HPLC of synthetic polymers

Summary Statistical copolymers of styrene and acrylonitrile have been separated by high performance precipitation chromatography according to the increase in acrylonitrile content. The separation mechanism was strongly depending on the combination of stationary phase and eluent composition. By applying a typical normal phase gradient from n-heptane (precipitation) to dichloromethane the polymers were adsorbed, after dissolution of the polar groups of the stationary phases. The elution curves became broader with increasing acrylonitrile content of the polymer. By the addition of 20% methanol to the dichloromethane the adsorption could be minimized and copolymer elution was dependent solely on solubility and independent of stationary phase polarity. Separation according to chemical composition was also possible without precipitation with a large pore silica and a normal phase gradient from dichloromethane to the same eluent containing 2.75% methanol.Part I see reference 19     

HPLC of synthetic polymers characterization of polystyrenes by high performance precipitation liquid chromatography (HPPLC)

Summary The advantages and disadvantages of high performance precipitation liquid chromatography have been demonstrated for polystyrene homopolymers. Depending on the mobile phase composition at the dissolution point of the polymeric sample and surface properties of the stationary phase, elution is governed either by a solution process or by adsorption. A contribution by adsorption was noticed on silica as well as on reversed phases based on silica with a normal phase gradient of increasing polarity (heptane to dichloromethane). Elution was solely governed by solubility of the polymers on both types of stationary phase for polystyrenes with a molecular weight above 35 000 and reversed phase gradient of decreasing polarity (methanol to dichloromethane). Under these conditions an identical dependence of elution solvent composition on sample size was found as for turbidity titrations. Due to differences in the velocity of the eluent front and the polymeric sample with porous stationary phases the polymers can be eluted as colloidal solutions Non-porous stationary phases are superior in this respect because the velocities of eluent and solutes are identical.     

Probing the interaction mode in hydrophilic interaction chromatography

This work aims at characterizing interactions between a select set of probes and 22 hydrophilic and polar commercial stationary phases, to develop an understanding of the relationship between the chemical properties of those phases and their interplay with the eluent and solutes in hydrophilic interaction chromatography. "Hydrophilic interaction" is a somewhat inexact term, and an attempt was therefore made to characterize the interactions involved in HILIC as hydrophilic, hydrophobic, electrostatic, hydrogen bonding, dipole-dipole, π-π interaction, and shape-selectivity. Each specific interaction was quantified from the separation factors of a pair of similar substances of which one had properties promoting the interaction mode being probed while the other did not. The effects of particle size and pore size of the phases on retention and selectivity were also studied. The phases investigated covered a wide range of surface functional groups including zwitterionic (sulfobetaine and phosphocholine), neutral (amide and hydroxyl), cationic (amine), and anionic (sulfonic acid and silanol). Principal component analysis of the data showed that partitioning was a dominating mechanism for uncharged solutes in HILIC. However, correlations between functional groups and interactions were also observed, which confirms that the HILIC retention mechanism is partly contributed by adsorption mechanisms involving electrostatic interaction and multipoint hydrogen bonding. Phases with smaller pore diameters yielded longer retention of solutes, but did not significantly change the column selectivities. The particle diameter had no significant effect, neither on retention, nor on the selectivities. An increased water content in the eluent reduced the multipoint hydrogen bonding interactions, while an increased electrolyte concentration lowered the selectivities of the tested columns and made their interaction patterns more similar.     

聚硅氧烷包夹聚合硅基高效液相色谱固定相的表征及其色谱性能

利用元素分析、红外光谱、扫描电镜及汞压吸附法对制备的聚硅氧烷包夹硅基反相高效液相色谱固定相进行了表征,给出了该固定相的结构信息及其与色谱性能的关系;用滴定法测定了该固定相表面硅羟基数目;考察了该固定相对碱性化合物的分离性能;由表征和色谱性能考察结果可知,该固定相表面的羟基基本被覆盖,因此,可在碱性流动相中长期使用。     

Separation of Proteins on Polar Bonded Phases by Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) with a polar bonded phase (“Acetamide”) developped for size exclusion chromatography (SEC) is described. Retention of proteins depends on the surface area of the stationary phase, the pH and ionic strength of the eluent. For efficient separation the pore diameter should be 25 nm or more. The surface area should be large to achieve retention even at low ionic strength. Separation is only possible with a gradient from high to low ionic strength. Gradient volumes of 10 empty column volumes with column lengths above 15 cm are recommended. Selectivity can be optimized via pH adjustment. The advantage of this column packing is its applicability for two different separation modes: SEC and HIC.     

Liquid chromatography of macromolecules at the critical adsorption point. II. Role of column packing: Bare silica gel

Liquid chromatography of macromolecules at the critical adsorption point (LC CAP) presents a potentially very powerful method for molecular characterization of complex polymers. However, LC CAP applicability is limited due to various experimental problems. The pore sizes and surface chemistry of the column packings belong to the most important weak points of the method. The LC CAP behavior of poly(methyl methacrylate)s was investigated using bare silica gels of 6, 12, and 100 nm pore sizes and with various amounts of surface silanols. Tetrahydrofuran as the adsorption suppressing liquid and toluene as the adsorption promoting liquid were mixed to form the “nearly critical” eluents. Both pore size and surface chemistry of silica were found to strongly influence the retentive characteristics of the system in the critical adsorption area. Macromolecules that were large enough to be excluded from the packing pores hardly followed the LC CAP rules: their retention volumes changed irregularly with the polymer molar mass and their recovery dropped sharply. The narrow pore silica gel-packed column governed the elution patterns of the whole column set composed of silica gels with different pore sizes. This makes the conventional LC CAP characterization of common polymers with broader molar mass distribution impractical and even not feasible. A hybrid column system was proposed containing narrow pore nonadsorptive column added in series to the meso- and macroporous LC CAP silica gels. This narrow pore column would allow separation of gas, impurities, and system peaks from the polymer peaks. The possible successive changes of the surface of silica gel, e.g., due to formation of silanols by hydrolysis or due to irreversible adsorption of some admixtures from the sample or eluent may make the LC CAP irrepeatable. Pronounced peak broadening was observed in the critical adsorption area and this effect increased strongly with the polymer molar mass. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 1363–1371, 1998     

Evaluation of silica from different vendors as the solid support of anion‐exchange chiral stationary phases by means of preferential sorption and liquid chromatography

Chromatographic performance of a chiral stationary phase is significantly influenced by the employed solid support. Properties of the most commonly used support, silica particles, such as size and size distribution, and pore size are of utmost importance for both superficially porous particles and fully porous particles. In this work, we have focused on evaluation of fully porous particles from three different vendors as solid supports for a brush‐type chiral stationary phase based on 9‐O‐tert‐butylcarbamoyl quinidine. We have prepared corresponding stationary phases under identical experimental conditions and determined the parameters of the modified silica by physisorption measurements and scanning electron microscopy. Enantiorecognition properties of the chiral stationary phases have been studied using preferential sorption experiments. The same material was slurry‐packed into chromatographic columns and the chromatographic properties have been evaluated in liquid chromatography. We show that preferential sorption can provide valuable information about the influence of the pore size and total pore volume on the interaction of analytes of different size with the chirally‐modified silica surface. The data can be used to understand differences observed in chromatographic evaluation of the chiral stationary phases. The combination of preferential sorption and liquid chromatography separation can provide detailed information on new chiral stationary phases.     

Characterization of the properties of stationary phases for liquid chromatography in aqueous mobile phases using aromatic sulphonic acids as the test compounds

We investigated the effects of the concentration of naphthalene sulphonic acids (NSAs) as anionic test compounds in the injected sample and of the salt additives to the mobile phase on ion-exclusion. The retention behaviour of NSAs sensitively reflects even minor changes in the ionic and hydrophobic interactions and can be useful for predicting the effects of the stationary phases in reversed-phase chromatography of polar and ionic compounds, both small ones and biopolymers, e.g., oligonucleotides. We studied chromatographic properties of several stationary phases intended for separations in aqueous mobile phases: a C18 column end-capped with polar hydrophilic groups, a densely bonded C8 column doubly end-capped with short alkyl groups, a short alkyl stationary phase designed to keep full pore accessibility in highly-aqueous mobile phases and a Bidentate column with “bridged” C18 groups attached to the silica hydride support. The chemistry and pore structure of various types of column packing materials and of the salt additives to the mobile phase affect the proportion of the pore volume non-accessible to anions due to ion-exclusion and consequently the peak asymmetry and hydrophobic selectivity in reversed-phase chromatography of organic acids. We also addressed the problems connected with the determination of column hold-up volume in aqueous mobile phases. The accessibility of the stationary phase for anionic compounds in contact with the sample zone is affected by ion-exclusion due to repulsive interactions with the negatively charged surface in the pores of the stationary phase. The accessible part of the stationary phase increases and consequently the migration velocity along the column decreases with increasing concentration of the sample in the zone moving along the column. Because of a limited access to the stationary phase, its capacity can be easily overloaded. The combination of the column overload and ion-exclusion effects may result in fronting or tailing peak asymmetry. To explain this behaviour, we proposed a modified Langmuir model, respecting the variation of the column capacity due to the effects of sample concentration on ion-exclusion.     

Naphthalene sulphonic acids--new test compounds for characterization of the columns for reversed-phase chromatography

Non-substituted naphthalene sulphonic acids are strong acids, which are completely ionised in aqueous and aqueous-organic solutions. Because of repulsive electrostatic interactions, they are more or less excluded from the pores of the column packing materials commonly used in reversed-phase chromatography. The ionic exclusion can be suppressed by increasing the ionic strength of the mobile phase. In aqueous sodium sulphate solutions, very good selectivity was observed for isomeric naphthalene di- and tri-sulphonic acids, allowing reversed-phase separations of these strongly ionic compounds without addition of ion-pairing reagents to the mobile phase. The retention of the isomeric acids increases proportionally to the dipole moment, which can be explained by its effect on increasing exposure of the naphthalene ring to hydrophobic interactions with the non-polar stationary phases. Chromatographic behaviour of isomeric naphthalene di- and trisulphonic acids was investigated on 25 different columns for reversed-phase chromatography. The elution order of the isomers is the same on all the columns, but very strong stationary phase effects were observed on the retention and on the band asymmetry, depending on polar interactions with residual silanol groups and other polar adsorption centres in the stationary phases. These effects are independent of the organic solvents, as the tests are performed in purely aqueous mobile phases and allow classification of the columns into several groups.     

Optimal conditions for the reversed-phase chromatography of proteins

Summary Optimum stationary phases for reversed phase chromatography should be based on a silica with a large average pore diameter, preferably above 30nm. To exclude anormalous elution behaviour of proteins, octadecyl groups should be bonded to the surface and the residual silanol groups should be reacted with a highly active silanization agent like bistrimethylsilyl acetamide. The total amount of bonded carbon should not exceed 5% w/w. Because of the low diffusion coefficients of proteins, the particle diameter should be as small as possible. Protein retention can hardly be influenced by changes of the organic modifier or by temperature. Because of solubility and viscosity, however, acetonitrile is to be prefered. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Study of Microstructure of High-Surface-Area Polyacrylonitrile Activated Carbon Fibers

High-surface-area polyacrylonitrile (PAN) activated carbon fibers having different pore size distribution activated by KOH were investigated. Nitrogen adsorption, XRD, SEM, and TEM were used to characterize the microstructure of PAN-ACFs. The specific surface area of samples was calculated from the standard BET method, and micropore surface area and volume were obtained from the Horvath-Kawazoe equations. The average pore size and characteristic energy were calculated by the Dubinin-Radushkevich equation according to the multistage adsorption mechanism. The whole pore size distribution was calculated by employing the regularization method according to the density functional theory, which is based on a molecular model for the adsorption of nitrogen in porous solids. It was shown that the isotherms were type I, the pore size was around 0.4-0.8 nm, and the mesorpore size was around 2-4 nm. The XRD pattern showed that PAN-ACFs activated by KOH are of amorphous material composed of very small crystallites. The SEM and TEM results showed that the monograph differs with differing activation degree, and the network is uniform or disordered. That all of these methods are in good agreement with one another. Copyright 2001 Academic Press.     

The role of accessibility in the characterization of porous solids and their adsorption properties

This paper addresses the role of accessibility for adsorption in porous solids on the adsorption properties including Henry constant, adsorption isotherms and isosteric heat of adsorption. The relevant parameters are the accessible volume, the accessible geometrical surface area and the accessible pore size and its associated volume. This concept will be demonstrated to be important and calls for the need to consider adsorption characteristics in the most coherent and consistent manner. It is particularly reinforced by the limitations inherent in the conventional ways in determining the void volume, surface area and pore size. We provide a number of examples to support this; the challenge that faces us is the development of consistent experimental procedures to determine these accessible quantities. We define the accessible pore size as the size of the largest sphere that rests on three closest solid atoms in such a manner that any probe particle residing in that sphere would have a non-positive solid-fluid potential energy. For each accessible pore size there is an associated accessible pore volume, giving rise to a new accessible pore size distribution (APSD). This is distinct from the classical pore size distribution commonly used in the literature, and in our definition of accessible pore size, a zero pore size is possible. It is also emphasized that the accessible quantities that we introduce here are dependent on the choice of molecular probe, which is entirely consistent with the concept of molecular sieving.     

A simple method for measuring excess adsorption isotherms of organic eluent components on reversed‐phase packing materials

A simple frontal analysis method has been developed for the reliable measurement of excess adsorption isotherms of an organic component on reversed‐phase adsorbents in a series of programmed concentration steps. In the present method, a peak, which is produced by refractive index change in column eluate, is detected at 589 nm; it represents the elution volume of the boundary. The method is applied to the measurement of the excess adsorption isotherms of organic eluent components from water on commercially available reversed‐phase stationary phases. The results are in good agreement with the previously reported isotherms. We also measure the excess adsorption isotherms of organic eluent components from solutions containing electrolytes. There are not any interference peaks on the elution traces. The method is thus reliably applicable to the evaluation of the excess adsorption of organic eluent components in practical systems.     

Epoxy-functionalized large-pore SBA-15 and KIT-6 as affinity chromatography supports

Mesoporous silica supports are proposed as an alternative to polymeric stationary phases for fast affinity chromatography due to their better mechanical strength compared to polymers. Ideal supports should combine high surface area and large pore size to allow a high loading capacity of large molecules, such as proteins, and favor their accessibility. Increasing the pore size of large-surface area micelle-templated silicas (SBA-15, KIT-6) has been achieved by swelling the micelles by the addition of organic molecules and increasing synthesis time and temperature. The pore size of hexagonal silica mesostructured SBA-15 has been increased up to 35 nm. These materials could find therefore application as affinity chromatography for immunoextraction.     

Partitioning of star branched polymers into pores at three chromatography conditions

The partitioning of star branched polymers into a slit pore at three different chromatography conditions, namely, size exclusion chromatography (SEC), liquid chromatography at the critical condition (LCCC), and liquid adsorption chromatography (LAC) have been investigated with lattice Monte Carlo simulations. Two different chain models are used: random walks (RW) that have no excluded volume interaction and self-avoiding walks (SAW) that have excluded volume interaction. The simulation data obtained for the two chain models are compared to illustrate the effect of excluded volume interactions on the partitioning of star branched polymers. The two most outstanding effects observed due to the introduction of excluded volume interactions are: (i) stars with a high number of arms can be excluded from the pore at condition corresponding to the LCCC of the linear polymers; (ii) the partition coefficient of stars in LAC mode is not dependent only on the total number of monomers on the chain. These effects illustrated by the current study should be taken into account when interpreting experimental chromatography data for branched polymers.     

高效液相色谱快速分离生物大分子的大孔硅质固定相的研制及应用

介绍了不同孔径的大孔硅胶基质的制备、二醇固定相的合成及其对蛋白质的色谱分离；考察了焙烧温度对硅胶孔隙度的影响；探讨了流动相中盐浓度和ｐＨ对蛋白质保留时间、柱效、分离度的影响以及硅胶孔径对键合密度和分离性能的影响。该种大孔固定相相对于常规多孔固定相具有小的比表面积、柱容量低、从而有利于生物大分子的微量分离分析。     

Concentration Effects in Size Exclusion Chromatography Under Equilibrium Stationary Conditions

Abstract

In size exclusion chromatography the elution volume increases with increasing concentration of injected polymer solutions. Several particular processes contribute to this concentration effect. Under model equilibrium stationary conditions, where the phases remain immobile, it is possible to eliminate dynamic phenomena connected with the higher viscosity of polymer solution in the chromatographic zone. The only factors operative here are the effect of changes in the effective size of macromolecules in solution with a change in concentration and the effect of the varying accessible pore volume with varying concentration. The ratio of these two contributions has been investigated both theoretically and experimentally. Theoretical calculations indicated that with both phenomena operating simultaneously, the elution volumes may increase or decrease with increasing concentration according to the given experimental conditions, as a result of the coupled influence of molecular parameters of the samples under study, of the thermodynamic quality of the solvent and of the distribution coefficient on the given porous material. Experimental results showed, however, that at least under stationary conditions secondary exclusion is probably operative to a less important degree.     

Application of size exclusion chromatography with surfactant eluent to the study of polymer–surfactant interactions: oligomeric and micellar chromatographic effects

Complexation of sodium dodecyl sulphate (SDS) with a wide range of molecular weights of poly(ethylene glycol) (PEG) and poly(ethylene oxide) (PEO) has been studied by size exclusion chromatography using aqueous SDS eluent. A multi-angle laser light scattering detector and a differential refractometer were applied to give direct measurement of the molecular weight of complexes without reference to elution volume, since the latter is not a reliable indicator of the complex size. Background light scattering from micellar eluents hampered quantitative size measurements, but was minimal in sub-micellar eluent, where saturated binding was observed for polymers larger than 1000 g mol⁻¹. Multiple peaks and voids were observed in the elution profiles of low molecular weight polymers (up to a mass of 600 g mol⁻¹) in eluent at micellar concentrations. Several sources contribute to this behavior, including micellar chromatographic separation of the PEG oligomers due to their different distribution coefficients between the micellar and water phases. Preliminary results are reported for distribution coefficients of individual oligomers in a 600 g mol⁻¹ PEG sample. Three distinct binding behaviors are observed with increasing degree of polymerization of PEG: no interaction for small glycols, equilibrium partitioning of intermediate oligomers in and out of micelles, and binding of micelles to the larger polymers.     

Immersion Calorimetry: Molecular Packing Effects in Micropores

Repeated and controlled immersion calorimetry experiments were performed to determine the specific surface area and pore‐size distribution (PSD) of a well‐characterized, microporous poly(furfuryl alcohol)‐based activated carbon. The PSD derived from nitrogen gas adsorption indicated a narrow distribution centered at 0.57±0.05 nm. Immersion into liquids of increasing molecular sizes ranging from 0.33 nm (dichloromethane) to 0.70 nm (α‐pinene) showed a decreasing enthalpy of immersion at a critical probe size (0.43–0.48 nm), followed by an increase at 0.48–0.56 nm, and a second decrease at 0.56–0.60 nm. This maximum has not been reported previously. After consideration of possible reasons for this new observation, it is concluded that the effect arises from molecular packing inside the micropores, interpreted in terms of 2D packing. The immersion enthalpy PSD was consistent with that from quenched solid density functional theory (QSDFT) analysis of the nitrogen adsorption isotherm.     

Two-dimensional thin-layer chromatography of selected coumarins

Polar-bonded stationary phases (CN-silica and Diol-silica) are used with nonaqueous eluents in adsorption mode or with aqueous eluents in partition mode. This enables the application of these systems in two-dimensional separations because of the different selectivity and application to the separation of closely related compounds of similar physicochemical properties and retention behaviour. Similarly, multiphase plates, connected with C18 strips and silica layers, are used with aqueous and nonaqueous eluents. Such layers were applied for the separation of selected coumarins. Thus, differences in separation selectivity are applied for the separation of coumarin fractions from plant extracts of the Apiaceae family by two-dimensional thin-layer chromatography.