Comprehensive analysis of the effective and intra-particle diffusion of weakly retained compounds in silica hydrophilic interaction liquid chromatography columns.

A detailed analysis of intra-particle volumes and layer thicknesses and their effect on the diffusion of solutes in hydrophilic interaction liquid chromatography (HILIC) was made. Pycnometric measurements and the retention volume of deuterated mobile phase constituents (water and acetonitrile) were used to estimate the void volume inside the column, including not only the volume of the mobile phase but also part of the enriched water solvent acting as the stationary phase in HILIC. The mobile phase (hold-up) volume accessible to non-retained components was estimated using a homologous series approach. The joint analysis of the different approaches indicated the formation of enriched water layers on the hydrophobic silica mesopore walls with a thickness varying significantly with mobile phase composition. The maximal thickness of the enriched water layers, which corresponded to the minimum void volume accessible to unretained solutes, marked a transition in the retention behavior of the studied analytes. Discrepancies between deuterated solvent measurements and pycnometry were explained by the existence of an irreplaceable water layer adsorbed on the silica surface. Regarding the diffusion behavior in HILIC, peak parking experiments were used to interpret the influence of the acetonitrile content on the effective diffusion coefficient Deff. A systematic decrease in Deff and molecular diffusion Dm was observed with decreasing acetonitrile concentration, primarily attributed to variations in mobile phase viscosity. Notably, Deff/Dm remained nearly unaffected by variations in mobile phase composition. Finally, the effective medium theory was used to make a comprehensive analysis of Dpart/Dm to study the contribution to band broadening when the solute resides in the mesopores. The obtained data unveiled a curvature with a minimum corresponding to conditions of maximum water-layer thickness and retention. For the weakly retained compounds (k' < 0.5) the Dpart/Dm-values were found to be relatively high (order of 0.35-0.5), which directly reflects the high γsDs/Dm-values that were observed (order 0.35-7).

Detailed analysis of the effective and intra-particle diffusion coefficient of proteins at elevated pressure in columns packed with wide-pore core-shell particles.

To determine the efficiency that can be obtained in a packed-bed liquid-chromatography column for a particular analyte, a correct determination of the molecular and effective diffusion coefficients (Dm and Deff) of the analyte is required. The latter is usually obtained via peak parking experiments wherein the flow is stopped. As a result, the column pressure rapidly dissipates and the measurement is essentially conducted at ambient pressure. This is problematic for analytes whose retention depends on pressure, such as proteins and potentially other large (dipolar) molecules. In that case, a conventional peak parking experiment is expected to lead to large errors in Deff. To obtain a better estimate ofDeff, the present study reports on the use of a set-up enabling peak parking measurements under pressurized conditions. This approach allowed us to report, for the first time, Deff for proteins at elevated pressure under retained conditions. First, Deff was determined at a (average) pressure of about 105 bar for a set of proteins with varying size, namely: bradykinin, insulin, lysozyme, ß-lactoglobulin, and carbonic anhydrase in a column packed with 400 Å core-shell particles. The obtained data were then compared to those of several small analytes: acetophenone, propiophenone, benzophenone, valerophenone, and hexanophenone. A clear trend between Deff and analyte size was observed. The set-up was then used to determine Deff of bradykinin and lysozyme at variable (average) pressures ranging from 28 bar to 430 bar. These experiments showed a decrease in intra-particle and surface diffusion with pressure, which was larger for lysozyme than bradykinin. The data show that pressurized peak parking experiments are vital to correctly determine Deff when the analyte retention varies significantly with pressure.

On-column modification for the creation of temperature-responsive stationary phases.

Temperature-responsive liquid chromatography (TRLC) offers an alternative for retention and selectivity optimisation in HPLC. This approach thereby exploits temperature gradients on stimuli-responsive stationary phases and forfeits the necessity for solvent gradients, allowing analyses to be performed using aqueous mobile phases. Consequently, it can be employed as a green alternative to reversed-phase separations. However, current production to obtain temperature-responsive columns inherently require dedicated column packing processes with polymer-modified particles. To facilitate the development of temperature-responsive phases, a flow-through modification procedure was developed allowing on-column modification of aminopropyl silica columns. Three columns were manufactured using this novel flow-through approach, which achieved identical column efficiencies compared to existing TRLC column. Demonstrating the possibility of bypassing the dedicated packing processes without losing efficiency. Additionally, it was observed that flow-through produced columns yielded higher retention at elevated temperatures despite their reduced carbon load. Further investigation of the carbon load revealed the presence of stationary phase gradients, whose influence was studied via novel developed retention experiments, which revealed a negligible change reduction in retention upon a change of polymer modification from 19.8% to 14.5%. However, further decrease from 14.5% to 12.3% resulted in a larger change. Interestingly, a further enhancement in apparent plate numbers was observed when operating the column under a reversed flow, yielding an increasing stationary phase gradient. This on-column modification procedure demonstrates the potential for modification of existing (commercial) packed columns to achieve temperature-responsive phases without loss of efficiency or retention. Thus, not only facilitating accessibility to temperature-responsive phases, but also aiding with development of further generations of temperature-responsive phases by removing the need for packing optimisation. Additionally, a novel experiment was set up to easily investigate the effect of inhomogeneous stationary phases retention.

Computational Fluid Dynamics Study of the Dispersion Caused by Capillary Misconnection in Nano-Flow Liquid Chromatography.

It is well known that high-speed/high-efficiency separations in nano-flow liquid chromatography (LC) are very sensitive to the quality of the connections between the column and the rest of the instrument. In the present study, two types of connection errors (capillary misalignment and the occurrence of an inter-capillary gap) have been investigated using computational fluid dynamics. Interestingly, it has been found that large degrees of capillary misalignment (assuming an otherwise perfect contact between the capillary end-faces) can be afforded without introducing any significant dispersion over the entire range of investigated relative misalignment errors (0 ≤ ε/dcap ≤ 75%), even at the largest flow rates considered in nano-LC. On the other hand, when an inter-capillary gap is present, the dispersion very rapidly increases with the radial width Dc of this gap (extra variance ∼Dcn with n even reaching values above 4). The dependency on the gap length Lc is however much smaller. Results show that, when Dc ≤ 30 µm and Lc ≤ 200 µm, dispersion losses can be limited to the order of 1 nL2 at a flow of 1.5 µL/min, which is generally very small compared to the dispersion in the capillaries (20 µm i.d.) themselves. This result also reconfirms that zero-dead volume connectors with a sufficiently narrow bore can in theory be used without compromising peak dispersion in nano-LC, at least when the capillaries can be matched perfectly to the connector in- and outlet faces. The results are also indicative of the extra dispersion occurring inside microfluidic chips or in the connections between a microfluidic chip and the outer world.

Fundamental investigation of the dispersion caused by a change in diameter in nano liquid chromatography capillary tubing.

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 σ2conn, 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, σ2conn converges to a limiting value σ2conn,∞ which varies to a close approximation with the square of flow rate. Under the investigated nano-LC conditions, the σ2conn,∞-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.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Measurement of the molecular diffusion coefficient and the effective longitudinal diffusion under supercritical fluid chromatography conditions in packed bed columns.

The improvement of supercritical fluid chromatography (SFC) instrumentation enhanced its reliability and utility over the past decade. The further development of high speed and high resolution separations is however obstructed by the lack of accurate models for axial dispersion in SFC. This work is a first step to tackle this by developing more reliable methods to measure molecular (Dmol) and longitudinal diffusion (Deff) in SFC, as these affect all aspects of separation efficiency. In the present contribution, we report on an improved method, to enable more flexible, reliable and accurate measurements of Dmol in SFC using commercial instrumentation. A two-column variant of the stopped-flow experiment is proposed as an adapted set-up for measuring the effective longitudinal diffusion coefficient Deff in SFC-conditions. Using the set-ups for a number of test-compounds, it has been found that Deff, and the coefficients describing its constituent sub-processes (cf. particle diffusion Dpart and surface diffusion γsDs), all vary in a linearly proportional way with the bulk diffusion coefficient Dmol within a high degree of accuracy. It has also been found that Deff decreases much more sharply with increasing retention factor compared to LC. By applying the effective medium theory, it was found that the relative surface diffusion coefficient γsDs/Dmol decreases strongly with retention factor for the investigated solutes and column, in contrary to what is typically observed in reversed phase liquid chromatography. Results indicate that this might be related to a change in retention behavior of the analytes. Obviously, more analytes and conditions need to be explored to complete this picture and the extend range of applicability of these observations.

Diffusion coefficients of an extensive set of pharmaceutical compounds in supercritical fluid chromatography over a wide range of mobile phase compositions.

Diffusion data are essential for adequate analysis of the kinetic separation performance of any chromatographic system. Unfortunately, for Supercritical Fluid Chromatography (SFC), very little data is available of the diffusion coefficients in mobile phases typically used in contemporary methods, i.e. with a non-negligible amount of polar modifier such as methanol. In this work, a relative simple method which only requires minor modifications to a standard commercially available SFC instrument is used to determine the diffusion coefficient of an extensive set of pharmaceutical compounds in the range of 10-50 vol% of modifier (methanol) in CO2. By using a traditional SFC column, the solute is first separated from the sample solvent plug, before entering a long capillary, where the band broadening can be linked to its diffusion coefficient using the Taylor-Aris equation. By using two UV-detectors, before and after the capillary, the effect of the dispersion in the column can be eliminated and the true volumetric flow rate determined. It was found that in the investigated range of conditions, the change in mobile phase viscosity in a first approximation allows to predict the variation in diffusion coefficient. Chemical structure and more particularly functional groups can however have a significant effect on the diffusion coefficient.

Theory of separation performance and peak width in gradient elution liquid chromatography: A tutorial.

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.

Review of recent insights in the measurement and modelling of the B-term dispersion and related mass transfer properties in liquid chromatography.

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 Deff (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 Deff-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.

Detailed computational fluid dynamics study of the parameters contributing to the viscous heating band broadening in liquid chromatography at pressures up to 2500 bar in 2.1 mm columns.

Over the past years viscous heating band broadening occurring in high pressure liquid chromatography has been studied extensively. In the present numerical study, we investigate the fine details of this band broadening contribution under extreme high-pressure conditions (2500 bar). To analyze the results, we first show that viscous heating leads to two clearly distinguishable band broadening effects, one originating from the radial differences in the species migration velocity and the other from the axial variations. It was found that the radial contribution is independent of the intrinsic band broadening of the bed (i.e. band broadening in absence of viscous heating) while it strongly depends on the radial dispersion coefficient and the retention enthalpy of the analytes. On the other hand, the axial contribution is strongly dependent on the bed intrinsic band broadening and it is found to be 4 to 5 times lower than the radial contribution. We also show the strong effect of the endfittings on the temperature gradients inside the column thus on the resulting viscous heating band broadening.

Modelling of analyte profiles and band broadening generated by interface loops used in multi-dimensional liquid chromatography.

Currently, the shape and variance of the analyte band entering the second dimension column when injected from an open loop interface in two-dimensional liquid chromatography is not fully understood. This is however important as it is connected to several other variables encountered when developing 2D-LC methods, including the first dimension flow rate, the sampling (modulation) time and the loop volume. Both numerical simulation methods and experimental measurements were used to understand and quantify the dispersion occurring in open tubular interface loops. Variables included are the analyte diffusion coefficient (Dmol), loop filling and emptying rates (Ffill & Fempty), loop inner diameter or radius (Rloop) and loop volume (Vloop). For a straight loop capillary, we find that the concentration profile (as measured at the loop outlet) depends only on a single dimensionless parameter tempty*=VloopFempty·DmolRloop2 and the ratio of the filling and emptying flow rates Fempty/Ffill. A model depending only on these two parameters was developed to predict of the peak variance resulting from the filling and emptying of a straight capillary operated in the first-in-last-out (FILO) modulation mode. Comparison of the concentration profiles and the corresponding variances obtained by either numerical simulation or experiments with straight capillaries shows the results generally agree very well. When the straight capillary is replaced by a tightly coiled loop, significantly smaller (20-40%) peak variances are observed compared to straight capillaries. The magnitude of these decreases is not predicted as well by simulations, however the simulation results are still useful in this case, because they represent an upper boundary (i.e., worst-case scenario) on the predicted variance.

Investigation of the potential of mixed solvent mobile phases in temperature-responsive liquid chromatography (TRLC).

Temperature-responsive liquid chromatography (TRLC) allows for extensive retention and selectivity tuning through temperature in HPLC. This is mainly achieved through the use of a stationary phases comprising of a temperature-responsive polymer which undergoes a reversible change from hydrophilic to hydrophobic behaviour upon increasing the temperature. The approach can allow for reversed phase type separations to be achieved with purely aqueous mobile phases, whereby the retention is controlled through temperature instead of mobile phase composition. Despite the promising nature of such form of retention control under isocratic mobile phase conditions, TRLC can suffer from excessive retention of highly apolar solutes even at lower column temperatures whereby the polymer is considered hydrophilic. This is related both to a residual apolarity of the polymer chain and due to the high log P's and low water solubility of higly apolar compounds. While it was known that elution in TRLC doesn't necessarily has to be performed under purely aqueous conditions and that the use of organic co-solvents to the water is possible, the impact thereof on the temperature responsive behaviour itself had not yet been investigated in a systematic way. Therefore in this work the advantages and drawbacks of the use of the organic co-solvents methanol and acetonitrile in TRLC is assessed on two types of temperature reponsive phases: poly-N-N-propylacrylamide (PNNPAAm) and poly-N-isopropylacrylamide (PNIPAAm). The influence of organic co-solvents is investigated with two representative test mixtures (comprising 4 parabens and 5 apolar steroids).

Implementations of temperature gradients in temperature-responsive liquid chromatography.

Temperature Responsive Liquid Chromatography (TRLC) offers an alternative and environmentally friendly way to perform reversed-phase like separations. Its use of temperature responsive polymers to control retention based on column temperature, instead of the fraction of organic modifier in the mobile phase mobile, eliminates the need for solvent composition gradients and allows, for example, for purely aqueous separations. In principle this temperature induced retention should allow for gradient elutions to be performed using downward temperature gradients to control retention and refocus the analyte peaks. Yet, the unavailability of dedicated commercial temperature controlling systems allowing suitable temperature control in TRLC limits implementations thereof often to isothermal or step gradient applications. In this work we study the potential of 1) a simple yet programmable water bath and of 2) a modified HPLC system allowing column temperature programming through controlled mixing of a warm and cold mobile phase streams. The performance of both systems was evaluated under both isocratic and gradient applications, resulting in a more thorough understanding of the influence of temperature gradients in TRLC. This knowledge is then applied to a sample of phenolic solutes, illustrating that, although both systems have some flaws, both are able to impose temperature gradients in TRLC resulting in significantly reduced retention and enhanced refocusing of the analyte peak.

Computational fluid dynamics study of potential solutions to alleviate viscous heating band broadening in 2.1 millimeter liquid chromatography columns.

We report on a numerical simulation study of a number of potential column technology solutions to minimize the plate height contribution (Hvh) originating from the use of ultra-high pressures and their concomitant viscous heating effect. Looking as far as possible into the future of UHPLC, all main results are obtained for the case of a 2500 bar pressure gradient. However, to generalize the result, a correlation is given that can be used to interpolate the results to lower pressures with some 10% accuracy. For the considered case of a 2.1mm column, a liquid flow rate of 0.45 ml/min, an analyte with retention factor k(25°C)=3 and a retention enthalpy chosen such that ΔHR/R= -1000 K, it is found that, in order to keep the global plate height as measured at the column outlet (Hvh,glob,out) below 1 µm, the bed conductivity would need to be raised to λbed=2.4 W/m•K, i.e., 4 times higher than a typical packed bed of fully-porous or core-shell silica particles. An equivalent effect on the band broadening could be obtained if it would be possible to replace the steel column wall with a low conductivity material. In this case, a wall conductivity of 0.25 W/m•K, i.e., 64 times smaller than the conductivity of steel, would be needed to keep Hvh,glob,out below 1 µm. Results are also interpreted based on contour plots of the axial and radial velocity variation of a retained analyte.

Assessment of the resolving power of hydrophobic interaction chromatography for intact protein analysis on non-porous butyl polymethacrylate phases.

This study reports on the assessment of the separation performance of hydrophobic interaction chromatography for intact protein analysis using non-porous butyl polymethacrylate phases. The maximum peak capacity in inverse gradient mode was reached at a volumetric flow rate which was significantly (10-20 times) higher than the flow rate yielding the minimum plate height in isocratic mode, as the gradient volume dominates the peak-capacity generation. The flow rate yielding the maximum peak capacity increased with decreasing gradient volume, i.e., steeper gradients, and also depends on the magnitude of the mass-transfer contribution to peak dispersion (affected by particle size and molecular diffusion coefficient of proteins) at these high flow rates. The maximum peak capacity using a 100 mm long column packed with 4 µm particles for steep 7.5 min gradients was determined to be 60. Increasing the column length by coupling columns leads to better gradient performance than increasing the gradient duration for gradients of 60 min and longer. Using a coupled column system (2 × 100 mm long columns packed with 4 µm particles), the maximum peak capacity was determined to be 105, which was 33% higher compared to that of a single column while applying a similar gradient volume. Decreasing the particle size to 2.3 µm leads to higher peak capacities even though the column was operated at lower volumetric flow rate. The maximum peak capacity obtained with the 2.3 µm column was 128% higher than was obtained with the coupled column. Even at suboptimal conditions, the 2.3 µm column yields a higher peak capacity (14%) than when using two coupled columns packed with 4 µm at optimal conditions (gradient time of 120 min and a flow rate of 0.5 mL/min).

Implications of dispersion in connecting capillaries for separation systems involving post-column flow splitting.

It is common practice in liquid chromatography to split the flow of the effluent exiting the analytical column into two or more parts, either to enable parallel detection (e.g., coupling the separation to two destructive detectors such as light scattering and mass spectrometry (MS)), or to accommodate flow rate limitations of a detector (e.g., electrospray ionization mass spectrometry). In these instances the user must make choices about split ratio and dimensions of connecting tubing that is used between the split point and the detector, however these details are frequently not mentioned in the literature, and rarely justified. In our own work we often split the effluent following the second dimension (2D) column in two-dimensional liquid chromatography systems coupled to MS detection, and we have frequently observed post 2D column peak broadening that is larger than we would expect to result from dispersion in the MS ionization source itself. For the present paper we describe a series of experiments aimed at understanding the impact of the split ratio and post-split connecting tubing dimensions on dispersion of peaks exiting an analytical column. We start with the simple idea - based on the principle of conservation of mass - that analyte peaks entering the split point are split into two parts such that the analyte mass (and thus peak volume) entering and exiting the split point is conserved, and directly related to the ratio of flow rates entering and exiting the split point. Measurements of peak width and variance after the split point show that this simple view of the splitting process - along with estimates of additional dispersion in the post-split tubing - is sufficient to predict peak variances at the detector with accuracy that is sufficient to guide experimental work (median error of about 10% over a wide range of conditions). We feel it is most impactful to recognize that flow splitting impacts apparent post-column dispersion not because anything unexpected happens in the splitting process, but because the split dramatically reduces the volume of the analyte peak, which then is more susceptible to dispersion in connecting tubing that would not cause significant dispersion under conditions where splitting is not implemented. These results will provide practitioners with a solid basis on which rational decisions about split ratios and dimensions of post-split tubing can be made.

Through-pore polymerization in polar high-performance liquid chromatography columns allowing scanning electron microscopy based imaging of the packing order.

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.

Methods to determine the kinetic performance limit of contemporary chromatographic techniques.

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.

Effect of the feed injection method on band broadening in analytical supercritical fluid chromatography.

The behavior of a novel type of SFC injector, the feed injector, was investigated. In SFC, the sample compounds are usually diluted in a solvent which has a higher elution strength than the mobile phase, which leads to solvent mismatch upon injection and evidently band broadening. The feed injector differs from standard injectors as the sample, contained in the sample needle or loop, is not switched in line with the mobile phase flow, but directly injected/added to the mobile phase flow (F). The subsequent mixing of sample and mobile phase flows inherently results in a dilution of the sample, thus reducing the solvent mismatch. However, for a given injection/feed flow rate Ffeed, the total volume in which the sample is contained increases with a factor (Ffeed + F)/Ffeed. In addition, to ensure that all of the loaded sample is injected on the column, an additional overfeed volume (Vov) needs to be injected after the sample plug. To better understand the effect of these operating parameters, a wide range of injection conditions was investigated by varying the Ffeed/F-ratio, Vov, overfeed solvent etc. under SFC conditions. It was found that an optimal Ffeed/F exists which is independent of F and decreases with increasing solvent strength dependency of the sample compound. Decreasing Vov has a beneficial effect on peak dispersion but can only be varied over a certain range to ensure the full injection of the loaded sample. On the other hand, it was found that a much larger gain could be made by switching the overfeed solvent to one more compatible with the CO2-based mobile phase. Further reduction of the band broadening could be achieved by applying partial sample injections.