Elution Mechanism Research Articles

In situ adsorption studies at silica/solution interfaces by attenuated total internal reflection fourier transform infrared spectroscopy: examination of adsorption models in normal-phase liquid chromatography

ATR-FT-IR spectroscopy was employed to the study the adsorption of ethyl acetate and 2-propanol to the surface of thin silica sol-gel films in contact with n-heptane solutions. In situ vibrational spectra of silica-adsorbed species provided information regarding the mechanisms of solute retention and elution in normal-phase chromatography. Previous normal-phase chromatographic studies of ethyl acetate adsorption revealed nonlinear isotherms which were explained by both bilayer and adsorbate delocalization models. Infrared spectra of ethyl acetate at the silica surface versus concentration showed that nonlinear adsorption can be attributed to site heterogeneity, where adsorption to free silanols and surface-adsorbed water can be distinguished. Least-squares modeling of the data produced resolved spectra for the two sites and adsorption equilibrium constants that differed by about an order of magnitude. Adsorption of 2-propanol was best modeled by a single Langmuir isotherm showing no significant difference in adsorption energy for the two sites; 2-propanol was shown to easily displace ethyl acetate from the silica surface. Ethyl acetate could also displace 2-propanol from the silica, and least-squares modeling again revealed two-adsorbed-component spectra for ethyl acetate that were indistinguishable from spectra obtained when ethyl acetate adsorbed directly onto the surface.

STEREOCHEMISTRY OF SIX-COORDINATE OCTAHEDRAL SILICON(IV) COMPLEXES CONTAINING 2,2-BIPYRIDINE

[Si(bpy)3]4+ (bpy = 2,2′-bipyridine), synthesized from Sil4 and 2′.-bipyndine. was optically resolved by a chromatographic method using an SP-Sephadex C-25 column as an adsorbent and a 0.16M aqueous solution of sodium (2R,3R)-(-)-O,O′-dibenzoyltartrate as an eluent. The optical isomers were characterized by measurement of their electronic absorption, circular dichroism, and 1H NMR spectra. The chromatographic resolution of [SKbpyb]4+ was also attempted with an aqueous solution of potassium [(2R,3R)-(+)-tartrato]antimonate(III). sodium (2R,3R)-(+)-hydrogentartrate, and sodium (2R,3R)-(+)-tartrate as eluents. Force-field calculations were used to elucidate the chromatographic elution mechanism. [Si(OH)2(bpy)2]I2was also synthesized from Sil4 and 2′,-bipyridine. The optical resolution of this complex was achieved with sodium [(2R,3R)-(+)-tartrato]antimonate(III).

Effect of hydrogen bromide doping on capillary gas chromatographic analysis of tributyltin and triphenyltin halides

Tributyltin (TBuT) and triphenyltin (TPhT) compounds have been used as effective antifoulants in the marine environment. Trace levels of the trisubstituted tin halides (chloride and bromide) were separated unsatisfactorily on a capillary GC column (without any treatment). The halides, especially TPhT halides, are very polar, and when injected onto a GC column, most are adsorbed on the active sites on the column surface and do not elute smoothly from the column. In this paper, an apolar capillary GC column with flame photometric detection was used to investigate the direct GC analysis of TBuT and TPhT halides. Spiking of the column used was carried out by doping 1 µl of dilute methanolic HBr solution into the GC system prior to the injection of a standard or sample solution. Under the acidic conditions of doping, the peaks of TBuT and TPhT appeared. HBr doping was effective and showed excellent results; sharper and more symmetrical peaks and larger peak areas could be obtained by increasing the HBr concentration. Using a DB-1 capillary column and doping with HBr, trace levels of TBuT and TPhT halides were determined efficiently. Tripentyltin halide was used as an internal standard. Calibration graphs for TBuT and TPhT halides were linear in the range 0.1–0.6 µg ml–1. Chlorides and bromides showed almost identical results. The mechanism of halide elution and peak sharpening by acid doping and the resulting halogen exchange reaction during GC analysis is considered from a chromatographic viewpoint.

High-performance liquid chromatographic separation of some 2- and 4-hydroxy derivatives of 2'-benzoylbenzoic acid on Florisil and silica gel columns

A series of 2'-benzoylbenzoic acid derivatives including some pairs of positional isomers and other related compounds were chromatographed by HPLC using a normal-phase system with polar adsorbents (Florisil and silica) and 2-propanol, 1,4-dioxane and tetrahydrofuran as modifiers of the eluent. The results obtained were compared as log k′ Florisil vs. log k′ silica relationships to ascertain the differences in distribution and chemical character of surface-active centres of the investifated adsorbents and the mechanism of elution in different eluent systems

Characteristics of Superdex® prep grade media for gel filtration chromatography of proteins and peptides

Some chemical and chromatographic properties of Superdex® 30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade, all new composite media for separation of peptides and proteins, were investigated. The selectivity of Superdex 30 prep grade was studied by constructing calibration graphs ( K av vs log M r) of peptides and poly(ethylene glycol) (PEG) standards. The influence of the sodium chloride concentration of the mobile phase on the distribution coefficient of peptides demonstrates that Superdex 30 prep grade contains small amounts of charged groups. Titration results show that the amount of negatively charged groups (carboxyl groups) was about 1 μmol/ml gel for all three investigated Superdex media. A homologous series of n-alphatic (n=4–8) alcohols was used to study the hydrophobic interaction behaviour of Superdex media. The effects of temperature, salt concentration and methanol on K av of the alcohols were used to characterize the elution mechanism. Hydrophobic interactions increased with the amount of dextran coupled to the agarose bead. This means that of the three Superdex media, Superdex 200 prep grade is the least hydrophobic. The functional stability of Superdex 30 prep grade was studied by testing the separation of PEG standards and peptides during repeated cleaning-in-place (CIP) treatments with 1·0 m sodium hydroxide solution and 0·1 m hydrochloric acid. The separation behaviour of the PEG standards and the peptides was unaltered after the medium had been treated for a total contact time of 400 h in 0·1 m hydrochloric acid. However, a slight increase in retention volume was observed after treatment in basic conditions. This effect is a result of hydrolysis of covalently bonded dextran in the agarose pores. In basic conditions, the highest carbohydrate (dextran) leakage was observed for Superdex 30 prep grade and the lowest for Superdex 200 prep grade. In acidic conditions it was found that for all Superdex media, dextran and low amounts of 5-(hydroxymethyl)-2-furaldehyde (5-HMF) were released but the separation characteristics were unaltered after treatment in 0·1 m hydrochloric acid. The highest 5-HMF leakage was observed for Sepharose High Performance (the agarose bead on which Superdex media are based). The lowest amount was released from Superdex 30 prep grade.

Preliminary tests to select operating conditions for the accurate determination of stability constants by cation-exchange chromatography: the Cd 2+Cl − and Cd 2+NO 3− systems

Ion chromatography (IC) has been demonstrated to be a powerful tool for equilibrium constant determination, related to various cation-ligand systems. Nevertheless, no systematic research has been carried out to develop preliminary checks in order to verify whether the variation in ligand concentration in the eluent at constant ionic strength affects the exchange mechanism for the system of interest in the selected chromatographic column. In this paper, tests are proposed which allow one to determine beforehand the experimental conditions to be used in cation-exchange chromatography, whereby parallel mechanisms of elution (mainly in the reversed-phase mode) are avoided. In this way IC becomes an independent rather than an auxiliary means to obtain accurate β i values. Cd(II)Cl − and Cd(II)NO 3 − systems were considered and are discussed.

Elution Mechanisms of Cyclodextrins in Reversed Phase Chromatography

The results of a systematic experimental study of the retention mechanism of native cyclodextrins (CD) in reversed phase liquid chromatography are reported. They show that the behavior of [alpha]-CD differs strongly from that of the larger [beta]- and [gamma]-CD oligomers. [gamma]-CD shows an unexpectedly high hydrophobic surface for solvent interaction and a reduced interaction with the surface of C18 bonded silica. A study by molecular modeling supports the differences observed between [alpha]-CD, [beta]-CD, and [gamma]-CD. 37 refs., 9 figs., 1 tab.

The mechanism of elution of gold cyanide from activated carbon

Numerous articles have appeared on the mechanism of the adsorption of gold cyanide onto activated carbon. In contrast, little information is available on the mechanism of elution of the adsorbed gold. It is the objective of this article to formulate such a mechanism on the basis of batch and column elution tests without analyzing adsorbed species on the carbon directly. The presence of spectator cations (Mn+) enhances the formation of Mn+{Au(CN)2−}n ion pairs on the carbon, which in turn suppress the elution of gold cyanide. The dynamics of removal of these cations determine the horizontal position of the gold peak in an elution profile. When the concentration of cations in the eluant is high and no cyanide is present in the solution or on the carbon, very little desorption of gold is observed. The quantitative effect of the concentration of spectator cations on the equilibrium for desorption of aurocyanide can be estimated from the elution profiles for gold and cations. Free cyanide in the eluant, which causes some competitive adsorption of cyanide with aurocyanide, therefore plays a minor role at the elevated temperatures used in industry. A more important effect of cyanide is its reaction with functional groups on the carbon, the products of which passivate the surface for adsorption of aurocyanide, and thereby cyanide promotes the elution of aurocyanide. The degree of passivation, which is determined to a large extent by the temperature of pretreatment, also affects the elution of cations and the degradation/adsorption of cyanide itself. Reactivation of the carbon surface occurs when the adsorbed/decomposed cyanide is removed by the eluant. At high temperatures of pretreatment, such as used in practice, it is not necessary to include a reactivation term in the mathematical model for elution.

Study of the reproducibility and selectivity of coal tar pitch fractionation by extrography on alumina

The potential of extrography on alumina as a fractionation and characterization technique of coal tar pitches has been evaluated in terms of reproducibility and selectivity of separations. Parameters controlling extrography were carefully selected and standard conditions were adopted by two different laboratories in a reproducibility study. Results relating to mass balances of extrography, selectivity of separation, thermal behaviour of the separated fractions and coke characteristics were comparable for interlaboratory runs. Selectivity of the separations was followed by chemical gas chromatography-mass spectrometry (g.c.-m.s.), nuclear magnetic resonance (n.m.r.), Fourier transform infrared (FT-i.r.) spectroscopy, thin layer chromatography-FID (t.l.c.-FID) and thermal thermogravimetric (TGA) and differential thermal analysis (DTA)] characterizations of both fractions and subfractions collected. Their derived cokes were studied by optical microscopy to determine their respective degree of anisotropy to relate chemical structure, thermal reactivity and coke quality. Concerning the mechanism of extrographic elution, analytical data suggest an extractive mechanism for the first eluted fraction, and dominant chromatographic processes for the more polar fractions.

Study of size distribution and stability of liposomes by high performance gel exclusion chromatography

Applications of high performance liquid chromatography on a gel exclusion column (HPLC-GEC) for the characterization of liposomes are presented following a review of the conventional uses of gel exclusion chromatography (GEC). The practical aspects of HPLC-GEC concerning the choice of the gel and the column operation are examined. A procedure for column calibration with vesicles is proposed, in order to obtain the selectivity curve of the TSK-G6000 PW gel; this appears to be the most convenient gel for sizing of liposomes. The mechanism of elution is discussed as a function of the particle size and composition. Methods involving this type of column and different detection modes (light scattering, refractive index, fluorescence and radioactivity) or their combination, were investigated in detail either to analyse the polydispersity of liposomes or their size stability in terms of aggregation and/or fusion.

Effect of column length and elution mechanism on the separation of proteins by reversed-phase high-performance liquid chromatography

Different theories have been proposed for the elution of proteins in reversed-phase high-performance liquid chromatography. To establish the correct elution mechanism, the effects of column length and the concentration of the organic solvent on column efficiency and the elution of high- and low-molecular-weight compounds were examined. It was concluded that protein elution principally involves the same retention process as for low-molecular-weight compounds, although the influence of partition is small under steep gradient conditions. In accordance with this, wide-pore packings in a short column (35 mm) gave excellent separations of proteins and were usable with a wide range of gradients.

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn 2+, Fe 2+, Fe 3+, Ni 2+, Co 2+, Ca 2+, Sr 2+, Zn 2+ and Cu 2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.

The chemical characterisation of zirconium molybdate gels

The stability of zirconium molybdate gel is pH dependent and related to its point of zero charge (p.z.c.). The p.z.c. (in log [H +] units) increases with decreasing ionic radius of the alkali metal cation associated with the gel. The stability and p.z.c. of zirconium molybdate gels are determined by potentiometric titration and pH-stat techniques. Incorporation of cerium(IV) in the gel matrix improves the elution properties of the gel. This improvement is due to the interaction between the molybdate moieties and the higher Lewis acidity of the cerium—zirconium oxide substrate compared with the standard hydrous zirconium substrate. Zirconium molybdate gels are used in a new type of technetium-99m generator. Evidence for the proposed mechanism of elution of technetium-99m from the gels is provided by structural information generated from thermogravimetric (TG) and differential thermal analysis (DTA), combined with Fourier transform-infrared (FT-IR) spectroscopy and X-ray diffraction (XRD) spectrometry.

Elution Kinetics of Lysine from a Strong Cation-Exchange Resin with Ammonia Water

Abstract The elution mechanism of lysine with ammonia water was investigated. In alkaline pH regions of the liquid phase, the effect of Donnan membrane equilibrium on the elution of lysine adsorbed onto the resin was found to be a predominant factor in determining the equilibrium kinetics in terms of the mass action law for adsorption.

Electrophoretic elution and adsorption: Investigations using microporous membrane immunoadsorbents

This report describes an initial investigation into electrophoretic elution and adsorption techniques which employ microporous membrane adsorbents. To probe the mechanism of elution, electrophoretic elution rates were measured as a function of key process parameters. The elution rate trends qualitatively obeyed the reaction-migration theory developed previously for packed column adsorbents. A quantitative theoretical description of the process is presented. Electrophoretic elution was observed to preserve the activity of a chemically-sensitive monoclonal antibody immunoadsorbent: when eluted using glycine buffer at pH 2.5, anti-bovine serum albumin immunomembranes lost 85% of their antigen binding capacities within three cycles, but essentially full binding capacity was retained over 15 cycles of electrophoretic elution. Electrophoretic adsorption is a technique whereby an electric field is used to facilitate adsorption. Compared with crossflow adsorption, electrophoretic adsorption increased antigen adsorption yields 10-fold (67% vs. 6.6%) while reducing adsorption times by a factor of eight (45 min vs. 6 hr). Adsorption data were interpreted using the numerical reaction-migration theory. Process scale-up and optimization strategies are discussed.

Investigation of the Neutral Filter Elution Technique: I. Effects of Pore Density and Pore Diameter on Elution Rate at pH 9.6

To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (Mr 1.15 x 10(8), E. coli (Mr 2.7 x 10(9)), and Chinese hamster lung fibroblasts (V79, Mr 2-4 x 10(10)). DNA eluted through 15% sucrose atop the filter in a biphasic pattern. The elution rate of the initial component correlated (r greater than 0.97) exponentially with 1/Mr for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 microns. Using this relationship between elution rate and Mr, we estimated Mn of polydisperse, X-irradiated (253 Gy) samples of DNA from E. coli or V79 cells to be 3.15 +/- 1.46 and 1.42 +/- 0.33, respectively, compared to expected values of 2.93 and 3.52 (10(8) Da). The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E. coli. The rate of elution of DNA from unirradiated E. coli was unrelated to pore density or diameter. While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with Mn ca. 10(8) Da appears to be valid quantitatively.

Non-exclusion phenomena in size exclusion chromatography with inorganic porous packing: a thermodynamic treatment

Diverse theoretical expressions have been used to describe the deviations in solute retention in size exclusion chromatography (s.e.c.) with mixed eluents. Most of them are based on the Flory-Huggins (FH) thermodynamic model of polymer solutions. Recently, and using a modified FH theory, we have calculated distribution coefficients K p, in s.e.c. with organic packings. A thermodynamic extension of FH formalism to a rigid gel (i.e. silica-based) in a binary solvent has allowed us to describe the elution mechanisms of a polymer molecule in these systems. The approach used is based on the determination of the polymer concentration ratio between the mobile and quasi-stationary phases, taking into account exclusively FH binary interaction parameters between polymer solute and two components of the eluent. The contribution of preferential solvation of the polymer at a given solvent (eluent) composition has also been analysed. This assumption has been verified using chromatographic data of different polystyrene standards in benzene-methanol, butanone-heptane and carbon tetrachloride-methanol on Spherosil as column packing. Assuming a cylindrical profile for the pores and considering the influence of the preferential solvation of the polymer in each phase, a good correlation between experimental and predicted values of K p has been obtained.

The stability of a polymer-supported rhodium complex in the batch hydroformylation of 1-hexene. II. Effect of reaction conditions

The stability of a polymer-supported rhodium complex has been studied in a batch process for hydroformylation of 1-hexene, using [Rh(CO) 2Cl] 2 as the catalytic precursor and poly(vinylbenzyl diethylenetriamine) as the polymeric ligand. It has been found that the reaction conditions, temperature, pressure, solvent and reaction time etc., have considerable influence on the degree of rhodium elution. Elution of rhodium is serious under normal conditions but low in certain blank experiments. These results indicate that rhodium elution is probably related to the substitution of a low molecular ligand for the polymeric ligand in the catalytic cycle. Several possible mechanisms of rhodium elution based on these results are proposed.

Gas—liquid chromatography in qualitative analysis: XVIII. Study of the dependence of the retention behaviour of polyoxyethylene glycol columns on the nature of the support and phase loading

The retention behaviour of fourteen solutes, possessing a wide range of polarity, and five n-alkanes was investigated using various percentage loadings ofpolyethylene glycol 400 as stationary phase on two diatomaceous supports of different adsorptivity. Retentions were measured as Kova´ts retention indices and specific retention volumes, in order to compare the dependences of absolute and relative retentions on phase loading. For all the solutes examined the values of the retention index decrease with a reduction in phase loading, suggesting a decrease in phase polarity. In contrast, the polar test solutes display slight increases in specific retention whereas those of the n-alkanes increase enormously as the phase loading is decreased and thereby giving rise to the observed large shifts in retention index. In order to explain the phase loading dependence of retention index with polar stationary phases an elution mechanism involving a significant contribution of gas—liquid interfacial adsorption is proposed.

Ion chromatography of transition metals on an iminodiacetic acid bonded stationary phase

A stationary phase synthesized by covalent binding of iminodiacetic acid to a porous silica support was investigated for the separation of transition metals by ion chromatography. Eluents containing carboxylic acids either alone or in combination with stronger complexing agents were studied in order to examine the elution mechanisms of alkali, alkaline earth and transition metal ions. It was found that both complexation reactions and ion-exchange mechanisms occurred, their rate being governed by the stability of metal complexes. These investigations were used to find optimum conditions for the determination of transition metal ions in alkali and alkaline earth metal-rich matrices, e.g., sea water.