High performance liquid chromatography column packings with deliberately broadened particle size distribution: relation between column performance and packing structure

The effect of the addition of 25%, 50% and 75% (weight percent, wt%) of larger particles (resp. 3 and 5 μm) to a commercial batch of 1.9 μm particles has been investigated as an academic exercise to study the effects of particle size distribution on the kinetic performance of packed bed columns in a magnified way. Comparing the performance of the different mixtures in a kinetic plot, it could be irrefutably shown that the addition of larger particles to a commercial batch of small particles cannot be expected to lead to an improved kinetic performance. Whereas the addition of 25 wt% of larger particles still only has a minor negative effect, a significantly deteriorated performance is obtained when 50 or 75 wt% of larger particles are added. In this case, separation impedance number increases up to 200% were observed. Studying the packing structure through computational packing simulations, together with the experimental determination of the external porosity, helped in understanding the obtained results. This showed that small particles tend to settle in the flow-through pores surrounding the larger particles, leading to very high packing densities (external porosities as low as 32% were observed) and also negatively influencing the column permeability as well as the band broadening (because of the broadened flow-through pore size range).     

Influence of particle diameter distribution on protein recovery in the expanded bed adsorption process

The General Rate model has been developed and solved to describe protein adsorption in an expanded bed. The model takes into account axial and local variation of particle size distribution (PSD), external and intra-particle mass transfer resistances, and dispersion in liquid phase. The influence of PSD on breakthrough profiles has been analysed. The simulation results show that for a significantly high expanded bed the lower part of the breakthrough curve profiles, calculated for local particle size distribution (LPSD) and for axial average particle size distribution (APSD) are very similar. However, the upper part of breakthrough profiles calculated for LPSD approaches inlet concentration much more slowly than those calculated for APSD. The retention times of the lower part of uptake curves calculated with average particle diameter are constantly shorter than those obtained from LPSD. For the calculation of the dynamic capacity (DC), the LPSD can be replaced by APSD for large expanded bed heights. Using breakthrough profiles calculated for average particle size, DC values are constantly underestimated.     

Relationship between the particle size distribution of commercial fully porous and superficially porous high-performance liquid chromatography column packings and their chromatographic performance

The separation efficiency and kinetics of several commercial HPLC particle types (both fully porous and superficially porous) have been investigated using a pharmaceutical weakly basic N-containing compound as a test molecule. A strong trend between the particle size distribution (PSD) of the particles and the typically employed “goodness of packing”-parameters was observed. The relative standard deviation of the PSD of the tested particles ranged between 0.05 and 0.2, and in this range, a near linear relationship between the A-term constant, the h_min-value and the minimal separation impedance was found. The experimental findings hence confirm the recent observations regarding the relationship between the narrow PSD of the recently commercialized porous-shell particles and their superior efficiency and kinetic performance. The outcome also suggests that the performance of the current generation of fully porous particle columns could be significantly improved if the PSD of these particles could be reduced.     

Mass transfer mechanism in liquid chromatography columns packed with shell particles: Would there be an optimum shell structure?

The mass transfer mechanisms in columns packed with old (55 μm Zipax and 5 μm Poroshell) and recently commercialized shell particles (2.7 μm Halo-C(18) and Kinetex-C(18)) were investigated from a physico-chemical point of view. Combining a model of diffusion in heterogeneous packed beds (effective medium theory) with values of the heights equivalent to a theoretical plate (HETPs derived from the first and second central moments of the elution profiles) and of the peak variances provided by the peak parking method, we demonstrate that columns packed with current shell particles perform better than those packed with fully porous particles in resolving low molecular weight compounds because the eddy diffusion term of the van Deemter equation of the former is markedly smaller. The calculation of eddy diffusion in column beds suggests that the smaller A terms are due to smaller trans-column velocity bias in columns packed with shell particles. We also show that the mass transfer of large molecules (e.g., proteins) is faster when the internal volume accessible to the analyte increases. Therefore, it is suggested that shell particles made of concentric layers with average pore sizes increasing with increasing diameter would provide columns with higher efficiency.     

Chromatography in pore networks II — The role of structure and adsorption in the band broadening

Summary The complex intraparticle structure typical of chromatographic column packings has been analyzed by use of an equivalent network model which emphasizes pore size distribution and connectivity. Special attention is given as to the way in which diffusion and adsorption interact and display modified peak spreading characteristics according to the morphology of the pore space. This study reveals a very significant increase in the column band broadening over that expected from physical adsorption which can arise from particular distributions of pore sizes. This has implications for designing packings which take advantage of the separating power due to adsorption but do not compromise the resolution of the chromatographic system.     

Native and modified alumina,titania and zirconia in normal and reversed-phase high-performance liquid chromatography

Summary Chromatographic properties of silica, alumina, titania and zirconia have been investigated in normal phase mode in the separation of test mixtures of basic, neutral and acidic compounds. In contrast to silica the chromatographic behaviour revealed the basic properties of the alumina, titania and zirconia surfaces. Therefore, separation of basic compounds on these packings seems very promising. Lypophilic packings have been synthesized by modification of titania, zirconia and alumina with organosilanes and polymers and tested for the separation of basic compounds and proteins. High hydrolytic stability of the modified packings was observed during separations with strong alkali and acidic eluents.     

Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments

A series of experiments and measurements demonstrate the importance of minimizing the extra-column band broadening contribution of the instrument used. The combination of several measures allowed the achievement of the full potential efficiency of three Kinetex-C₁₈ columns, using a conventional liquid chromatograph. The first measure consists in minimizing the extra-column volume of the instrument, without increasing much its back pressure contribution, by changing the needle seat volume, the inner diameter and length of the capillary connectors, and the volume of the detector cell of a standard instrument (Agilent 1100). The second measure consists in injecting a volume of weak eluent (less than half the elution strength of the mobile phase) right after the sample, before the sample had time to reach the column. Experimental results show that these changes could provide most of the resolution expected from the true column performance. After the changes were made, the resolutions of the 2.1 mm ×$\times$ 50 mm, 4.6 mm ×$\times$ 50 mm, and 4.6 mm ×$\times$ 100 mm Kinetex-C₁₈ columns for compounds having retention factors close to 1 were increased by about 180, 35, and 30%, respectively. The resolutions obtained are then similar to those measured with advanced instruments like the Agilent 1200, the Agilent 1290 Infinity HPLC, and the Acquity chromatographs.     

Relative peak broadening and pore size distribution of the stationary phase in exclusion chromatography

Summary It is shown experimentally that in size exclusion chromatography theh-values of a homologous series versus the relative molecular mass of the samples and the pore size distribution of the packing material determined by exclusion chromatography exhibit the same maximum.Part of the Ph.D. Thesis of W. Werner, University of Saarbrücken, Saarbrücken 1976.     

Relationship between trans-column eddy diffusion and retention in liquid chromatography: Theory and experimental evidence

Experimental results demonstrate that trans-column eddy diffusion depends on the retention of compounds. The combination of elution profiles recorded in different points of the exit column cross-section and of the height equivalent to a theoretical plate (HETP) of small molecules clearly show a strong link between retention and column performance in liquid chromatography. These results validate a new model of trans-column eddy diffusion in packed columns. The contribution to the column HETP of the trans-column eddy diffusion term decreases with increasing retention factor from k′ = 0 to k′ = 3 above which it becomes negligible. The best column performance in RPLC is observed for the most retained compounds. This is due to the combination of the lack of a residual trans-column eddy diffusion contribution and the vanishing contribution of the instrument to band broadening.     

Temperature-programmed high performance liquid chromatography separation of mono- and divinyl chlorophyll forms from marine phytoplankton

Summary Divinyl chlorophyll a (a₂) and divinyl chlorophyll b (b₂) are chemotaxonomic marker pigments for the marine prochlorophytes, and can be used to study their distribution in marine samples. In this paper we report a baseline resolution of mono- and divinyl forms of chlorophylls a and b employing polymeric ODS stationary phases at sub-ambient temperatures. The simultaneous resolution of mono- and divinyl forms of chlorophylls a and b, chlorophylls c₁, c₂, c₃, Mg 3,8-divinylphaeoporphyrin a₅ monomethyl ester (MgDVP), and phytol-substituted chlorophylls c was achieved when a temperature step gradient was employed during the analysis. An example is given of the utility of the protocol in oceanic field samples.     

Overloaded elution band profiles of ionizable compounds in reversed‐phase liquid chromatography: Influence of the competition between the neutral and the ionic species

The parameters that affect the shape of the band profiles of acido‐basic compounds under moderately overloaded conditions (sample size less than 500 nmol for a conventional column) in RPLC are discussed. Only analytes that have a single pK_a are considered. In the buffer mobile phase used for their elution, their dissociation may, under certain conditions, cause a significant pH perturbation during the passage of the band. Two consecutive injections (3.3 and 10 μL) of each one of three sample solutions (0.5, 5, and 50 mM) of ten compounds were injected on five C₁₈‐bonded packing materials, including the 5 μm Xterra‐C₁₈ (121 Å), 5 μm Gemini‐C₁₈ (110 Å), 5 μm Luna‐C₁₈(2) (93 Å), 3.5 μm Extend‐C₁₈ (80 Å), and 2.7 μm Halo‐C₁₈ (90 Å). The mobile phase was an aqueous solution of methanol buffered at a constant _W^WpH of 6, with a phosphate buffer. The total concentration of the phosphate groups was constant at 50 mM. The methanol concentration was adjusted to keep all the retention factors between 1 and 10. The compounds injected were phenol, caffeine, 3‐phenyl 1‐propanol, 2‐phenyl butyric acid, amphetamine, aniline, benzylamine, p‐toluidine, procainamidium chloride, and propranololium chloride. Depending on the relative values of the analyte pK_a and the buffer solution pH, these analytes elute as the neutral, the cationic, or the anionic species. The influence of structural parameters such as the charge, the size, and the hydrophobicity of the analytes on the shape of its overloaded band profile is discussed. Simple but general rules predict these shapes. An original adsorption model is proposed that accounts for the unusual peak shapes observed when the analyte is partially dissociated in the buffer solution during its elution.     

Performance of new prototype packed columns for very high pressure liquid chromatography

The reduced heights equivalent to a theoretical plate (HETP) of naphtho[2,3-a]pyrene were measured at room temperature on two sets of new prototype columns designed to be used in very high pressure liquid chromatography (VHPLC). The mobile phase used was pure acetonitrile. The columns are 50, 100, and 150 mm long. Those of the first set are 2.1 mm I.D., those of the second set, 3.0 mm I.D. The performance of these new columns were compared to those of the first generation of VHPLC columns, commercially available in 2.1 mm I.D. The prototype and commercial columns behave similarly at low reduced linear velocities (ν<5$\nu <5$), when the heat effects are negligible. At high flow rates, the shorter prototype columns have a twice better efficiency and less steep C-branches than the commercial columns. In contrast, the C-branch of the 150 mm long prototype columns are slightly steeper than those of the commercial columns. The important contribution to the reduced HETP that is due to the heat effects at high flow rates can in part be accounted for by a band broadening model governed by a flow mechanism with the shortest prototype columns. The sole heat effects cannot, however, explain the mediocre reduced HETPs of the 2.1 and 3.0 I.D. 150 mm long prototype columns. It seems that radial heterogeneity of the flow rate of the long prototype columns is significantly larger than that of the short columns. The contribution of the packing heterogeneity adds up to that of the heat effects to yield a poor column efficiency when sub-2μm$\text{sub-}2\mu \text{m}$ are packed into thin, long column tubes.     

On the use of ionic liquids as mobile phase additives in high-performance liquid chromatography. A review

The popularity of ionic liquids (ILs) has grown during the last decades in several analytical separation techniques. Consequently, the number of reports devoted to the applications of ILs is still increasing. This review is focused on the use of ILs (mainly imidazolium-based associated to chloride and tetrafluoroborate) as mobile phase additives in high-performance liquid chromatography (HPLC). In this approach, ILs just function as salts, but keep several kinds of intermolecular interactions, which are useful for chromatographic separations. Both cation and anion can be adsorbed on the stationary phase, creating a bilayer. This gives rise to hydrophobic, electrostatic and other specific interactions with the stationary phase and solutes, which modify the retention behaviour and peak shape. This review updates the advances in this field, with emphasis on topics not always deeply considered in the literature, such as the mechanisms of retention, the estimation of the suppressing potency of silanols, modelling and optimisation of the chromatographic performance, and the comparison with other additives traditionally used to avoid the silanol problem.     

On crystal chemistry of mercury chromates. Details of fragmentation and packing in crystal structures

In the crystal structures of the minerals edoylerite, deanesmithite, and wattersite and in the structures of the compounds HgCrO₄, Hg₃O₂CrO₄ (analog of the mineral schuetteite), and Pb₂HgO₂CrO₄, there are atomic groups or fragments linked by relatively strong (covalent) bonds. The anion fragments represented by CrO₄ tetrahedra are condensed into pairs and chains, in which adjacent tetrahedra are related by symmetry centers. The cation fragments form various patterns from zigzag ribbons of [Hg₄S₄] rings to ribbons and frameworks with [Hg₆O₂] r-octahedra, [Hg₂CrO] and [Pb₂HgO] triangles, and [Hg₆Cr₂O₄] (stella quadrangula) groups. The symmetry of the fragments and their assemblies is analyzed. Analysis of the reference crystal-forming planes has revealed cation sublattices (close to the F-cubic sublattice in two cases), determining the typical features of the structures.Original Russian Text Copyright © 2004 by S. V. Borisov, S. A. Magarill, and N. V. PervukhinaTranslated from Zhurnal Strukturnoi Khimii, Vol. 45, No. 3, pp. 471–479, May–June, 2004.     

Thermodynamic analysis of equilibria between binary liquid and solid solutions in ternary systems. Application to systems in which two guests compete for sites in the same host,tetrakis(4-methylpyridine)nickel(II) isothiocyanate

A thermodynamic analysis is presented of the equilibria between liquid solutions of guest A in guest B and solid solutions of HA in HB, where HA and HB are isomorphous 1:1 compounds of these guests with the host H. (The treatment is applicable whether or not HA and HB are inclusion compounds.) The mole ratio of A to B in the liquid,R ₁, is generally different from the same ratio in the solid,R _s. Data on many systems have indicated a linear relation between InR ₁, and InR _s, but to date no theoretical basis has been forthcoming. The present analysis shows that this relation is usually sigmoidal in shape but, with certain restrictions, is nearly linear. The slope and intercept are interpreted in terms of the equilibrium constant for the displacement of A from HA by B and the deviations from ideality in the liquid and solid phases. If the deviations from ideality in the liquid phase are known or can be estimated, those for the solid phase can be determined, and thermodynamic equilibrium constants and standard free energy changes for the displacement of A by B calculated. These methods were applied to available data for the following pairs of guests with the host Ni(4-mepy)₄(NCS)₂:p-xylene with each ofp-dibromobenzenp-xylene-d₆,p-xylene-d ₁₀,p-bromotoluene,p-chlorotoluene,p-dichlorobenzene,p-fluorotoluene, ethylbenzene, toluene, and benzene, and the pairsp-dichlorobenzene/p-chlorotoluene and ethylbenzene/ toluene.     

Adsorption mechanism of acids and bases in reversed-phase liquid chromatography in weak buffered mobile phases designed for liquid chromatography/mass spectrometry

The overloaded band profiles of five acido-basic compounds were measured, using weakly buffered mobile phases. Low buffer concentrations were selected to provide a better understanding of the band profiles recorded in LC/MS analyses, which are often carried out at low buffer concentrations. In this work, 10 microL samples of a 50 mM probe solution were injected into C(18)-bonded columns using a series of five buffered mobile phases at (SW)pH between 2 and 12. The retention times and the shapes of the bands were analyzed based on thermodynamic arguments. A new adsorption model that takes into account the simultaneous adsorption of the acidic and the basic species onto the endcapped adsorbent, predicts accurately the complex experimental profiles recorded. The adsorption mechanism of acido-basic compounds onto RPLC phases seems to be consistent with the following microscopic model. No matter whether the acid or the base is the neutral or the basic species, the neutral species adsorbs onto a large number of weak adsorption sites (their saturation capacity is several tens g/L and their equilibrium constant of the order of 0.1 L/g). In contrast, the ionic species adsorbs strongly onto fewer active sites (their saturation capacity is about 1g/L and their equilibrium constant of the order of a few L/g). From a microscopic point of view and in agreement with the adsorption isotherm of the compound measured by frontal analysis (FA) and with the results of Monte-Carlo calculations performed by Schure et al., the first type of adsorption sites are most likely located in between C(18)-bonded chains and the second type of adsorption sites are located deeper in contact with the silica surface. The injected concentration (50 mM) was too low to probe the weakest adsorption sites (saturation capacity of a few hundreds g/L with an equilibrium constant of one hundredth of L/g) that are located at the very interface between the C(18)-bonded layer and the bulk phase.     

Multi-location peak parking method: an important new tool for the study of mass transfer kinetics in liquid chromatography

The peak parking (PP) method probes the longitudinal diffusion coefficient of a compound at a single location along the chromatographic column. We extended to a so-called multi-location peak parking (MLPP) method, in which a large number of axial locations along the column are selected in order to check the validity of the conventional PP method and to reveal possible defaults in the structure of the packed bed or pitfalls of the PP and the MLPP methods. MLPP was applied to a series of HILIC columns, including a 5.0 μm Venusil, a 3.0 μm Luna-diol, three 2.7 μm Halo, and a 1.7 μm Kinetex columns. The results demonstrate that the MLPP method may reveal local heterogeneities in the axial diffusion of small retained low molecular weight compounds along the column. Most importantly, experiments show that the sample zone should not be parked in the entrance of the column (i.e., at <1/10 th of the column length). The abrupt drop in the flow rate considerably affects the peak shape and prevents scientists from using the conventional PP method. Practical solutions to cope with that problem are proposed and their success/failure are discussed.     

A sensitive fluorescence reagent for the determination of aldehydes from alcoholic beverage using high-performance liquid chromatography with fluorescence detection and mass spectrometric identification

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl carbonylhydrazine (DBCEEC) followed by high-performance liquid chromatography with fluorescence detection and APCI-MS identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl functional group, which resulted in a sensitive fluorescence tagging reagent DBCEEC. DBCEEC could easily and quickly labeled aldehydes. The maximum excitation (300 nm) and emission (400 nm) wavelengths did not essentially change for all the aldehyde derivatives. Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M + (CH₂)_n]⁺ in positive-ion mode (M: molecular weight of DBCEEC, n: corresponding aldehyde carbon atom numbers). The collision-induced dissociation of protonated molecular ion formed fragment ions at m/z 294.6, m/z 338.6 and m/z 356.5. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10 to 15-fold molar reagent excess. Separation of the derivatized aldehydes had been optimized on ZORBAX Eclipse XDB-C₈ column with aqueous acetonitrile as mobile phase in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.01-10 nmol mL⁻¹ with coefficients of >0.9991. Detection limits obtained by the analysis of a derivatized standard containing 0.01 nmol mL⁻¹ of each aldehyde, were from 0.2 to 1.78 nmol L⁻¹ (at a signal-to-noise ratio of 3).