Ion chromatographic separation of car☐ylic acids prediction of retention data

The retention behavior of biologically relevant monovalent (formic, acetic, propionic, lactic and pyruvic) and divalent (oxalic, malonic, succinic, fumaric, maleic and tartaric acids) car☐ylic acids together with inorganic analytes (chloride and sulphate) has been studied. The separation was performed on a latex-based strong anion-exchange resin using carbonate buffer systems in suppressed IC. The retention behaviour of analytes was investigated at different pH values and [HCO₃⁻]+[CO₃²⁻] concentrations. A theoretical model, involving ion-exchange equilibria of sample and eluent anions, was derived and applied to the chromatographic data obtained. Chromatographic ion exchange selectivity values were determined and retention data were calculated for the anions using different carbonate eluent conditions. The average of errors between the predicted and the measured retention volumes of the analytes studied does not exceed 4.0%. The study effectively characteristics the behaviour of different analytes under elution conditions of practical importance.     

Analysis of aliphatic car☐ylic acids and amino acids in effluents of landfills, composting plants and fermentation plants by ion-exclusion and ion-exchange chromatography

Short chain aliphatic acids, including volatile fatty acids (VFAs), di-/tricar☐ylic acids, hydroxy- and keto-acids were analyzed in landfill leachates and related water samples by two independently operated ion-exclusion chromatographic systems, differing mainly in the retention characteristics of the separation columns (Merck Polyspher OA-HY, Dionex HPICE AS6), and in the detection mode (UV absorbance at 210 nm, conductivity). The amino acid content of the samples was determined by ion chromatography. Because methods for amino acids analysis are widely standardized, the main efforts were undertaken to optimize the determination of car☐ylic acids. The VFAs (7 compounds) contributed between 33% and 89% to the sample's dissolved organic carbon (DOC) content. The DOC proportions of the multifunctional acids (9 compounds) ranged from 1.1–49%. Between 0.9% and 13% of the DOC content was apportioned to amino acids. Main components were alanine, valine and leucine. The analytical efficiencies of the ion-exclusion chromatography systems were compared and the specific application properties are discussed.     

Ion-exclusion chromatography of aliphatic car☐ylic acids on a cation-exchange resin by elution with polyvinyl alcohol

Ion-exclusion chromatography of aliphatic car☐ylic acids of different acidity (pK_a) and hydrophobicity was investigated on a polystyrene-divinylbenzene (PS-DVB) based strongly acidic cation-exchange resin in the H⁺ form and conductivity detection by elution with polyvinyl alcohol (PVA). When water was used as an eluent, the resolution of the car☐ylic acids was very low and the peak accompanied a fronting depending on their hydrophobicities. Therefore, to improve the peak shape and the peak resolution, aqueous eluents containing PVAs (degrees of polymerization, n=500, 1500and2000) with many OH groups were tested for the ion-exclusion chromatographic separation of the car☐ylic acids. When aqueous eluents containing PVA were used, the fronting was decreased dramatically by the effect of increased hydrophilicity of the PS-DVB cation-exchange resin surface due to adsorption of OH group in PVA. The high resolution ion-exclusion chromatographic separation without the fronting and highly sensitive conductimetric detection of the car☐ylic acids was accomplished successfully by elution with a 0.2% PVA (n=1500)-10% methanol-water.     

Continuous desorption rate measurement from a shallow-bed of poly(styrene-divinylbenzene) particles with correction for experimental artifacts

A 0.50 mm high bed, containing ca. 3 mg of the nominally non-porous poly(styrene-divinylbenzene) (PS-DVB) sorbent Hamilton PRP-infinity, is located in a valve. After the bed is pre-equilibrated with a (7/3) methanol/water solution of naphthalene (NA), the valve is switched and (7/3) methanol/water solvent flows continuously through the bed at a high linear velocity. This causes NA to desorb into a constantly refreshed solvent, creating a "shallow-bed" contactor with an "infinite bath" kinetic condition. The effluent from the bed passes through a UV-absorbance detector which generates the observed instantaneous desorption rate curve for NA. The same experiment is performed using the solute phloroglucinol (PG), which is not sorbed by PRP-infinity and serves as an "impulse response function marker" (IRF-Marker). The resulting peak-shaped IRF curve is used in two ways (i.e. subtraction and deconvolution) in order to correct the observed instantaneous rate curve of NA for the following experimental artifacts: hold-up volume of the bed and valve, transit-delay time between the bed and the detector and instrument bandbroadening of the NA zone. The cumulative desorption rate curve, which is a plot of moles NA desorbed versus time, is obtained by integration. It is found to be accurately described by the theoretical equation for homogeneous spherical diffusion. The diffusion coefficient of NA inside the PRP-infinity particles (5.0+/-0.6) x 10(-11) cm2/s, agrees with the literature value that was obtained from the sorption rate of NA into the same particles. This constitutes virtually conclusive evidence for diffusion control of intra-particle kinetics of NA in the PS-DVB matrix of PRP-infinity and related polymers. The influence of both sorbent and solute properties on the method is evaluated.     

Studies on the combination of ion chromatography-particle-beam mass spectrometry with capillary columns

A particle beam interface was investigated for coupling ion chromatography with mass spectrometric detection. Several prerequisites must be fulfilled, including mobile phases containing volatile buffers and high amounts of organic solvents at low flow-rates. Microcolumns with inner diameters between 130 and 1000 μm (packed with a silica-based anion-exchange material) in combination with a microflow aerosol generator meet these requirements. Organic solvetns in the mobile phase lead to considerable changes in separation selectivity, so that the retention order can be partly reversed in comparison with aqueous mobile phases. The performance of the interface and the mass spectrometric detection has been studied for a series of inorganic anions as well as for aminopolycar☐ylic acids and their metal complexes. The detection limits are between 10 and 100 ng injected and are significantly poorer than those for conductivity detection. On the other hand, the possibility of operating the detector at pre-selected masses greatly improves the selectivity of the analysis and helps to confirm peaks from a non-selective conductivity detector. On-line and off-line preconcentration techniques allow the detection of anions in drinking water at ppb levels.     

Comparison of the chromatographic behavior of monolithic capillary columns in capillary electrochromatography and nano-high-performance liquid chromatography

Porous monoliths based on N,N-dimethylacrylamide (DMAA) or methacrylamide (MAA) were prepared inside fused silica capillaries as stationary phases for nano-chromatography. The columns were characterized in terms of flow rate and backpressure and showed, e.g. differences as a function of the salt concentration added to the polymerization mixture. When the columns were investigated for the separation of uncharged (polar hydroxylated aromatic compounds) and charged (amino acids) analytes under pressure driven conditions (pLC), differences to the previously observed behavior under voltage driven conditions (CEC) were observed. Whereas the non-charged analytes showed similar behavior in both cases--thus, corroborating the previous assumption of a mainly chromatographic separation mode driven by hydrophilic interactions in CEC--the charged amino acids did not. Assuming that the separation was governed by chromatographic phenomena in the pLC mode and by both chromatographic and electrophoretic effects in the CEC mode, the experiments allowed deconvoluting the two contributions. In particular, the charged amino acids appeared to interact with the stationary phases mainly by electrostatic interactions modified by some hydrophilic effects.     

Donor-acceptor complex chromatography (DACC) Part I. Introduction and nomenclatur

Summary Liquid chromatographic separations by the formation of donor-acceptor complexes (DAC) are reviewed. The article treats these examples, where donor or acceptor compounds are coated on, or chemically bonded to, a matrix. The effect of chemical structure of donors and acceptors, the effect of temperature and solvent composition of the mobile phase on retention is discussed.     

Donor-acceptor complex chromatography (DACC) part II. Review of papers

Summary Liquid chromatographic separations by the formation of donor-acceptor complexes (DAC) are reviewed. The article treats these examples, where donor or acceptor compounds are coated on or chemically bonded to a matrix. The effect of chemical structure of donors and acceptors, the effect of temperature and solvent composition of the mobile phase on retention is discussed.     

Solute retention and the states of water in polyethylene glycol and poly(vinyl alcohol) gels

The states of water sorbed in a cross-linked polyethylene glycol (PEG) gel, TSKgel Ether-250, and cross-linked poly(vinyl alcohol) (PVA) gels of different pore sizes, TSKgel Toyopearl HW-40S, 50S, 55S and 75S, were investigated by means of differential scanning calorimetry (DSC). It was found that there were three types of water in these hydrogels, non-freezing water, freezable bound water and free water. The amount of water that functions as the stationary phase in the column packed with the each gel was also estimated by a liquid chromatographic method. The estimated amount of the stationary phase water is in good agreement with the sum of the amount of non-freezing water and that of freezable bound water for HW-40S, 50S and 55S, while it agrees with the amount of only non-freezing water for HW-75S and Ether-250. This means that the stationary phase water consists of non-freezing water and freezable bound water for HW-40S, 50S and 55S, while only non-freezing water functions as the stationary phase in HW-75S and Ether-250 gels. This result can be attributed to the difference in the structure of the gels; the PVA gels containing PVA at relatively high concentrations, HW-40S, 50S and 55S, have a homogeneous gel phase, whereas HW-75S and Ether-250 have a heterogeneous gel phase consisting hydrated polymer domains and macropores with relatively hydrophobic surface. The freezable bound water in Toyopearl HW-40S, 50S and 55S can be regarded as a component of a homogeneous PVA solution phase, while that in HW-75S and Ether-250 may be water isolated in small pores of the hydrophobic domains. The results obtained by the investigation on the retention selectivity of these hydrogels in aqueous solutions supported our postulated view on the structures of the hydrogels.     

Hydrophobic and cation exchange mechanisms in the retention of basic compounds in a polymeric column

A cation exchange retention mechanism concomitant with the well-known hydrophobic partition mechanism in a polymeric column has been observed and investigated. This exchange process is attributed to ionization of some acidic sites present in the polymer column at basic mobile phase pH values. Several drugs of different basicity have been chromatographed on a polymeric PLRP-S column with methanol-water and acetonitrile-water mobile phases. The cation exchange between the protonated basic drug and the buffer cations (Na+, K+ and BuNH4+) is observed at the pH range where the protonated drug and the ionized sites of the column coexist. This process produces a shift of the retention versus pH plot of the base to pH values lower than those expected from the pKa of the base as well as a maximum in the plot at basic pH values. These effects are more pronounced for acetonitrile-water mobile phases.     

Sulfoacylated poly(styrene-divinylbenzene) copolymers as resins for cation chromatography. Comparison with sulfonated, dynamically coated and silica gel cation exchangers

Most important properties of an ion chromatographic resin are influenced by the resin matrix, the type of functional group and the ion-exchange capacity. Highly crosslinked PS-DVB resins of 5 microm diameter have been functionalized by sulfoacylation, by sulfonation and by dynamic coating over a wide range of exchange capacities. These materials allow a study of the influence of different ion-exchange sites and capacities. The influence of the degree of functionalization on resin performance is completely reverse for sulfoacylated and sulfonated resins. The HETP values for all observed analytes (Cu, Pb, Zn, Ni, Co, Cd, Mn, Ca, Mg) in a tartaric acid elution system decrease for sulfoacylated resins with increasing capacity, for sulfonated resins with decreasing capacity. The performance of sulfoacylated exchangers is better than for dynamically coated ones and far better than for sulfonated resins. The performance of silica gel based cation-exchangers such as BioSil CAT is in most cases better than observed for sulfoacylated resins.     

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     

Relative retention of the fibroblast growth factors FGF-1 and FGF-2 on strong cation-exchange sorbents

The isocratic retention of two heparin-binding fibroblast growth factors, FGF-1 (acidic FGF) and FGF-2 (basic FGF), was compared on a set of six preparative strong cation-exchange adsorbents. The FGFs comprise a solute pair that are structurally equivalent, yet differ in protein parameters of potential importance in cation-exchange chromatography, such as isoelectric point, net charge, and the number and distribution of basic amino acids. The cation-exchange adsorbents comprise a diverse set of materials in common use for protein purification, with physical and chemical properties that have been characterized and described previously. Isocratic k' values for the two proteins obtained on each adsorbent at several different [NaCl] are compared with one another and with corresponding data for hen egg lysozyme, which is also strongly retained on cation-exchangers. Of the six adsorbents examined, three showed strong retention of both FGFs, with equivalent k' values for FGF-1 and FGF-2. Three others, which showed weaker overall retention for the FGF pair, showed much larger retention differences between FGF-1 and FGF-2. The trends in retention order among the stationary phases are very similar to those seen previously with other unrelated proteins. However, retention differences between the two FGFs, and between the FGFs and lysozyme, do not correlate well with simple charge properties such as net charge, indicating, as in some previous studies, the importance of local regions on the protein surface in determining retention. These observations are interpreted in terms of the structural features of the proteins and the physicochemical properties of the adsorbents.     

High-performance liquid chromatographic separation of detritylated oligonucleotides on highly cross-linked poly-(styrene-divinylbenzene) particles.

Detritylated oligonucleotides were separated by reversed-phase high-performance liquid chromatography on highly cross-linked polystyrene-based particles having a mean particle diameter of 2.3 microns. The addition of poly(vinyl alcohol) during polymerization, which resulted in the presence of hydroxyl groups on the surface of the poly(styrene-divinylbenzene) beads, was necessary to obtain baseline resolution of phosphorylated oligodeoxyadenylic acids with a chain length of up to 30 bases. The impact of temperature was investigated and optimum resolution was achieved at 40 degrees C. At pH 7.0, the retention times of oligonucleotides were found to depend on the ratio of bases and to increase in the order of C less than G less than A less than T. Under the same conditions, it was possible to separate phosphorylated from dephosphorylated oligonucleotides, the former being eluted earlier. Recoveries ranged from 92 to 100%.     

Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer

The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and alpha-chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 microm, 1000 A) and a monolithic column (UNO S1). Both columns are strong cation exchangers, carrying -SO3(-)-groups linked to a hydrophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100-200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20000 g mol(-1), which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer.     

An information theory-based evaluation of stationary phases developed for optical resolution of amino acids

Summary Naphthylethylurea multiple-bonded chiral stationary phases prepared for the optimal resolution of p-bromophenylcarbamyl derivatives of enantiomeric amino acids are examined from the viewpoint of information theory. The criterion used is the function of mutual information (FUMI) which describes the precision of analysis as a function of peak shape (area and width), overlap and noise level. The length of the polyethyleneamine spacer in the chiral stationary phases is evaluated by the precision (FUMI) calculated from the peak shape and overlap. The best column is defined as the one which gives the highest precision. The temperature effect on the precision is also examined.     

Preparation and applications of weak acid cation exchanger based on monodisperse poly(ethylvinylbenzene-co-divinylbezene) beads

New technology is reported here for the synthesis of an effective weak acid-cation exchanger for ion chromatography. Monodisperse macroporous poly(ethylvinylbenzene-co-divinylbezene) (PEVB-DVB) beads of 5 microm diameter were prepared by a two-step swelling and polymerization method. Then carboxyl groups were introduced by polymerization of maleic anhydride with unreacted vinyl groups on the resin beads, followed by hydrolysis of the maleic anhydride groups. A column packed with the carboxylate beads was used to separate alkali and alkaline earth metal ions in a single isocratic run. Separations were found to be better than those with similar resin particles that are simply coated with maleic acid. The columns containing the new particles were 100% compatible with solvents commonly used for HPLC. Additionally, the prepared column was stable and could be used for a long time in a wide range of pH. The column gave good resolution, low detection limits and good repeatability for the separation of common cations. Satisfactory results were also obtained for separations of organic amines and of common cations in rainwater.     

The use of ligand exchange for the separation of aromatic acids on synthetic cation exchangers

Summary A very good separation of the mixture of eight aromatic acids has been obtained by ion-exchange chromatography using the ligand-exchange technique, on a column containing a synthetic cation exchanger and trivalent metal ion (Al³⁺, Fe³⁺, or Ce³⁺).     

Investigations on the chromatographic behavior of hybrid reversed-phase materials containing electron donor-acceptor systems. I. Contribution of sulfur-aromatic interactions

The influence of sulfur-aromatic interactions on the chromatographic separation behavior of hybrid RP phases, containing thiol-groups and/or embedded sulfide-groups (S-RP) has been investigated. To allow a precise outline of this new interaction mode, a wide variety of S-RP phases with different alkyl chain length, with and without residual thiol-groups and silanol-endcapping were prepared and tested in comparison to some conventional monomerical as well as polymerical n-alkyl type RP phases. The solute test sets employed in this study comprised the classical chromatographic column tests from Engelhardt and Tanaka as well as test assemblies containing polycyclic aromatic hydrocarbons, stilbene-based cis/trans isomers and functional isomers of benzene. In general, a pronounced strong planar recognition ability as well as a strong increase in the retention for aromatic compounds have been noticed. It was furthermore found that not only the sulfur atom incorporated into the alkyl chain, but also the residual thiol-groups of the 3-propylthiol silica backbone contribute to the overall retention behavior of these novel S-RP type phases.