The impact of extra-column band broadening on the chromatographic efficiency of 5 cm long narrow-bore very efficient columns

Small columns packed with core-shell and sub-2 μm totally porous particles and monolith columns are very popular to conduct fast and efficient chromatographic separations. In order to carry out fast separations, short (2-5 cm) and narrow-bore (2-2.1 mm) columns are used to decrease the analyte retention volume. Beside the column efficiency, another significant issue is the extra-column band-spreading. The extra-column dispersion of a given LC system can dramatically decrease the performance of a small very efficient column. The aim of this study was to compare the extra-column peak variance contribution of several commercially available LC systems. The efficiency loss of three different type 5 cm long narrow bore, very efficient columns (monolith, sub-2 μm fully porous and sub-2 μm core-shell packing) as a function of extra-column peak variance, and as a function of flow rate and also kinetic plots (analysis time versus apparent column efficiency) are presented.     

Accurate measurements of peak variances: importance of this accuracy in the determination of the true corrected plate heights of chromatographic columns

The true efficiency of a column is derived from the differences between the variances of the peak profiles of the same compound recorded in the presence and the absence of the chromatographic column. These variances are usually derived using one of three methods: (1) the retention time of the peak apex and its half-height width; (2) the moments of the best fit between the experimental data and a hybrid response function, e.g., an exponentially convoluted Gaussian; or (3) the exact moments of the experimental band profiles. Comparisons of the results of these methods show that the first method is always inaccurate because all the band profiles recorded are strongly tailing. The peak fit method is accurate only for 4.6mm I.D. columns operated with instruments having low extra-column volume but fails for short narrow-bore columns due to the severe tailing of peaks passing through the complex channels of the extra-column volumes and to the inaccuracies in the fit of experimental data to the selected function. Although far better, the moment method may be inaccurate when the zero dead volume union used to measure the extra-column peak variances has a higher permeability than the column, causing the upstream part of the instrument to operate under comparatively low pressures.     

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.     

Study of peak shape and efficiency in butyl acrylate-based monolithic columns for capillary electrochromatography

The study and modelling peak shape of in capillary electrochromatography (CEC), obtained using butyl acrylate (BA)-based monolithic columns, is described in this article. A modified-Gaussian model, which is a function of conventional experimental parameters: retention time (t_R), peak height (H₀) and standard deviation (σ₀) at the peak maximum, and left (A) and right (B) halfwidths, was used to describe the peaks of thiourea and several polyaromatic hydrocarbons compounds, which were eluted under several applied voltages. A mean relative error below 2% in the prediction of peak shape is obtained. Based on peak parameters, an easy and reliable estimation of global chromatographic performance, efficiency and peak capacity of BA-based monoliths was also considered. A comparison in terms of chromatographic performance of BA-based monoliths in CEC with CE mode and RPLC microparticulate columns was also performed.     

Improved computer algorithm for characterizing skewed chromatographic band broadening : II. Results and comparisons

A newly developed method using an exponentially modified Gaussian peak shape model produces results that are more precise and less subject to baseline noise than previous methods for characterizing chromatographic band broadening. The method requires only precisely measurable experimental peak parameters: peak retention time, peak height, peak area, and peak centroid (first moment). Accuracy and precision of the new method were compared with other digital approaches by using computer-synthesized peaks and experimental chromatographic data from many HPLC columns. The proposed method offers a reasonable compromise between accuracy, precision, and convenience. A rapid visual estimate of peak skew can be made by inspecting peak shape and referring to a calibration plot involving peak parameters. Peak variance and skew data from this method are also useful for finding column dispersion corrections in size-exclusion chromatography calibrations.     

Achieving high peak capacity production for gas chromatography and comprehensive two-dimensional gas chromatography by minimizing off-column peak broadening

By taking into consideration band broadening theory and using those results to select experimental conditions, and also by reducing the injection pulse width, peak capacity production (i.e., peak capacity per separation time) is substantially improved for one dimensional (1D-GC) and comprehensive two dimensional (GC×GC) gas chromatography. A theoretical framework for determining the optimal linear gas velocity (the linear gas velocity producing the minimum H), from experimental parameters provides an in-depth understanding of the potential for GC separations in the absence of extra-column band broadening. The extra-column band broadening is referred to herein as off-column band broadening since it is additional band broadening not due to the on-column separation processes. The theory provides the basis to experimentally evaluate and improve temperature programmed 1D-GC separations, but in order to do so with a commercial 1D-GC instrument platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a resistively heated transfer line is coupled to a high-speed diaphragm valve to provide a suitable injection pulse width (referred to herein as modified injection). Additionally, flame ionization detection (FID) was modified to provide a data collection rate of 5kHz. The use of long, relatively narrow open tubular capillary columns and a 40°C/min programming rate were explored for 1D-GC, specifically a 40m, 180μm i.d. capillary column operated at or above the optimal average linear gas velocity. Injection using standard auto-injection with a 1:400 split resulted in an average peak width of ～1.5s, hence a peak capacity production of 40peaks/min. In contrast, use of modified injection produced ～500ms peak widths for 1D-GC, i.e., a peak capacity production of 120peaks/min (a 3-fold improvement over standard auto-injection). Implementation of modified injection resulted in retention time, peak width, peak height, and peak area average RSD%'s of 0.006, 0.8, 3.4, and 4.0%, respectively. Modified injection onto the first column of a GC×GC coupled with another high-speed valve injection onto the second column produced an instrument with high peak capacity production (500-800peaks/min), ～5-fold to 8-fold higher than typically reported for GC×GC.     

How to utilize the true performance of monolithic silica columns

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

Study of elution behaviour with gradient voltage in CEC using methacrylate monolithic columns

A theoretical study on the retention behaviour and chromatographic performance of neutral solutes using a lauryl methacrylate‐based monolithic column under voltage gradient mode in CEC was carried out. Through a flexible mathematical function based on a modified Gaussian model, the peak shape of compounds was firstly fitted under constant and gradient voltage. Using the peak shape parameters and retention time, the estimation of global chromatographic performance, efficiency and peak capacity under several voltage conditions was performed. The influence of voltage gradient on the separation efficiency is discussed and simple equations are presented to calculate retention and peak widths under voltage gradient conditions. A comparison in terms of chromatographic performance of a test mixture of neutral solutes under constant and gradient voltage modes was also carried out. The experiments carried out under gradient voltage showed better efficiencies (172 000 plates/m) and lower peak widths than those obtained under constant voltage (52 000 plates/m).     

Chromatographic characterization of molecularly imprinted polymers

Recent efforts in the investigation of chromatographic characterization of molecularly imprinted polymers (MIPs) have focused mainly on the nature of heterogeneous binding sites. More data on the thermodynamics than on the kinetic features of MIP columns have been published. The present article addresses the sources of peak broadening and tailing, which are the main drawbacks often associated with imprinted polymers in chromatography for practical applications. With use of the theory of nonlinear chromatography, the peak properties of a MIP column, including the retention and peak broadening and tailing, can be well interpreted. Efforts to improve chromatographic efficiency using MIPs prepared by approaches different from the conventional method, including covalent imprinting and the format of uniformly sized spherical microbeads, are reviewed and discussed. This review leads to the conclusion that nonlinear chromatography theory is useful for characterizing chromatographic features of MIP columns, since a MIP is essentially an affinity-based chromatographic stationary phase. We expect more theoretical and experimental studies on the kinetic aspects of MIP columns, especially the factors influencing the apparent rate constant, as well as the analysis of the influences of mobile-phase composition on the chromatographic performance. In addition to revealing the affinity interaction by molecular recognition, slow nonspecific interactions which may be inherited from the imperfect imprinting and may be involved in the rebinding of the template to MIPs also need to be characterized. Figure The peak broadening and tailing associated often with molecularly imprinted polymers (MIPs) in column chromatography for practical applications can be well characterized by the theory of nonlinear chromatography.     

Comparison of monolithic silica and polymethacrylate capillary columns for LC

Organic polymer monolithic capillary columns were prepared in fused-silica capillaries by radical co-polymerization of ethylene dimethacrylate and butyl methacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in the presence of various amounts of porogenic solvent mixtures and different concentration ratios of monomers and 1-propanol, 1,4-butanediol, and water. The chromatographic properties of the organic polymer monolithic columns were compared with those of commercial silica-based particulate and monolithic capillary and analytical HPLC columns. The tests included the determination of H-u curves, column permeabilities, pore distribution by inversed-SEC measurements, methylene and polar selectivities, and polar interactions with naphthalenesulphonic acid test samples. Organic polymer monolithic capillary columns show similar retention behaviour to chemically bonded alkyl silica columns for compounds with different polarities characterized by interaction indices, Ix, but have lower methylene selectivities and do not show polar interactions with sulphonic acids. The commercial capillary and analytical silica gel-based monolithic columns showed similar selectivities and provided symmetrical peaks, indicating no significant surface heterogeneities. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra-column contributions. With 0.3 mm ID capillary columns, corrections for extra-column volume contributions are sufficient, but to obtain true information on the efficiency of 0.1 mm ID capillary columns, the experimental bandwidths should be corrected for extra-column contributions to peak broadening.     

Peak half-width plots to study the effect of organic solvents on the peak performance of basic drugs in micellar liquid chromatography

The addition of the anionic surfactant sodium dodecyl sulphate (SDS) to hydro-organic mixtures of methanol, ethanol, propanol or acetonitrile with water yielded enhanced peak shape (i.e. increased efficiencies and symmetrical peaks) for a group of basic drugs (β-blockers) chromatographed with a Kromasil C18 column. The effect can be explained by the thin layer of surfactant associated to the hydrocarbon chain on the stationary phase in the presence of the organic solvents, which covers the free silanols on the siliceous support avoiding their interaction with the cationic basic drugs. These instead interact with the anionic head of the surfactant increasing their retention and allowing a more facile mass transfer. The peak shape behaviour with the four organic solvents (methanol, ethanol, propanol and acetonitrile) was checked in the presence and absence of SDS. The changes in peak broadening rate and symmetry inside the chromatographic column were assessed through the construction of peak half-width plots (linear relationships between the left and right half-widths at 10% peak height versus the retention time). The examination of the behaviour for a wide range of compositions indicated that the effect of acetonitrile in the presence of SDS is different from ethanol and propanol, which behave similarly. Acetonitrile seems to be superior to the alcohols in terms of peak shape, which can be interpreted by the larger reduction in the adsorbed surfactant layer on the C18 column. However, the decreased efficiencies observed at increasing surfactant concentration in the mobile phase should be explained by the reduction in retention times, more than by a change in the stationary phase nature.     

Peak broadening in isothermal runs due to large retention gaps in capillary GC?

A simple formula is derived which allows estimation of the band broadening due to longitudinal diffusion in retention gaps for isothermal runs with the column held at the injection temperature. The broadening is strongly dependent on the linear gas velocity in the retention gap. If the internal diameters of the retention gap and the separation column are similar, the retention gap may have a length exceeding 100 m, even if the separation column is only 10 m long. A 0.5 mm I.D. retention gap attached to a 0.3 mm I.D. separation column (of interest for automatic on-column injection) may be several meters long. Retention gaps of 0.3 mm I.D. may be used for on-column injections into separation columns with I.D's. down to about 0.1 mm.     

Estimation of chromatographic parameters on two silica-based columns connected in series under nonaqueous reversed phase liquid chromatographic conditions

Empirical equations were produced to relate important chromatographic parameters on two silica-based columns serially linked, in isocratic nonaqueous RP HPLC, to retention times and peak widths of the separated compounds on the individual columns. These equations were derived because the experimental data seemed to deviate from the values expected, applying basic chromatographic theoretical equations. The chromatographic parameters studied were retention time, peak width, resolution, number of theoretical plates, capacity factor, and separation factor. In addition, empirical linear relationships were produced for the estimation of the above mentioned parameters of the serial systems, in direct and reverse order, relating them to those obtained on each column, separately. The experimentally obtained values were in good agreement with those estimated by the derived equations.     

Effect of extra-column volume on practical chromatographic parameters of sub-2-μm particle-packed columns in ultra-high pressure liquid chromatography

Effects of extra-column volume on apparent separation parameters were studied in ultra-high pressure liquid chromatography with columns and inlet connection tubings of various internal diameters (id) using 50-mm long columns packed with 1.8-μm particles under isocratic conditions. The results showed that apparent retention factors were on average 5, 11, 18, and 41% lower than those corrected with extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns, respectively, when the extra-column volume (11.3 μL) was kept constant. Also, apparent pressures were 31, 16, 12, and 10% higher than those corrected with pressures from extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns at the respective optimum flow rate for a typical ultra-high pressure liquid chromatography system. The loss in apparent efficiency increased dramatically from 4.6- to 3.0- to 2.1- to 1.0-mm id columns, less significantly as retention factors increased. The column efficiency was significantly improved as the inlet tubing id was decreased for a given column. The results suggest that maximum ratio of extra-column volume to column void volume should be approximately 1:10 for column porosity more than 0.6 and a retention factor more than 5, where 80% or higher of theoretically predicted efficiency could be achieved.     

Influence of the errors made in the measurement of the extra-column volume on the accuracies of estimates of the column efficiency and the mass transfer kinetics parameters

The influences of the errors made in the measurement of the extra-column volume of an instrument on the accuracies of the estimates made of the column efficiency and of the parameters of the mass transfer kinetics were investigated from an experimental point of view. A standard HP1090 apparatus (extra-column volume, approximately 50 micro L) was used to measure the efficiency of a Sunfire-C(18) RPLC column (column hold-up volume, approximately 1.50 mL). The first and second moments of the peaks of phenol (a retained compound) and of thiourea (a practically non-retained compound) were measured at six different temperatures between 22 and 78 degrees C, for flow rates between 0.10 and 4.70 mL/min (i.e., for linear velocities between 0.025 and 1.179 cm/s). Each series of measurements was successively made with the instrument being fitted with and without the column. The experimental HETP data must be corrected for the solute dispersion in the connected tubes in order properly to assess the true column efficiency. Even with a modern, high performance instrument, the dispersion of a non-retained compound is essentially due to the band broadening phenomena that take place in the extra-column volumes, the sum of all these extra-column band broadening contributions accounting for more than 80% of the total band broadening measured. The contribution of the sampling device is particularly deleterious since, for a 2 mu L injection, the maximum solute concentration in the peak that enters into the column is nearly ten-fold lower than that of the sample. Nevertheless, the impact of the extra-column volumes on the estimates of the kinetic parameters (e.g., molecular diffusion coefficient D(m) and effective particle diffusivity D(e)) remains negligible. Obviously, the relative error made on the column efficiency of a retained compound depends much on its retention factor. It decreases from 8 to 1% when the retention factor increases from 5 to 17.     

Practical method for the definition of chromatographic peak parameters in preparative liquid chromatography

A practical method was established for the definition of chromatographic parameters in preparative liquid chromatography. The parameters contained both the peak broadening level under different amounts of sample loading and the concentration distribution of the target compound in the elution. The parameters of the peak broadening level were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The concentration distribution of the target compound was described by the heat map of the elution profile. The most suitable stationary phase should exhibit the narrower peak broadening and it was best to broaden to both sides to compare to the peak under analytical conditions. Besides, the concentration distribution of the target compounds should be focused on the middle of the elution. The guiding principles were validated by purification of amitriptyline from the mixture of desipramine and amitriptyline. On the selected column, when the content of the impurity desipramine was lower than 0.1%, the recovery of target compound was much higher than the other columns even when the sample loading was as high as 8.03 mg/cm³. The parameters and methods could be used for the evaluation and selection of stationary phases in preparative chromatography.     

The system evaluation strategy in practice,allowing direct estimation of peak heights and limits of detection in HPLC

Summary The novel system evaluation strategy recently developed describes an HPLC system in terms of two parameters, that ideally are independent of the test conditions. These describe the peak height response, or dilution, occurring in the column and the system. Limits of detection (LOD’s) can thus also be calculated. System parameters, (e.g. cell length, external dispersion), chromatographic parameters (e.g. retention, column length, flow-rate), and sample parameters (e.g. sample volume & absorptivity), are all included. Consequently this approach is applicable to the chromatographer and LC system designer alike. The system evaluation strategy was previously demonstrated under typical chromatographic conditions, on two HPLC systems, and systematic changes were studied. In principle this is applicable to any compound under any chromatographic conditions, on any column. This is now to be investigated further, under a wide range of conditions, and applied to the calculation of an LOD in a real LC analysis.     

Estimation of the band broadening contribution of HPLC equipment to column elution profiles

Summary Methods to determine the contribution of the chromatographic equipment to the total band broadening which involve replacing the column by a union or a capillary tube are not suitable as they involve a fundamental change in the chromatographic system. The linear extrapolation method, based on the estimation of the relative influence of the instrument variance on solutes with different capacity factors, is a more attractive alternative method since the column remains in the chromatographic system. This method is only valid when a number of conditions are satisfied. By meeting these conditions the error in the instrument variance by using the linear extrapolation method was determined. At the same time, ways to minimise these errors were studied. Use of the linear extrapolation method in combination with conventional columns of 4.6 mm i.d. appears to yield inaccurate results. In combination with microbore columns the method can be used, provided the columns have a maximum length of 5cm and contain a packing material with a particle size of 2 or 3μm. The error in the determined instrument variance is then of the order of 2μl².