Comparison of the retention characteristics of aromatic and aliphatic reversed phases for HPLC using linear solvation energy relationships

The similarities and differences in retention characteristics of aromatic and aliphatic phases have been elucidated by the use of linear solvation energy relationships (LSERs). Three aromatic phases and three aliphatic phases were investigated in a series of mobile phases. The results of LSERs on a polymer-based aromatic phase, poly(styrene-divinylbenzene) resin (PRP-1) are very different from those on either silica- or zirconia-based aromatic and aliphatic phases. Retention on all aromatic and aliphatic phases except PRP-1 is dominated by two factors: the solute size and hydrogen bond acceptor basicity. On the other hand, in addition to these two major contributions, retention on PRP-1 is markedly influenced by the solute hydrogen bond donor acidity. We believe that PRP-1 exhibits a more adsorption-like retention mechanism than do the other phases. With the inorganic oxide-based phases, the aromatic phases are less retentive than the aliphatic phases but show a larger dependence on molecular polarizability. The enhanced polarizability of aromatic phases is the likely cause of some differences in their chromatographic selectivity relative to the aliphatic phases.     

Interactions between dansyl amino acids and human serum albumin using high-performance liquid chromatography: mobile-phase pH and temperature considerations

The reversed-phase liquid chromatography (RPLC) retention mechanism of a series of dansyl amino acids was investigated over a wide range of mobile-phase pH and column temperatures using human serum albumin (HSA) as a chiral stationary phase. Thermodynamic constants for the transfer of a solute from the mobile to the HSA stationary phases were determined. Different van't Hoff plot shapes were observed with different mobile-phase pH values, indicating a change in the retention mechanism. Enthalpy-entropy compensation revealed that the solute retention mechanism was independent of the compound molecular structure, the same at four pH values (5.5, 6, 6.5, and 8), but changed at pH = 7 and 7.5. Differential scanning calorimetry was used to show phase transition in the HSA stationary phase at pH = 7 and 7.5. A new theory was presented to explain that the HSA protein structure balance between a disordered and an ordered solid-like state. Variations of column temperature and mobile-phase pH tend to cause this phase transition between these two states, explaining the observed thermodynamic constant variations with pH and temperature.     

HSA-solute interactions, enantioselectivity, and binding site geometrical characteristics

Recently, a theoretical model was proposed to study the existence of pockets of acetonitrile (ACN) called clusters in a hydroorganic mixture. The proposal used ACN as an interaction organic modifier between D,L-dansyl amino acids and their binding site in human serum albumin at site II. This solute binding is governed by primary and secondary interactions. The primary interactions are under the dependence of the solute solvation by ACN clusters and electrostatic interactions. Following this first step, the solute engages strong short-range interactions with the residues of site II. Using a biochromatographic approach, the solute binding, i.e., the solute retention, was divided into two dielectric constant (epsilon) ranges. In the first range, epsilon > epsilon c (epsilon c is the critical dielectric constant); the primary and secondary nonstereoselective electrostatic interactions were the major contributions to the variation in the solute binding with the ACN fraction in the mixture. In the second range, epsilon < epsilon c, the solute retention variation with the ACN fraction was governed by its solvation by the ACN clusters and also by the secondary hydrophobic stereoselective interaction. The mathematical model developed provided the determination of the surface charge density of site II as well as the cluster number that solvates each solute.     

Validated method for the determination of idazoxan in human plasma by liquid chromatography with tandem mass spectrometric detection

The effect of perchlorate anion as a mobile phase modifier on the retention of dansyl norvaline and dansyl tryptophan enantiomers on a human serum albumin (HSA) column was studied by varying the chaotropic agent concentrations. The thermodynamic parameters for the transfer of a solute from the mobile to the HSA stationary phases were determined from linear van't Hoff plots. An enthalpy-entropy compensation study revealed that the type of interaction between the solute and HSA was independent of the molecular structure. The parabolic variations observed with the enthalpic and entropic terms of dansyl amino acid transfer in relation to the concentration of perchlorate anion were considered to be the result of the change from reversed to normal-phase conditions for this chromatographic system.     

Separation of oligomers of nonylphenyl ethylene oxide [correction of nonlphenylethylene oxide] on a porous graphitized carbon column

The retention of nonylphenyl ethylene oxide oligomer surfactants was determined on a porous graphitized carbon (PGC) column using water--methanol mixtures as eluents. Linear correlations were calculated between the logarithm of the capacity factor (k') and the methanol concentration in the eluent. To test the validity of the hypothesis that in the case of homologous series of solutes the intercept and slope values are intercorrelated linear correlation was calculated between the two chromatographic parameters. To elucidate the role of the length of the polar ethylene oxide chain in the retention linear correlations were calculated between the chromatographic parameters and the number of ethylene oxide groups per molecule. Nonylphenyl ethylene oxide oligomers were well separated on the PGC column. Significant linear relationships were found between the corresponding chromatographic parameters indicating that the solutes behave as a homologous series of compounds. The retention of surfactants increased linearly with increasing number of ethylene oxide groups per molecule indicating hydrophilic interactions between the solutes and the surface of PGC support.     

Surface segregation of phosphorus, carbon, and sulfur in commercial low-carbon grades of steel

Surface segregation behavior of solute atoms has been studied on low-carbon steels used in producing galvannealed sheet steels for automotive body panel applications. Samples of cold-rolled low-carbon steels with different amounts of carbon and phosphorus in solution were heated in a vacuum chamber and their surface chemistries analyzed by Auger electron spectroscopy. For the steels studied here, one or more of the elements carbon, phosphorus, and sulfur accumulated significantly at the surface within a temperature window of 300 to 973 K. As the temperature was increased, carbon appeared on the surface first, followed by phosphorus, and then sulfur. Each succeeding segregating element displaced the previous one from the surface. The free solute concentration in the bulk and the temperature were critical factors controlling the amount of solute accumulation at the surface. Once segregated, the solute atoms remained on the surface as the samples cooled. Carbon and/or boron in steels retarded the transport of phosphorus to the surface. The implications of these findings in understanding the galvannealing behavior of these steels are discussed.     

Correlation of the intercept and slope of the retention equation ln k' = ln k'w - SC in MECC with the solvatochromic parameters of solutes

The intercept (ln k'w) and slope (S) of the retention equation in k' = ln k'w - SC in micellar electrokinetic capillary chromatography (MECC) have been studied through linear solvation energy relationships (LSER). It has been observed that the ln k'w is dependent on the properties of micelle and solute but independent of the properties of organic modifier, while the slope (S) is related to the organic modifier and the solute. The LSER results show that the ln k'w of solute is primarily determined by its molecular size and hydrogen bond acceptor (HBA) basicity, whereas the S of solute is primarily determined by its properties in terms of the type of organic modifier, of which molecular size is the most important factor. Additionally, the LSER results show that the interactions of micelle-water and water-organic modifier are also important factors for the retention of solute.     

Evaluation of temperature effects on selectivity in RPLC separations using polybutadiene-coated zirconia

The effect of temperature on selectivity in RPLC method development has been evaluated on polybutadiene-coated zirconia. We find that the influence of temperature on selectivity depends strongly on solute type. For solutes of similar structure such as polyaromatic hydrocarbons, temperature has almost no effect on selectivity; however, for solutes with very different functional groups such as chlorophenols, temperature changes did significantly affect selectivity. We feel that simple mixtures with one dominant retention mechanism-e.g., solvophobic retention-will not be helped appreciably by adjusting temperature. However, in complex mixtures with polar and ionizable solutes, optimization by varying the temperature may well be fruitful.     

Synthesis and characterization of new zirconia-based polymeric cation-exchange stationary phases for high-performance liquid chromatography of proteins

Ion-exchange chromatography is a major method used for large-scale protein separations. New zirconia-based polymeric cation-exchange HPLC stationary phases have been developed for protein separations. Two routes were employed for the synthesis. In one method, polyethyleneimine (PEI) was adsorbed onto porous zirconia particles and cross-linked with 1,4-butanediol diglycidyl ether (BUDGE). Succinic anhydride was then reacted with the remaining primary and secondary amine groups on PEI to afford anionic functionalities. The second method utilizes poly(acrylic acid) anhydride as both the crosslinker and the stationary phase. The resulting stationary phases act to separate proteins by a weak cation-exchange mechanism with a slight contribution to retention from hydrophobic interactions. In the presence of 20 mM phosphate buffer, Lewis acid/base interactions between the zirconia support and the proteins, which can significantly broaden the peaks, are sufficiently suppressed. The effects of ionic strength, mobile phase pH, and salt type are discussed. Protein mass recovery and loading capacity for protein separations on these phases have been evaluated. These weak cation-exchange stationary phases exhibit good stability under normal separation conditions for months and are stable in alkaline solution up to pH 10. In contrast to zirconia supports modified with small anionic species, these new phases have no limitation on the type of salt used as the eluent, and they exhibit unique selectivities. Therefore, they offer interesting alternatives for protein separations. To our knowledge, this work represents the first successful example of protein separations using porous zirconia-based polymeric phases under normal chromatographic conditions, which will definitely help make zirconia-based supports more useful for bio-separation.     

The effect of different amines added to eluents as silanol masking agents on the chromatographic behavior of some diuretics in reversed-phase high-performance liquid chromatography using C18 packings

A preliminary gradient separation in reversed-phase liquid chromatography of a mixture of 25 solutes (diuretics, probenecide, and atenolol) is carried out using several C18 columns and an aqueous phosphoric acid solution (pH 3.2)-acetonitrile mobile phase as a control. Using this separation, the chromatographic behavior of these solutes is studied using 11 water-soluble primary, secondary, and tertiary amine modifiers in the range of 0.7-7.5 mM and a Spherisorb C18 column. This study reveals the presence in the complex sample of two groups of solutes with positive (five typical solutes showed improvements in peak symmetry and retention) or negative responses using these amines as mobile phase modifiers. After experimentation in the presence of amines, these differences are related to solute structure. Hexylamine is found to be an effective masking agent of silanols because of its structure and small required concentration. On these bases, the silanophilic and hydrophobic character of typical solutes and several C18 packings are evaluated under isocratic elution and a relative effectiveness index for amines, and a method for their assessment is proposed. The role of the amine structure on solute retention and the importance of selecting amines of suitable hydrophobic character, molecular geometry, and concentration is discussed. A model of the formation and stabilization of the silanol-amine complex based on hydrophobic and ionic interactions is also proposed.     

An investigation of the effects of carbon loading and endcapping on the solid-phase extraction of beta-blockers onto C18 bonded silica gel

The effects of carbon loading and endcapping on the solid-phase extraction onto C18 bonded silica gel of a range of beta-blockers from aqueous buffer and from dog plasma has been investigated. The highest extraction efficiencies were obtained for those phases with carbon loadings of between 5 and 16% for phases without endcapping or 10.5-14% for endcapped material. With carbon loadings of 18 and 22% (plus endcapping) poor extractions from the matrix were obtained combined with further losses at the wash steps. Matrix effects were observed with dog plasma which accentuated the effects seen with buffer. These results are best explained by assuming that a cationic interaction of the secondary amino group present in the analytes with residual silanols on the silica surface is primarily responsible for the extraction of these analytes.     

Surface segregation and surface tension of liquid mixtures

A model has been developed in which surfaces are treated as separate phases with a thickness corresponding to a monolayer. It is argued that the surface tension of liquids is a measure of the excess surface chemical potential of the surface atoms relative to the bulk atoms. Equations for the calculation of the surface composition and surface tension of liquid mixtures are developed. Using only the surface tension and molar volume data of the pure components, excellent correspondence between calculated and experimental surface tension values was obtained. The method was also tested on liquid systems showing immiscibility. The surface coverage calculated from the present model is compared with that calculated using Gibbs adsorption equation. The surface coverage of the solute species increases with increasing solute concentration. However, depending on the surface properties of the system, the excess surface coverage may pass through a maximum value and then decrease with increasing solute concentration.     

Retention in reversed-phase chromatography: partition or adsorption?

A unified framework within the hermeneutics of the solvophobic theory is employed for the treatment of experimental data with nonpolar and weakly polar substances in reversed-phase chromatography (RPC), oil-water partitioning and adsorption on activated charcoal from dilute aqueous solution. This approach sheds light on the energetic similarities between such processes driven by the hydrophobic effect. Among several stationary phase models that have been proposed in the literature for the physical representation of alkyl-silica bonded phases, the isolated solvated hydrocarbon chains model is adopted for the retention in RPC since it represents most closely the stationary phase configuration and is not based a priori on a partition or adsorption mechanism as some other models are for the retention in RPC. Using the fundamental framework of the solvophobic theory, the free energy change per unit nonpolar surface area for octanol-water and hexadecane-water partitioning, retention in RPC as well as adsorption on activated charcoal from dilute aqueous solution at 25 degrees C are evaluated and they are found to be in good agreement with the corresponding experimental data. Furthermore, such quantities are very similar for all the above mentioned processes involving aqueous solution, in contradistinction to the predictions by the lattice theory. From the results it follows that these apparently disparate processes are subject to the same physicochemical principle. The present study demonstrates the capability of the solvophobic theory in describing the energetics of processes involving hydrophobic interactions, and exposes the difficulties in distinguishing between partition and adsorption mechanisms in RPC by using partition models based on the lattice approach. It is concluded that a clear distinction between partition and adsorption in RPC of nonpolar elites is not apparent from thermodynamic analysis.     

Effects of a cationic and hydrophobic peptide, KL4, on model lung surfactant lipid monolayers

We report on the surface behavior of a hydrophobic, cationic peptide, [lysine-(leucine)4]4-lysine (KL4), spread at the air/water interface at 25 degrees C and pH 7.2, and its effect at very low molar ratios on the surface properties of the zwitterionic phospholipid 1,2-dipalmitoylphosphatidylcholine (DPPC), and the anionic forms of 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and palmitic acid (PA), in various combinations. Surface properties were evaluated by measuring equilibrium spreading pressures (pi(e)) and surface pressure-area isotherms (pi-A) with the Wilhelmy plate technique. Surface phase separation was observed with fluorescence microscopy. KL4 itself forms a single-phase monolayer, stable up to a surface pressure pi of 30 mN/m, and forms an immiscible monolayer mixture with DPPC. No strong interaction was detected between POPG and KL4 in the low pi region, whereas a stable monolayer of the PA/KL4 binary mixture forms, which is attributed to ionic interactions between oppositely charged PA and KL4. KL4 has significant effects on the DPPC/POPG mixture, in that it promotes surface phase separation while also increasing pi(e) and pi(max), and these effects are greatly enhanced in the presence of PA. In the model we have proposed, KL4 facilitates the separation of DPPC-rich and POPG/PA-rich phases to achieve surface refinement. It is these two phases that can fulfill the important lung surfactant functions of high surface pressure stability and efficient spreading.     

The salubrious effects of ascorbic acid on cyclophosphamide instigated lipid abnormalities in fibrosarcoma bearing rats

A new class of silica gel-bound fluorene phases is described. The compounds are synthesized via reaction of fluorenyl lithium with omega-alkenyl bromides leading to 9-(5'-hexenyl)-9H- and 9-(9'-decenyl)-9H-fluorene (1 and 2), followed by hydrosilation reactions with different hydrosilanes. The resulting omega-functionalized silylalkyl fluorenes 3a(T0), 3b(T0), 4a(T0), 4b(T0) and 4c(M0) (Scheme 1) react with surface silanol groups of silica gel to generate the new fluorene phases 3a(Tn)(Qm)y, 3b(Tn)(Qm)y, 4a(Tn)(Qm)y, 4b(Tn)(Qm)y and 4c(M1)(Qm)y. The phases are characterized by employing 1H, 13C and 29Si solid-state nuclear magnetic resonance spectroscopy. Their applicability in high-performance liquid chromatography is proved by the Sander and Wise test (SRM 869). In contrast to conventional n-alkyl phases, pi-pi interactions are additionally involved in the separation process and, therefore, the retention times of the polycyclic aromatic hydrocarbons sample molecules depend on the ligand densities of the applied fluorene phases.     

Molecular imprint polymers as highly selective stationary phases for open tubular liquid chromatography and capillary electrochromatography

Chiral separations employing molecular imprint polymer (MIP) stationary phases in both open tubular liquid chromatography (OT-LC) and capillary electrochromatography (OT-CEC) are demonstrated. MIPs are highly crosslinked polymers containing spatial and functionality memory of template molecules which provide a higher degree of selectivity when used as stationary phases for chromatographic separations. Thin films of molecular imprinted polymers bonded to the inner walls of 25 microm ID fused-silica capillaries were prepared using an in situ polymerization technique developed in our laboratory that allows the use of conventional fused-silica capillaries with polyimide outer coatings. The success rate in preparing such open tubular columns was about 70%. Methacrylic acid and 2-vinyl pyridine were chosen as functional monomers, and either ethylene dimethacrylate or trimethylol propane trimethacrylate was used as the crosslinker. Toluene was employed as the porogen. Effects of polymerization conditions on column preparation and chromatographic performance were studied. Enantiomeric separations of D- and L-dansyl phenylalanines were achieved in both OT-LC and OT-CEC modes with good selectivity and efficiencies. Both types of separations may be performed on the same column using a single commercial instrument.     

Protein-protein interactions on weak-cation-exchange sorbent surfaces during chromatographic separations

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized protein coats 24% or less of the sorbent surface. It was found that columns on which proteins were immobilized still behaved as a cation-exchange chromatography sorbents, but their selectivity was different from the parent weak cation-exchange column. This was interpreted to mean that in addition to the normal electrostatic interactions expected in ion- exchange chromatography, protein analytes interact with immobilized protein on the sorbent surface. Anionic proteins were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It is concluded that these protein-protein interactions occur after proteins are captured by the primary interaction mechanism of the column, in this case, electrostatic interaction. Protein-protein interaction is a secondary, lateral process. These lateral interactions were observed between 4% and 24% surface saturation. The significance of this observation is that in preparative chromatography and the case of "fouled" columns, strongly adsorbed proteins could alter the elution characteristics of sample proteins being target for analysis or purification.     

Nucleation on ceramic particles in cast metal-matrix composites

In order to understand the nucleation on ceramic particles in the melts of metal-matrix composites (MMCs), the effect of segregation of solute on the surface of reinforcement particles in the melt has been analyzed as a function of particle temperature and the surface energy of the particle/liquid melt. The temperature of the particle in the melt, calculated analytically, was found to become close to the melt temperature within a very short time of contact between the particle and the melt. The solute concentration near the particle surface will, therefore, primarily be influenced by the surface energy of the particle and the melt. Based on this, the undercooling due to solute segregation around the particle and the chemical free-energy change due to the formation of the new solid phase on the particle were calculated in selected hypo- and hypereutectic Al-Si alloy melts containing (1) SiC particles or (2) graphite particles. The chemical free-energy change (driving force for nucleation) due to the formation of the new phase on the particle is lower for hypoeutectic compositions than for hypereutectic compositions in the aluminum-silicon alloy systems; this is due to the higher undercooling in the hypereutectic alloys due to solute segregation on the surface of the particle. This suggests that the formation of the primary phase on the surfaces of particles in the melt should be more favorable in the hypereutectic compositions than for hypoeutectic compositions. This also indicates that even when the particle temperature is not significantly lower than the liquidus temperature, nucleation on the particles can take place due to the segregation of the solute on the particles. Experimental observations of the microstructure of several cast metal-matrix composites, including Al-Si-SiC and Al-Si-graphite, show (1) the presence of silicon in contact with the reinforcement particles in hypereutectic alloys, suggesting that nucleation and growth of primary silicon under certain conditions occurs on silicon carbide and graphite particles, possibly due to solute segregation on the surface of the particles, and (2) the presence of reinforcement particles in the last-freezing interdendritic regions of the primary phases in hypoeutectic alloys, suggesting the absence of nucleation of primary phases on the reinforcement surface, as predicted by the analysis.     

Monodisperse hydrolyzed poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads as a stationary phase for normal-phase HPLC

The basic characteristics of a rugged, stable, and highly efficient polymeric stationary phase for normal-phase HPLC prepared by hydrolysis of porous monodisperse poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads have been studied and compared with those of bare Nucleosil silica and Nucleosil silica-diol. As a result of their overall chemical composition and the more than 10-fold larger surface coverage with hydroxyl groups, the polymer beads provide much higher retention of model solute molecules. In contrast to silica hydroxyls, all of the polymer surface diol groups are chemically equal and homogeneously distributed over all of the surface. In addition, they are less acidic than typical silanol functionalities. The exceptional selectivity of the polymeric column can be controlled by the composition of the mobile phase, as demonstrated with a broad spectrum of separations involving positional isomers of benzene derivatives, nonpolar isobutylene copolymers with minute composition differences, and hydrophilic poly(ethylene oxides) differing only in their chain length. Unlike silica phases, the properties of the polymeric stationary phrase are not affected by the presence of water in the mobile phase. As a result, repetitive gradient separations in eluents ranging in polarity from hexane through tetrahydrofuran to water can be easily accomplished.