for the evaluation of column, efficiency and porosity, are also employed. The obtained porosity information is compared and showed the same trend with the external porosity measurements obtained via inverse size exclusion approach, illustrating its potential application to study the micro-heterogeneity of packed HPLC columns and to guide the optimization of the packing process of HPLC columns. [ABSTRACT FROM AUTHOR]

• Flow segmentation can be used to measure radial dispersion coefficient (D rad) in practice. • Mass flow meter feedback can be used to correct flow rate mismatches. • Method validated by showing independence of relative central flow rate and tracer concentration. • Measured D rad -values higher than in most engineering literature reports but still within cited range. The present contribution reports on the practical implementation and validation of a new experimental method to determine the radial dispersion (D rad) in packed bed liquid chromatography columns, as well as on the results obtained with it. A first important validation was that the measured D rad -values were independent of the applied relative central flow rate (varied from 25% to 57%). The obtained D rad -values did not vary significantly when changing the concentration of the injected tracer to check potential mass overloading effects (25, 50 or 75 ppm of tracer for the acetophenone measurements; 12.5 and 25 ppm of tracer for the toluene measurements). And yet another important validation step was the observation that the D rad -values clearly converged to the value of D eff for velocities going to zero, as physically and theoretically expected. Plotting the obtained results as a plot of D rad /D mol versus the reduced velocity ν, a quasi-linear relationship is obtained. The slope of the curve (β = 0.38 and β = 0.46 for toluene and acetophenone, respectively) is significantly larger than the value that is most frequently cited in engineering literature. However, the obtained β-values and D rad /D mol -values still fall within the broad range of β- and D rad /D mol -values cited in literature. [ABSTRACT FROM AUTHOR]

• Alternative radial dispersion coefficient (D rad) measurement method is proposed. • Method is based on the use of flow segmentation frit technology. • Different D rad -determination read-out methods are possible and have been established. • Negligible effect of segmentation ring thickness proven via computational fluid dynamics. • Correction method established for flow rate mismatches between central inlet and outlet. We report on an alternative experimental method to determine the transversal or radial dispersion coefficient (D rad) in packed bed columns for liquid chromatography. The method uses a recently developed type of column end-fitting with an impermeable segmentation ring that splits the incoming flow in a central and peripheral part. Using this device and continuously sending a tracer-laden flow through the central inlet and a tracer-less flow through the peripheral inlet, a steady-state radial dispersion pattern is established which can be used to determine the degree of radial dispersion. The present part of the study lays the theoretical foundation for the method and shows the parameter sensitivity of the different possible data analysis variants. In addition, computational fluid dynamics simulations have been used to validate the established procedure (agreement between true and determined D rad -value better than 0.4%) as well as to study the effect of the most important error sources: the presence of the annular flow segmentation ring and the almost inevitable occurrence of mismatches between the central and the peripheral in- and outlet flows. In the latter case, a combined read-out method can be proposed that nearly perfectly compensates for the flow mismatch error (remaining error on the D rad -value smaller than 3%). [ABSTRACT FROM AUTHOR]

• HIC method development is demonstrated using a ternary salt system. • The ternary salt system included ammonium sulfate, sodium chloride, and phosphate buffer. • Prediction error increases with increase in gradient duration and did not exceed 12 %. • A method for optimizing ternary-gradient conditions is established using a genetic algorithm. • Application is demonstrated for critical-pair separation in a mixture of proteins. The use of a ternary mobile-phase system comprising ammonium sulphate, sodium chloride, and phosphate buffer was explored to tune retention and enhance selectivity in hydrophobic interaction chromatography. The accuracy of the linear solvent-strength model to predict protein retention with the ternary mobile-phase system based on isocratic scouting runs is limited, as the extrapolated retention factor at aqueous buffer conditions (k 0) cannot be reliably established. The Jandera retention model utilizing a salt concentration averaged retention factor (k ¯ 0) in aqueous buffer for ternary systems overcomes this bottleneck. Gradient retention factors were derived based on isocratic scouting runs after numerical integration of the isocratic Jandera model, leading to retention-time prediction errors below 11 % for linear gradients. Furthermore, an analytical expression was formulated to predict HIC retention for both linear and segmented linear gradients, considering the linear solvent-strength (LSS) model within ternary salt systems, relying on a fixed k 0. The approach involved conducting two gradient scouting runs for each of the two binary salt systems to determine model parameters. Retention-time prediction errors for linear gradients were below 12 % for lysozyme and 3 % for trypsinogen and α -chymotrypsinogen A. Finally, the analytical expression for a ternary mobile-phase system was used in combination with a genetic algorithm to tune the HIC selectivity. With an optimized segmented ternary gradient, a critical-pair separation for a mixture of 7 proteins was achieved within 15 min with retention-time prediction errors ranging between 0.7 and 15.7 %. [ABSTRACT FROM AUTHOR]

Dispersion (broadening, or spreading) of analyte zones (peaks) outside of chromatography columns can seriously erode the resolution provided by good columns. In this final instalment of the series we focus on the impact of elution mode (isocratic or gradient) and postcolumn flow splitting on the total level of extracolumn dispersion (ECD) in a liquid chromatography (LC) system, and demonstrate the use of a free, web-based calculator that can be used to guide decision making aimed at reducing ECD during method development. [ABSTRACT FROM AUTHOR]

Columns for liquid chromatography produce ever narrower peaks and analysts continue to push to smaller column diameters to save solvents and have better MS responses. The combined effect of both trends is that the instrument volumes outside the separation column (=extra-column volumes of the instrument) become a significant contributor to the overall dispersion. This currently occurs to such an extent that the current state-of-the-art chromatographic columns have become too good compared to the quality of most instruments. Extra-column dispersion also becomes very critical in the now rapidly emerging multi-dimensional LC application area. The present contribution reviews past and recent studies of the experimental and theoretical work conducted thus far to measure, predict and optimize extra-column dispersion. Emphasis is put on providing some general rules that can be followed to minimize extra-column dispersion in the most efficient way (i.e., without sacrificing too much of the available pressure or detection sensitivity). • An overview is provided of past and recent studies of the extra-column dispersion. • The different methodologies to measure extra-column dispersion are discussed. • The different instrument contribution to extra-column dispersion contribution are reviewed. • Trade-offs between dispersion and pressure drop or sensitivity are presented. • General rules to minimize extra-column dispersion are provided. [ABSTRACT FROM AUTHOR]