Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. I. Effect of the mobile phase

The high-performance liquid chromatographic (HPLC) behaviour of two different styrene-divinyl-benzene-based reversed-phase (RP) columns was evaluated using crude acetic acid extracts from normal and diabetic human pancreata as samples. Acetic acid gradients in water and acetonitrile gradients in triethylammonium phosphate (TEAP) and trifluoroacetic acid (TFA) were used as mobile phases, and comparisons were made with a silica-based C4 column. When two different polymeric RP columns were eluted with acetic acid gradients in water, surprisingly similar HPLC profiles of the pancreatic extracts were obtained. Elution of the polymer-based columns with acetonitrile gradients in TFA or TEAP resulted in changes in the polypeptide selectivity of these columns, in parallel with that of a silica-based C4 column eluted under similar conditions, indicating the general usability of polymeric columns for RP-HPLC of peptides and proteins. The pronounced difference in composition between normal and diabetic samples, which also was demonstrated after size-exclusion chromatography (SEC) on a silica-based and an agarose-based high-performance SEC column, was found to be related to the different ischaemia times for the two types of pancreata.     

Evaluation of a new polymeric stationary phase with reversed-phase properties for high temperature liquid chromatography

The performance of a polymeric stationary phase with reversed-phase properties (ET-RP1) was evaluated for LC separations at elevated temperature. The most significant observation was that the reduced plate height (h) decreased from 3.4 at 25 °C (optimal flow 0.5 mL/min) to 2.4 at 150 °C (optimal flow 2.5 mL/min) which is comparable to the efficiency obtained with silica-based reversed-phase columns of 4.6 mm ID operated at 0.8 mL/min. The phase showed no deterioration after long use at 150 °C within the pH range 1–9. Catalytic activity originating from the stationary phase material, e.g. as experienced on zirconium columns operated at elevated temperature, was absent. The performance of ET-RP1 is illustrated with the analysis of some pharmaceutical samples by LC and LC–MS. Operation at elevated temperature also allows to reduce the amount of organic modifier or to replace acetonitrile and methanol by the biodegradable ethanol.     

Selectivity in reversed-phase separations Influence of the stationary phase

The selectivity difference between 15 different stationary phases was measured using a large number of analytes at 2 or 3 different pH values (3, 7 and 10) with acetonitrile and methanol as the mobile phase modifiers. The packings discussed include standard C(8) and C(18) packings, packings with embedded polar groups, a phenyl packing, a pentafluoro-phenyl packing, an adamantylethyl packing and others. The major selectivity differences observed are discussed in detail. Specific effects such as pi-pi interactions on phenyl packings or hydrogen-bond interactions on phases with embedded polar groups are confirmed.     

Preparation and characterization of a magnesia-zirconia stationary phase modified with β-cyclodextrin for reversed-phase high-performance liquid chromatography

A new stationary phase of magnesia-zirconia composite matrix for high-performance liquid chromatography was first prepared by modification of β-cyclodextrin (β-CD) via fosfomycin as a spacer. Various modification procedures were attempted for achievement of successful modification. The modified composite was characterized by using coloration, elemental analysis, diffused reflectance FT-IR, surface area and pore size distribution. The separation of alkylbenzenes, polycyclic aromatic hydrocarbons, positional isomers of some acidic, basic and amphoteric disubstituted benzenes was studied on the new stationary phase. The effect of pH and methanol content in the mobile phase on retention and separation selectivity for the positional isomers were investigated. The chromatographic performance of CD modified magnesia-zirconia was compared with fosfomycin modified magnesia-zirconia as intermediate material and bare magnesia-zirconia as raw material. The results show that various retention mechanisms such as hydrophobicity, inclusion complexation and hydrogen bond interaction exist in the chromatography process of the packing modified with CD. The β-CD played the major role in the chromatographic property of this new stationary phase. The modified magnesia-zirconia exhibits superiority of separation for basic aromatics and high stability above pH 11.     

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.     

Poly(2-N-carbazolylethyl acrylate)-modified silica as a new polymeric stationary phase for reversed-phase high-performance liquid chromatography

Poly(2-N-carbazolylethyl acrylate) having terminal trimethoxysilyl groups was newly synthesized by radical polymerization and immobilized onto the silica surface (Sil-CEA). The chromatographic property of Sil-CEA was examined by applying polycyclic aromatic hydrocarbons as solutes. Poly(4-vinylpyridine)-modified silica (Sil-VP) and monomeric octadecylated silica (ODS) columns were used as the reference columns. Less sensitivity to molecular hydrophobicity and enhanced molecular planarity selectivity were obtained with Sil-CEA compared to ODS. On the other hand, high retention factors for the analyzed solutes and an opposite elution order for linear and disc-shaped solutes were obtained with Sil-CEA compared with Sil-VP. In this paper, the application for separation of tocopherols was also described.     

Studies on the special selectivity of the liquid crystal stationary phase used in gas chromatography: II. A new parameter for characterization of the liquid crystal stationary phase

In this paper, the special effect of liquid crystal stationary phase is expressed in terms of ΔI, the difference of the retention index of probed solute between mesomorphic phase and isotropic phase. A series of compounds with different structures and polarities are selected as probed solutes for the investigation of their retention behavior on liquid crystal PBOB and MPBOB. According to the differences of ΔI of various solutes, the rule of special selectivity on liquid crystal is explained.     

Synthesis and characterization of a novel resorcinarene-based stationary phase bearing polar headgroups for use in reversed-phase high-performance liquid chromatography

A novel silica-bonded stationary phase containing a functionalized resorcinarene selector was prepared by a straightforward synthesis. The complete modification of all resorcinic hydroxyl groups was achieved by reaction with isopropyl isocyanate. The derivatized resorcinarene selector was subsequently immobilized via the four alkenyl chains containing a terminal double bond by a free radical-induced reaction on mercaptopropyl-functionalized silica. A comprehensive characterization of the resulting bonded stationary phase was carried out by solid state NMR, IR and elemental analysis. The resulting selector is defined as a "polar headed" reversed phase since the highly ordered polar carbamate groups of the new stationary phase are located, compared to conventional polar embedded stationary phases, at a greater distance from the silica surface. Thus a new concept is introduced in the field of polar modified reversed-phase HPLC. The properties of the novel stationary phase are demonstrated by comparison with commercially available reversed phases.     

Excess isotherms as a new way for characterization of the columns for reversed-phase liquid chromatography

In this work, we present the excess isotherm of acetonitrile for stationary phases with different coverage density. Data obtained with the minor disturbance method were compared with (29)Si cross-polarization/magic-angle spinning NMR spectra to find dependence between acetonitrile adsorption on C18 chemically bonded stationary phases and coverage density of stationary phase. The preferential adsorption of acetonitrile on the bonded phase and the adsorption of water on the silica surface can be well correlated with the coverage density.     

Adsorption mechanism in reversed-phase liquid chromatography. Effect of the surface coverage of a monomeric C18-silica stationary phase

The effect of the bonding density of the octadecyl chains onto the same silica on the adsorption and retention properties of low molecular weight compounds (phenol, caffeine, and sodium 2-naphthalene sulfonate) was investigated. The same mobile phase (methanol:water, 20:80, v/v) and temperature (T = 298 K) were applied and two duplicate columns (A and B) from each batch of packing material (neat silica, simply endcapped or C1 phase, 0.42, 1.01, 2.03, and 3.15 micromol/m2 of C18 alkyl chains) were tested. Adsorption data of the three compounds were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) were calculated using the expectation-maximization method. Results confirmed earlier findings in linear chromatography of a retention maximum at an intermediate bonding density. From a general point of view, the saturation capacity of the adsorbent tends to decrease with increasing bonding density, due to the vanishing space intercalated between the C18 bonded chains and to the decrease of the specific surface area of the stationary phase. The equilibrium constants are maximum for an intermediary bonding density (approximately 2 micromol/m2). An enthalpy-entropy compensation was found for the thermodynamic parameters of the isotherm data. Weak equilibrium constants (small deltaH) and high saturation capacities (large deltaS) were observed at low bonding densities, higher equilibrium constants and lower saturation capacities at high bonding densities, the combinations leading to similar apparent retention in RPLC. The use of a low surface coverage column is recommended for preparative purposes.     

An evaluation of the robustness of the Tanaka characterization protocol for reversed-phase liquid chromatography columns

The robustness of the popular 'Tanaka' LC column characterization protocol has been evaluated by the statistical tools of reduced factorial design, multiple linear regression and principal component analysis. These have shown that in order to obtain reliable and reproducible results, it is especially important to control the methanol content, the temperature and, in the case of the total ion-exchange capacity test (alphaB/P pH 7.6), the pH of the mobile phase. In particular, the hydrophobicity tests (kPB and alphaCH2) are sensitive to small changes in methanol content. Provided that the operating parameters for the Tanaka column characterization protocol are controlled within the following experimental limits, i.e. methanol content +/-0.5% v/v, temperature +/-3degreesC, pH +/-0.10 and buffer concentration +/-2.0 mM, it is feasible to distinguish between RP materials that possess selectivity differences larger than their batch-to-batch reproducibility. These experimental requirements can be easily met by current LC instrumentation. Hence, the validity of the Tanaka testing protocol for characterizing columns has been verified.     

A simple preparation of stationary phase of alkylamide reversed-phase liquid chromatograph for the separation of basic substances.

A simple preparation process of alkylamide phase for reversed-phase HPLC (RP-HPLC) is described. The process includes aminopropyltrimethoxysilane firstly reacted with octanoyl chloride, then the intermediate was coupled onto porous silica. The resultant bonded silica has a reproducible ligand surface concentration and homogenous bonded ligand distribution on the porous silica. Characterization of prepared packing was carried out with elemental analysis, solid-state 13C NMR and Fourier transform infrared (FT-IR). Chromatographic evaluations were carried out by using a mixture of organic compounds including acidic, basic and neutral analytes under methanol/water as binary mobile phase. The results showed that the stationary phase have excellent chromatographic properties and can be efficiently used for the separation of basic compounds.     

Liquid chromatography and characterization of ether-functionalized imidazolium ionic liquids on mixed-mode reversed-phase/cation exchange stationary phase

A new series [C_nO_mmim]Cl of imidazolium cation-based ionic liquids (ILs), with an ether functional group on the alkyl side-chain, has been prepared. The possibility of analyzing the ionic liquids by high performance liquid chromatography (HPLC) was investigated on mixed-mode reversed/cation exchange stationary phase with the aqueous-acetonitrile mobile phase. Elution parameters, such as retention factor, selectivity and column efficiency, were studied as functions of mobile phase composition and pH. The ILs were characterized by elemental analysis, and infrared, UV and ¹H, ¹³C NMR spectroscopy.     

新型吡咯烷键合反相高效液相色谱固定相的制备与研究（英文）

正A new ionic liquid-based high-performance liquid chromatography stationary phase is reported.A derivative of N-methyl pyrrolidinium tetrafluoroborate was covalently immobilized on the surface of silica particles to prepare silica-based N-methyl pyrrolidinium tetrafluoroborate(SilprMP BF4)stationary phase.The obtained ionic liquid-modified silica was evaluated and confirmed by elemental analysis,infrared spectroscopy,and thermogravimetric analysis.A column was packed with the modified particles.The retention behavior of aromatic compounds,alkyl benzenes,and acidic and basic compounds on the SilprMP BF4 stationary phase was studied under reversed-phase liquid chromatography conditions.The effect of the eluent pH on the separation of the acidic and basic compounds was also studied.The new stationary phase involves multiple retention mechanisms,such as electrostatic,hydrophobic,ion-dipole,and anion-exchange interactions,which might lead to multipurpose separation media.     

Preparation of pH-responsive stationary phase for reversed-phase liquid chromatography and hydrophilic interaction chromatography

Novel pH-responsive polymer-grafted silica was successfully synthesized through the radical "grafting from" polymerization on azo initiator-immobilized silica. The immobilization of azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4'-azobis(4-cyanovaleric acid chloride). The polymer-grafted silica was prepared by stirring suspension of the azo initiator-immobilized silica in anhydrous dioxane containing acrylic acid (AAc) and butyl acrylate (BA). The resulting polymer-grafted silica was demonstrated to be pH responsive to hydrophobic/hydrophilic property by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). In RPLC mode, the retention of aromatic compounds decreased with the increase in the pH of mobile phase. However, the opposite result was obtained in HILIC mode; the retention of soybean isoflavones was stronger with the mobile phase at higher pH. Finally, the separations of sulfonamides and soybean isoflavones were carried out in RPLC mode and the separation of some nucleotides was achieved in HILIC mode.     

Use of short columns and high flow rates for rapid gradient reversed-phase chromatography

Summary Rapid analyses were performed using reversed-phase liquid chromatography with short (20–100 mm) columns swept by fast yet shallow gradients, and the results compared with those obtained with 150 mm columns and slow gradients. The resolution losses incurred with shorter columns were minimised by employing elevated flow rates, to ensure that comparable mean retention factors were experienced by individual analytes during gradients run on different columns. This conserves gradient steepness. High quality performance was obtained with turn-around times of 5–10 minutes. An overall 5-fold enhancement in the rate of information generation was obtained. The relevance of instrumental parameters and of column and packing dimensions, upon the potential for improved performance is discussed. Some implications for the rapidly developing technique of capillary electrochromatography are briefly indicated.     

New method for the preparation of small diameter columns with polymeric stationary phases for open tubular liquid chromatography

The purpose of this report is to introduce a new method for use in coating polymeric stationary phase films on the inside wall of small bore diameter fused silica capillary tubing. This technique is being developed for use in fabricating capillary columns for open tubular liquid chromatography.