Extra-column Contributions Research Articles

Influence of metals in the column or instrument on performance in hydrophilic interaction liquid chromatography

A method is proposed for measuring the relative contribution of extracolumn and column effects to the detrimental interactions which occur between metal-sensitive solutes and the complete HPLC system. The method involves the substitution of a length of narrow bore silica tubing for the column and measuring the extracolumn contribution, which is subtracted from the total bandspreading measured with a column in place to yield the column contribution. The investigation focussed on HILIC separations, which have been relatively little studied compared with similar effects in RPLC. Metal-solute interactions can lead to tailing peaks and reduced sensitivity or even irreversible adsorption of particularly challenging solutes such as mono-, di- and triphosphorylated nucleotides, which show strong interactions between their phosphate groups and metals. A deactivated HILIC column, treated by a vapour deposition procedure gave generally good results when using high pH (pH 9.0) mobile phases, which suppress the effects of metals. The addition of metal complexing agents such as citrate at low millimolar concentration gave further improvements in peak shape at high pH, and even micromolar concentrations of citrate or medronic acid showed good results. These lower concentrations are more favourable for LC-MS. Addition of the higher concentration of citrate gave acceptable results for the nucleotides even at low pH (pH 3.0). With the standard UHPLC instrument used, loss of efficiency due to metal solute interactions was 25% or less, with most losses due to interactions with the column, although this result will depend on the condition and design of the instrument, which is easily assessed by the proposed procedure.

The counterintuitive role of extra-column volume in the determination of column efficiency and scaling of chromatographic processes

In industrial liquid separation processes chromatography often has a key function in the optimization of yield and purity. For the design of an industrial system, chromatographic processes are generally simulated using mathematical models, tested and optimized at laboratory level, and then scaled up to pilot and subsequently industrial scale. To describe the system, experimental data and model data need to be fitted and extra column contribution must be determined. This paper describes the influence of extra-column volume on overall separation efficiency for lab scale and its impact on the design of large scale systems.Measurement of extra-column contribution was investigated in terms of mean retention time and variance using two different methods the commonly used zero dead volume connector and as an alternative the zero length column. Further a technique is presented to estimate extra-column contribution to band broadening for different injection volumes, velocities, and tracers based on representative measurements.When scaling up, often contribution of extra-column volume from laboratory equipment is neglected assuming to be on the safe side, however column efficiency is often lower than efficiency measured for the entire chromatographic system. Relation between system efficiency and column efficiency was investigated using laboratory data and the lumped kinetic model. Depending on the ratio of extra-column volume to retention volume in the system, deduced column efficiency was up to 20% smaller than overall system efficiency. This ratio revealed the misleading nature of the term efficiency loss, when describing influence of extra-column volume on column efficiency. A scheme, which relates the relative variance of the system to the relative extra-column volume, provided an assessment of under- or overestimation of column efficiency. In this article it is shown how scaling up a system based on laboratory data, where extra-column volume contribution is not accounted for, may severely overestimate column efficiency. This overestimation results in underestimated column dimensions at pilot and industrial scale, and hence underperformance of the industrial system.

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis.

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300μm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2μm fully porous as well as the 2.7μm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2μm fully porous particle packed column for ultra-fast liquid chromatography.

Problems involving the determination of the column-only band broadening in columns producing narrow and tailed peaks

We have investigated which of the different existing peak variance read-out methods (including the effect of a deconvolution pre-treatment method) are most suited to eliminate the system contribution from the total observed band broadening observed in LC systems. Emphasis is put on the most demanding case, i.e., the measurement of non-retained component peaks, which typically are very narrow and tailed. The problem with such peaks is that the method that is generally considered to be the only mathematically correct method (i.e., the method of moments) leads to peak variance values that are so strongly dominated by the tail of the peak that they become highly exaggerated and practically meaningless (i.e., they are dominated by the peak width at 10 or 12σt, which corresponds to resolutions and peak purities that are so high they are never pursued in practice). Interestingly, filtering away the extra-column contribution from the entire peak shape using peak deconvolution (wherein not only the second order moment is corrected but also all other moments) produces corrected 4σt- and half height peak widths that are physically meaningful, i.e., the corrected values allow to make sufficiently accurate predictions of how the peak width at 4σt and at half height changes when the column length changes. This result now allows to navigate away from the classical method of moments to define the column plate height, and resort to plate heights based on the practically much more relevant 4σt- and 5σt-widths, provided theses are corrected via peak deconvolution.

Prediction of Peak Shape and Characterization of Column Performance in Liquid Chromatography as a Function of Flow Rate

Traditionally, column performance in liquid chromatography has been studied using information from the elution of probe compounds at different flow rates through van Deemter plots, which relate the column plate height to the linear mobile phase velocity. A more recent approach to characterize columns is the representation of the peak widths (or the right and left peak half-widths) for a set of compounds versus their retention times, which, for isocratic elution, give rise to almost linear plots. In previous work, these plots have been shown to facilitate the prediction of peak profiles (width and asymmetry) with optimization purposes. In this work, a detailed study on the dependence of the peak widths (or half-widths) on the flow rate is reported. A new approach to quantify the deterioration of column performance for slow and fast flow rates and to characterize chromatographic columns is proposed. The approach makes use of the width (or half-widths) for a set of compounds with similar interaction kinetics and does not require knowledge of the extra-column contributions to the total variance. The chromatographic data of two sets of compounds of different natures (sulfonamides and β-blockers), eluted from Spherisorb and Chromolith columns with acetonitrile-water mixtures, are used to illustrate the approach.

Simple equations to simulate closed-loop recycling liquid–liquid chromatography: Ideal and non-ideal recycling models

The ideal (the column outlet is directly connected to the column inlet) and non-ideal (includes the effects of extra-column dispersion) recycling equilibrium-cell models are used to simulate closed-loop recycling counter-current chromatography (CLR CCC). Simple chromatogram equations for the individual cycles and equations describing the transport and broadening of single peaks and complex chromatograms inside the recycling closed-loop column for ideal and non-ideal recycling models are presented.The extra-column dispersion is included in the theoretical analysis, by replacing the recycling system (connecting lines, pump and valving) by a cascade of Nec perfectly mixed cells. To evaluate extra-column contribution to band broadening, two limiting regimes of recycling are analyzed: plug-flow, Nec→∞, and maximum extra-column dispersion, Nec=1. Comparative analysis of ideal and non-ideal models has shown that when the volume of the recycling system is less than one percent of the column volume, the influence of the extra-column processes on the CLR CCC separation may be neglected.

The Effects of the Column Length on the Efficiency of Capillary Zwitterionic Organic Polymer Monolithic Columns in HILIC Chromatography

Organic polymer monolithic columns of different lengths have been prepared in 320 µm i.d. fused silica capillary by in situ radical polymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine as a zwitterionic functional monomer and bisphenol A glycerolate dimethacrylate as a crosslinking monomer in the presence of porogenic solvents. The zwitterionic monolithic columns are intended for separations of polar compounds in hydrophilic interaction chromatography (HILIC). The effects of the capillary column length, from 115 to 175 mm, on separation efficiency, were investigated under HILIC conditions, using 95:5 acetonitrile in water as the mobile phase. The extra-column contributions to band broadening significantly decrease the efficiency (apparent height equivalent to a theoretical plate), especially for weakly retained samples, and increase with diminishing column length. The experimental height equivalents of theoretical plate, HETP, were corrected for the extra-column contributions, which were determined for a series of columns by extrapolation to zero column length. On a 175 mm long column, the column efficiency, HETP = 16.5 μm, measured at the optimum linear flow velocity of 0.5 mm s−1, improved to HETP = 5 µm, after correction for extra-column contributions. For more strongly retained small polar compounds, interactions with zwitterionic groups and (or) water adsorbed inside the pores decrease the column efficiency at higher flow rates.

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.

Flow rate dependent extra-column variance from injection in capillary liquid chromatography

Efficiency and resolution in capillary liquid chromatography (LC) can be significantly affected by extra-column band broadening, especially for isocratic separations. This is particularly a concern in evaluating column bed structure using non-retained test compounds. The band broadening due to an injector supplied with a commercially available capillary LC system was characterized from experimental measurements. The extra-column variance from the injection valve was found to have an extra-column contribution independent of the injection volume, showing an exponential dependence on flow rate. The overall extra-column variance from the injection valve was found to vary from 34 to 23nL. A new mathematical model was derived that explains this exponential contribution of extra-column variance on chromatographic performance. The chromatographic efficiency was compromised by ∼130% for a non-retained analyte because of injection valve dead volume. The measured chromatographic efficiency was greatly improved when a new nano-flow pumping system with integrated injection valve was used.

Effect of the pressure on pre-column sample dispersion theory, experiments, and practical consequences

The effect of the pressure on the dispersion of a low molecular weight compound along 0.508 and 1.016mm i.d. × 50cm long open circular tubes was investigated theoretically and experimentally. The theoretical predictions were based on the early models of dispersion derived by Aris and Taylor (1953) and on the approximate model of Alizadeh for the time moments (1980). Experimentally, the system pressure was increased at constant flow rate (0.15mL/min) from less than 20 to nearly 1000bar by using a series of capillary tubes (25μm i.d. PEEKSIL tubes) of increasing flow resistances placed upstream the detection cell of a commercial very high pressure liquid chromatograph (vHPLC) but downstream the 50cm long tube. Theoretical and experimental results agree that the peak variance increases linearly with increasing pressure in the tube volume. The relative increase of the peak variance is 7% above that measured at low pressure (<20bar) for each 100bar increment in the tube volume. This result confirms that accurate measurements of the column efficiency corrected for extra-column contribution cannot be made by replacing the column with a zero dead volume union connector, because the pressures applied in the pre-column volume are significantly different in these two cases. This work shows also that increasing the pressure in the pre-column volume by increasing the flow rate affects the apparent column efficiency that is measured, independently of the direct effect of the flow rate. For a 2.1 × 50mm column packed with 1.3μm core–shell particles run with a classic Acquity system, the associated relative decreases of the column efficiency are expected to be −30%, −20%, and −5% for retention factors of 1, 3, and 10, respectively. The column HETP is no longer independent of its length.

Theory of Gradient Elution Liquid Chromatography with Linear Solvent Strength: Part 2. Peak Width Formation

The process of formation of the width (σ b) of a solute band migrating along a column and its effect on the width (σ) of a corresponding peak in a chromatogram are quantified along with the extra-column contributions (Δσ b and Δσ) to these parameters due to insufficiently narrow injection plugs. Previously unknown expressions for σ b and Δσ b as functions of the band migration distance and time were found. The negative gradients in the solvent strength cause the fronts of the solute bands to travel slower than their tails. This compresses the bands (reduces their widths). Previously unknown expressions describing the band compression process as functions of the band migration distance and time are found. The band compression tends to narrow the peaks. However, as shown here, the gradients that compress the bands also reduce their elution speeds. This tends to broaden the peaks (typically ignored phenomenon) and, as shown here, can cause a slight net peak broadening under normal conditions (in spite of general expectations that the gradients should narrow the peaks). On the other hand, as shown here, the gradients can significantly suppress the harmful effect of the extra-column peak broadening.

Effect of parallel segmented flow chromatography on the height equivalent to a theoretical plate. I—Performance of 4.6 mm × 30 mm columns packed with 3.0 μm Hypurity-C18 fully porous particles

The mass transfer kinetics in short and wide 4.6mm×30mm columns packed with 3.0μm Hypurity-C18 fully porous particles were measured for three different configurations of the inlet sample distribution and outlet sample collection: (1) both the inlet and outlet column endfittings are standard, (2) the inlet endfitting is standard while the outlet endfitting allows parallel segmentation of the exiting flow between a central and a peripheral region across the column diameter, and (3) both the inlet and outlet endfittings allow a parallel segmentation of the flow entering and exiting the column, respectively. The total reduced heights equivalent to a theoretical plate (HETP) were carefully measured, using the first and second central moments of the elution band profiles, obtained by its accurate numerical integration. The longitudinal diffusion term was measured at the lowest experimental reduced velocity applied. The solid–liquid mass transfer resistance was estimated from measurements of the intra-particle diffusivity using the Torquato's model of effective diffusion in packed beds. The trans-channel and short-range interchannel eddy diffusion HETP terms were taken from data obtained by solving numerically the Navier–Stokes equations and simulating advective-diffusive transport in computer-generated random sphere packings. The results clearly show that the trans-column eddy diffusion HETP term can account for up to 85% of the total intrinsic HETP (corrected for extra-column contributions) of the standard columns. Parallel segmented flow chromatography can reduce this contribution by half at high velocities, by eliminating most of the baseline peak tailing. This holds true irrespective of the retention factor of the analyte. It was found also that segmenting the inlet/outlet flow increases detection sensitivity by 25–50% for peaks with large to small retention factors, respectively. In practice, the advantage of parallel segmented flow chromatography in gradient elution (thin peak widths) is essentially limited by post-column bandspreading and diffusion in the dwell volume of the instrument used. Analyst should minimize post-column bandspreading (caused by connectors and detection cell volume) and synchronize the eluent composition in the peripheral and central inlet ports of the column, by using two separate pumps with appropriate dwell volumes.

Possibilities of retention prediction in fast gradient liquid chromatography. Part 1: Comparison of separation on packed fully porous, nonporous and monolithic columns

Due to ever increasing importance of short analysis times in modern HPLC practice, fast gradient elution is becoming widely used both in one-dimensional and two-dimensional separations, especially in the second dimension where the speed of separation is of primary importance. For method development and optimization, prediction of retention in gradient elution from the isocratic data is very important. This is principally possible using the well-established theory of gradient elution, especially for reversed-phase separations. Feasibility of these approaches has been proven in conventional liquid chromatography and commercial prediction software is available for this purpose. However, fast gradient chromatography employing short columns and gradient times in between 1 and 2min or even less, impose additional stringent demands on the accuracy of prediction, due to rapid changes in mobile phase composition and increased role of extra-column contributions and instrumental dwell volume on the retention. In the present work, possibilities of prediction of the gradient elution times from the isocratic data measured only at two mobile phase concentrations were investigated in fast chromatography of alkylbenzenes, phenylurea and triazine pesticides on five columns differing in lengths: two monolithic ones and three packed with particles of different size. In the second dimension, gradient times are strongly restricted by the short cycle frequency. The effects of the flow-rate on the elution times, on the accuracy of their prediction and on peak capacity were studied under time constraint conditions. Gradient theory can be useful for the optimization of fast gradients.

The current revolution in column technology: How it began, where is it going?

AbstractThis work revisits the exceptionally rapid evolution of the technology of chromatographic columns and the important progress in speed of analysis and resolution power that was achieved over the last ten years. Whereas columns packed with 10 and 5μm fully porous particles dominated the field for nearly thirty years (1975–2000), it took barely six years to see the commercialization of monolithic silica rods (2000), their raise to fame and decay to oblivion, the development of finer fully porous particles with size down to 1.7μm (2006), and of sub-3μm superficially porous particles (2006). Analysis times and plate heights delivered by columns packed with these recent packing materials have then been improved by more than one order of magnitude in this short period of time. This progress has rendered practically obsolete the age-old design of LC instruments. For low molecular weight compounds, analysts can now achieve peak capacities of 40 peaks in about 15s with a hold-up time of the order of 1.5s, in gradient elution, by operating columns packed with sub-3μm shell particles at elevated temperatures, provided that they use optimized high pressure liquid chromatographs. This is the ultimate limit allowed by modern instruments, which have an extra-column band broadening contribution of 7μL2 at 4.0mL/min and data acquisition rate of 160Hz. The best 2.1mm×50mm narrow-bore columns packed with 1.7μm silica core–shell particles provide peaks that have a variance of 2.1μL2 for k=1. Finally, this work discusses possible ways to accelerate separations and, in the same time perform these separations at the same level of efficiency as they have today. It seems possible to pack columns with smaller particles, probably down to 1μm and operate them with current vHPLC equipments for separations of biochemicals. Analyses of low molecular weight compounds will require new micro-HPLC systems able to operate 1mm I.D. columns at pressures up to 5kbar, which would eliminate the heat friction problems, and providing extra-column band broadening contributions smaller than 0.1μL2. Alternatively, a new generation of vHPLC systems with minimal extra-column contributions of less than 0.5μL2 could run 2.1mm I.D. columns if these latter were to be packed with high heat conductivity materials such as core–shell particles made with an alumina or gold core.

Measurement of the eddy diffusion term in chromatographic columns. I. Application to the first generation of 4.6 mm I.D. monolithic columns

Abstract The corrected heights equivalent to a theoretical plate (HETP) of three 4.6 mm I.D. monolithic Onyx-C 18 columns (Onyx, Phenomenex, Torrance, CA) of different lengths (2.5, 5, and 10 cm) are reported for retained (toluene, naphthalene) and non-retained (uracil, caffeine) small molecules. The moments of the peak profiles were measured according to the accurate numerical integration method. Correction for the extra-column contributions was systematically applied. The peak parking method was used in order to measure the bulk diffusion coefficients of the sample molecules, their longitudinal diffusion terms, and the eddy diffusion term of the three monolithic columns. The experimental results demonstrate that the maximum efficiency was 60 000 plates/m for retained compounds. The column length has a large impact on the plate height of non-retained species. These observations were unambiguously explained by a large trans-column eddy diffusion term in the van Deemter HETP equation. This large trans-rod eddy diffusion term is due to the combination of a large trans-rod velocity bias (≃3%), a small radial dispersion coefficient in silica monolithic columns, and a poorly designed distribution and collection of the sample streamlets at the inlet and outlet of the monolithic rod. Improving the performance of large I.D. monolithic columns will require (1) a detailed knowledge of the actual flow distribution across and along these monolithic rod and (2) the design of appropriate inlet and outlet distributors designed to minimize the nefarious impact of the radial flow heterogeneity on band broadening.

Fast and efficient size-based separations of polymers using ultra-high-pressure liquid chromatography

Ultra-high-pressure liquid chromatography (UHPLC) has great potential for the separations of both small molecules and polymers. However, the implementation of UHPLC for the analysis of macromolecules invokes several problems. First, to provide information on the molecular-weight distribution of a polymer, size-exclusion (SEC) columns with specific pore sizes are needed. Development of packing materials with large pore diameters and pore volumes which are mechanically stable at ultra-high-pressures is a technological challenge. Additionally, narrow-bore columns are typically used in UHPLC to minimize the problem of heat dissipation. Such columns pose stringent requirements on the extra-column dispersion, especially for large (slowly diffusing) molecules. Finally, UHPLC conditions generate high shear rates, which may affect polymer chains. The possibilities and limitations of UHPLC for size-based separations of polymers are addressed in the present study. We demonstrate the feasibility of conducting efficient and very fast size-based separations of polymers using conventional and wide-bore (4.6 mm I.D.) UHPLC columns. The wider columns allow minimization of the extra-column contribution to the observed peak widths down to an insignificant level. Reliable SEC separations of polymers with molecular weights up to ca. 50 kDa are achieved within less than 1 min at pressures of about 66 MPa. Due to the small particles used in UHPLC it is possible to separate high-molecular-weight polymers (50 kDa ≤ M r ≤ 1–3 MDa, upper limit depends on the flow rate) in the hydrodynamic-chromatography (HDC) mode. Very fast and efficient HDC separations are presented. For very large polymer molecules (typically larger than several MDa, depending on the flow rate) two chromatographic peaks are observed. This is attributed to the onset of molecular deformation at high shear rates and the simultaneous actions of hydrodynamic and slalom chromatography.

Kinetic investigation of the relationship between the efficiency of columns and their diameter

Abstract Many brands of packing materials made of fine particles are now available in both conventional (4.6 mm i.d.) and narrow-bore (2.1 mm i.d.) columns. It is a general observation that the efficiency of the former tends to be markedly higher than that of the latter. This report provides a detailed illustration of the characteristics of this enigma. The corrected reduced plate heights of three brands of columns packed with shell particles in 4.6 and 2.1 mm I.D. columns were measured. The brands were the 1.7 and 2.6 μm Kinetex-C 18 (Phenomenex, Torrance, CA, USA), the 2.7 μm Poroshell120-C 18 (Agilent Technologies, New Castle, DE, USA), and the 2.7 μm Halo-C 18 (Advanced Material Technologies, Wilmington, DE, USA). The extra-column contributions were minimized by optimizing the configuration of the instrument (injection volume < 1.0 μL, 115 μm needle seat capillary, 80 μm connecting tubes, no heat exchanger, 0.8 μL detection cell). The correct peak variances were derived from the numerical integration of the first and second order moments of the experimental band profiles. These experimental results confirm that the kinetic performance of narrow-bore columns is inferior to that of conventional columns for all three brands of shell particles. We demonstrate that this difference is accounted for by a contribution to the column HETP of the long-range eddy diffusion term that is larger in the 2.1 than in the 4.6 mm I.D. columns. While the associated relative velocity biases are of comparable magnitude in both types of columns, the characteristic radial diffusion lengths are of the order of 100 and 40 μm in the wall regions of narrow-bore and conventional columns, respectively.

Evaluation of 1.0 mm i.d. column performances on ultra high pressure liquid chromatography instrumentation

The present study focuses on the evaluation of 1.0 mm i.d. (internal diameter) columns on a commercial Ultra-High Pressure system. These systems have been developed specifically to operate columns with small volumes, typically 2.1 mm i.d., by reducing extra-column volume dispersion. The use of columns with smaller i.d. results in a reduced solvent consumption and required sample volume. The evaluation of the columns was carried out with samples containing neutral and pharmaceutical compounds. In isocratic mode, the extra-column volume produced additional band broadening leading to poor performances compared to equivalent 2.1 mm i.d. columns. By increasing the length of the column, the influence of the extra-column bandspreading could be reduced and 75,000 plates were obtained when four columns were coupled. In gradient mode, the effect of the extra-column contribution on efficiency was limited and about 80% of the performance of the 2.1 mm i.d. columns was obtained. Optimum conditions in gradient mode were further investigated by changing flow rate, gradient time and column length. A different approach of the calculation of peak capacity was also considered for the comparison of the influence of these different parameters.

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

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

Peak compression factor of proteins

An experimental protocol is proposed in order to measure with accuracy and precision the band compression factor G 12 2 of a protein in gradient RPLC. Extra-column contributions to bandwidth and the dependency of both the retention factor and the reduced height equivalent to a theoretical plate (HETP) on the mobile phase composition were taken into account. The band compression factor of a small protein (insulin, MW = 5.8 kDa) was measured on a 2.1 mm × 50 mm column packed with 1.7 μ m C 4-bonded bridged ethylsiloxane BEH-silica particles, for 1 μ L samples of dilute insulin solution ( < 0.05 g/L). A linear gradient profile of acetonitrile (25–28% acetonitrile in water containing 0.1% trifluoroacetic acid) was applied during three different gradient times (5, 12.5, and 20 min). The mobile phase flow rate was set at 0.20 mL/min in order to avoid heat friction effects (maximum column inlet pressure 180 bar). The band compression factor of insulin is defined as the ratio of the experimental space band variance measured under gradient conditions to the reference space band variance, which would be observed if no thermodynamic compression would take place during gradient elution. It was 0.56, 0.71, and 0.76 with gradient times of 5, 12.5, and 20 min, respectively. These factors are 20–30% smaller than the theoretical band compression factors (0.79, 0.89, and 0.93) calculated from an equation derived from the well-known Poppe equation, later extended to any retention models and columns whose HETP depends on the mobile phase composition. This difference is explained in part by the omission in the model of the effect of the pressure gradient on the local retention factor of insulin during gradient elution. A much better agreement is obtained for insulin when this effect is taken into account. For lower molecular weight compounds, the pressure gradient has little effect but the finite retention of acetonitrile causes a distortion of the gradient shape during the migration of its breakthrough front along the column. This phenomenon should be taken into account in the theoretical models.