Elution behavior of polyethylene and polypropylene standards on carbon sorbents

The elution behavior of linear polyethylene and isotactic, atactic and syndiotactic polypropylene was tested using three different carbon column packings: porous graphite (Hypercarb), porous zirconium oxide covered with carbon (ZirChrom-CARB), and activated carbon TA 95. Several polar solvents with boiling points above 150°C were selected as mobile phases: 2-ethyl-1-hexanol, n-decanol, cyclohexylacetate, hexylacetate, cyclohexanone, ethylene glycol monobutyl ether and one non-polar solvent, n-decane. Polyethylene standards were completely or partially adsorbed in all tested sorbent/solvent systems. Polypropylene standards were partially adsorbed on Hypercarb and carbon TA95, but did not adsorb on ZirChrom-CARB. ZirChrom-CARB retained polyethylene pronouncedly when 2-ethyl-1-hexanol, cyclohexylacetate or hexylacetate were used as mobile phases at temperature 150 or 160°C, while all three basic stereoisomers of polypropylene eluted in size exclusion mode in these sorbent/solvent pairs. This is very different from the system Hypercarb/1-decanol, which separated polypropylene according to its tacticity. The opposite elution behavior of polyethylene and polypropylene in system ZirChrom-CARB/2-ethyl-1-hexanol (polypropylene eluted, polyethylene fully adsorbed) enabled to realize separation of blends of polyethylene and polypropylene. Ethylene/1-hexene copolymers were separated according to their chemical composition using system Hypercarb/2-ethyl-1-hexanol/1,2,4-trichlorobenzene.     

A Comparison of the Retention of Homologous Series and Other Test Solutes on an Ods Column and a Hypercarb Carbon Column

Abstract

When compared with an ODS column having good surface coverage and operated with pure methanole eluent, the Hypercarb column exhibits similarities in some respects and dissimilarities in others. Methylene homologues separations look similar on ODS and on Hypercarb. Their In k' increase linearly with carbon number, although much steeper on Hypercarb. The free sorption enthalpy of methylene groups is about three times higher on the carbon column. The apparent phase ratio is lower with Hypercarb by a factor of about 100. Consequently, the retention of smaller size solutes is lower than on ODS, and the sample load capacity is smaller, too. n-Alkanes and other long chain alkyl compounds exhibit an unusual behavior on Hypercarb. With increasing sample size, their peaks show a strongly fronting profile, possibly indicating a change in ret-     

Carbonaceous sorbents for high‐temperature interactive liquid chromatography of polyolefins

The elution behavior of polyethylene (PE) and the three stereoisomers of polypropylene (PP) was studied on porous graphite along with three other carbon‐based sorbents, carbon‐clad zirconia particles, activated carbon, and exfoliated graphite in a systematic way in this work. Decahydronaphthalene, 1,2,3,4‐tetrahydronaphthalene, 1,3,5‐trimethylbenzene, tetrachloroethylene, xylene and p‐xylene were used as mobile phases. While PE is adsorbed to various extents on all the tested carbonaceous sorbents from the majority of the solvents, PP is fully adsorbed only in selected cases. Testing alcohols (C7–C9) as mobile phase with Hypercarb? indicates that all stereoisomers of PP are selectively adsorbed and desorbed when a solvent gradient alcohol→1,2,4‐trichlorobenzene is used at 160°C. The retention of all stereoisomers of PP increases with the polarity of the alcohol. Linear PE is retained on Hypercarb? even from 1,2‐dichloro‐ and 1,2,4‐trichlorobenzene, when a temperature below 120°C is applied, while it is not retained from these solvents at higher temperatures. All stereoisomeric forms of PP are not adsorbed under the same conditions. Some of the tested new sorbent/solvent systems have potential to be applied in routine analysis of industrially synthesised polyolefins.     

Mixed-mode chromatography for fractionation of peptides, phosphopeptides, and sialylated glycopeptides

A mixed-mode chromatographic (MMC) sorbent was prepared by functionalizing the silica sorbent with a pentafluorophenyl (PFP) ligand. The resulting stationary phase provided a reversed-phase (RP) retention mode along with a relatively mild strong cation-exchange (SCX) retention interaction. While the mechanism of interaction is not entirely clear, it is believed that the silanols in the vicinity of the perfluorinated ligand act as strongly acidic sites. The 2.1 mm x 150 mm column packed with such sorbent was applied to the separation of peptides. Linear RP gradients in combination with salt steps were used for pseudo two-dimensional (2D) separation and fractionation of tryptic peptides. An alternative approach of using linear cation-exchange gradients combined with RP step gradients was also investigated. Besides the attractive forces, the ionic repulsion contributed to the retention mechanism. The analytes with strong negatively charged sites (phosphorylated peptides, sialylated glycopeptides) eluted in significantly different patterns than generic tryptic peptides. This retention mechanism was employed for the isolation of phosphopeptides or sialylated glycopeptides from non-functionalized peptide mixtures. The mixed-mode column was utilized in conjunction with a phosphopeptide enrichment solid phase extraction (SPE) device packed with metal oxide affinity chromatography (MOAC) sorbent. The combination of MOAC and mixed-mode chromatography (MMC) provided for an enhanced extraction selectivity of phosphopeptides and sialylated glycopeptides peptides from complex samples, such as yeast and human serum tryptic digests.     

Molecular statistical calculation of thermodynamic characteristics of adsorption of O-, S-, and Se-containing heteroadamantanes on graphitized thermal carbon black

The thermodynamic characteristics of adsorption (TCA) on the basal face of graphite have been calculated in terms of the semiempirical molecular statistical theory of adsorption for molecules of O-, S-, and Se-containing heteroadamantanes of different structure and isostructural cyclohexane derivatives. The influence of the nature, number, and position of heteroatoms in the adamantane framework on the TCA values was studied in detail, which made it possible to predict the retention of the compounds considered on the surface of graphitized thermal carbon black under the conditions of equilibrium gas adsorption chromatography. The introduction of each subsequent heteroatom into a polyheteroadamantane molecule makes a non-additive contribution to the TCA values. The contributions of various fragments to the total adsorption energy were calculated by the TCA of isostructural heterocyclohexane molecules, and the most preferential orientations of the framework molecules of the adsorbates relative to the planar graphite surface were determined.     

Microcolumn liquid chromatography on carbon sorbents

Summary The retention volume dependences on the molar volume were found for aromatic compounds using liquid microcolumn chromatography. The contributions of the functional groups in molecules of these compounds to the total retention value were calculated from the capacity factor values. The comparison of capacity factor values for carbon sorbents and octadecyl-silicagel has shown that for several microcolumn separations the carbon sorbent column has a better selectivity and resolution than the octadecyl-silicagel column.     

HPLC Determination of Glyphosate,Aminomethylphosphonic Acid,and Glufosinate Using a Hypercarb Porous Graphite Adsorbent

A method for the HPLC determination of glyphosate, aminomethylphosphonic acid, and glufosinate using the gradient separation of analytes on a Hypercarb porous graphitized carbon adsorbent and an aqueous solution of ammonium formate and ammonia as a mobile phase is proposed. Analytes are detected using quadrupole and three-quadrupole mass spectrometers. In order to increase the retention of the analytes, the chromatographic column is washed with water before the injection of a sample solution. This procedure results in a three- to fourfold increase in the retention factors of the analytes in comparison with the analogues described in the publications.     

Chiral ion-pair chromatography on porous graphitized carbon using N-blocked dipeptides as counter ions

Two newly synthesized chiral di-anionic counter ions were tested for enantiomeric resolution of a set of amino alcohols on porous graphitized carbon, Hypercarb. Z-L-Aspartyl-L-proline dissolved in methanol baseline resolved nine of 12 tested racemates. One of its diastereoisomers, Z-L-aspartyl-D-proline was also tested but resulted in low separation factors, <1.1. Sodium hydroxide was added to the mobile phase in order to titrate the counter ion to its mono- or di-anionic form. Results show that the di-anionic form was found to be superior to the mono-anionic form regarding enantioselectivity. Increased content of the counter ion in the mobile phase, with constant ratio between counter ion and sodium hydroxide concentration, decreased retention but only slightly affected enantioselectivity. Increased retention and enantioselectivity were observed with decreased column temperature. Resolution factors >3 were obtained between the enantiomers in atenolol and metoprolol with a total retention time of less than 15 min. Further, all four stereoisomers of an analogue to metoprolol were separated using Hypercarb and a mobile phase of 5 mM Z-L-aspartyl-L-proline and 9 mM sodium hydroxide in methanol.     

Gradient HPLC Separation of Alkylphosphonic Acids on a Hypercarb Porous Graphitic Carbon Adsorbent with an Aqueous Formic Acid Solution as the Mobile Phase

An approach to the gradient HPLC separation of alkylphosphonic acids on a Hypercarb porous graphitic carbon adsorbent with an aqueous formic acid solution as a mobile phase is proposed. Analytes were detected using a quadrupole mass spectrometer. To increase the retention of the analytes, the chromatographic column was washed with water before the injection of a sample solution. This procedure results in a three- to fourfold increase in the retention factors of alkylphosphonic acids in comparison with the analogs described in the publications.     

Direct Dynamic Protein-Affinity Selection Mass-Spectrometry

Mobile phase chromatographic conditions for the selective separation of metoprolol from related amino alcohols have been evaluated using Hypercarb as support and carbon dioxide with addition of methanol as mobile phase. The objective for the presented study was to show the unique ability of the porous graphitized carbon surface to separate closely structurally related substances. Experimental results, using Hypercarb and “chromatographic normal phase conditions” (SFC), are presented with focus on how to control retention and to improve peak performance. A high concentration of basic aliphatic amine additive was required in order to elute the amine analytes as symmetrical peaks. N,N-Dimethyloctylamine was preferred over triethylamine since the retention was markedly shorter though the selectivity and resolution were virtually the same. The selectivity can be optimized by altering the temperature of the column. A high selectivity was demonstrated between metoprolol and two homologues with one and two extra methylene groups inserted between the secondary nitrogen atom and the carbon atom with a hydroxyl group attached. The mobile phase flow rate effect the column efficiency only to a minor extent as the steepness of the van Deemter curve for metoprolol was virtually flat in the range studied: 0.5–3.0 mL min^?1.     

Liquid chromatographic analysis of nucleosides and their mono-, di- and triphosphates using porous graphitic carbon stationary phase coupled with electrospray mass spectrometry

Analysis of nucleosides and nucleotides is desirable in many biological studies, but the task is analytically challenging due to the high polarity of the analytes. In this study, resolution of mixtures containing nucleosides and their mono-, di- and triphosphates was achieved using a porous graphitic carbon (PGC) stationary phase, Hypercarb, under conditions suitable for liquid chromatography/mass spectrometry (LC/MS). Different organic mobile phases and modifiers were evaluated and the separation of 16 nucleosides and nucleotides was optimized using gradient elution with a water/acetonitrile mobile phase containing ammonium acetate and diethylamine as modifiers. The ammonium acetate concentration proved to be critical for retention and diethylamine was found to improve the peak shapes of di- and triphosphates for mass spectrometric detection. A variety of silica-based columns designed for polar compound separation were also tested using optimized LC conditions and compared with results obtained with the Hypercarb column. Only the Hypercarb column provided separations suitable for accurate quantitation of mixed nucleosides and their phosphates.     

Reversed-Phase High-Performance Liquid Chromatography of Hemin-Based Peptides on a Sorbent Modified with Zinc 4,5-Carboxyphthalocyaninate

The chromatographic retention of some hemin-based peptides on the Diasphere-110-C₁₈ sorbent modified with zinc octa-4,5-carboxyphthalocyaninate was studied. It was demonstrated that this stationary phase is more selective for hemin-peptides than Nucleosil-C₁₈ and Silasorb-600. The conditions of the separation of mixtures of hemin-based peptides were determined, and the parameters of their chromatographic separation were calculated. It was shown that the retention parameters are related to the structure of hemin-based peptides.     

Molecular statistical calculation of thermodynamic characteristics of adsorption of polymantane molecules on the basis face of graphite

The thermodynamic characteristics of adsorption (TCA) of polymantane molecules and their some derivatives on the basis face of graphite were calculated for the first time in terms of the atom-atomic approximation of the semiempirical molecular statistical theory of adsorption. The graphite surface exhibits high structural selectivity to isomeric polymantanes. A model for adsorption of cage molecules on the planar surface was proposed. The model is based on the idea that contributions of atoms of the adsorbate molecule to the total adsorption energy can be discriminated according to the distance of these atoms from the surface of a solid. Advantages and limitations of using the data on adsorption of molecules of isomeric alkanes, including rotamers, for the analysis of equilibrium TCA values of isomeric polymantanes on graphite are discussed. The possibility of separation of representatives of the polymantane hydrocarbon family by gas adsorption chromatography on columns with graphitized thermal carbon black was suggested on the basis of the calculated TCA values and logarithmic retention indices.     

Analysis of triacylglycerols on porous graphitic carbon by high temperature liquid chromatography

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30-70 degrees C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC-ELSD and LC-APCI-MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.     

Retention behavior of peptides in hydrophilic-interaction chromatography

Selected hydrophilic interaction chromatography (HILIC) columns packed with bare silica, bridge-ethyl hybrid silica, or an amide sorbent chemistry were utilized for an investigation of chromatographic behavior and separation selectivity of tryptic peptides. Retention model was proposed allowing for retention prediction of peptides with correlation coefficient R(2)~0.92-0.97 for various columns. The values of optimized amino acid retention coefficients were compared to those obtained for reversed-phase liquid chromatography (Gilar et al., Anal. Chem. 2010, 82, 265-275) and used to elucidate the impact of different amino acid on peptide HILIC retention. In contrast to reversed-phase chromatography, where presence of Phe, Trp, Ile, and Leu amino acid residues in sequence strongly promoted, and presence of hydrophilic His, Lys and Arg residues strongly reduced peptide retention, the effects of these amino acid residues in HILIC were opposite (His, Lys and Arg promote, Phe, Trp, Ile and Leu demote peptide retention in HILIC). Retention coefficient optimized for pH experiments illustrated the impact of silanols on HILIC retention.     

Liquid chromatography analysis of monosubstituted sulfobutyl ether-beta-cyclodextrin isomers on porous graphitic carbon

The retention behaviour of the three positional isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was investigated on a porous graphitic carbon (PGC) column. The influence of the mobile phase composition (nature and concentration of organic and electronic modifiers) was studied as well as the effect of column temperature. These hydrophilic and anionic analytes were highly retained on the PGC stationary phase compared to octadecyl bonded phases. The retention is mainly governed by a reversed-phase mechanism with electronic interaction playing a secondary role. An increase in solute retention and efficiency with temperature was observed. Successful isocratic separation with satisfactory baseline resolution of the three isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was achieved at 75 degrees C on a Hypercarb column by using ammonium acetate as electronic modifier in water-acetonitrile (83:17). The chromatographic methodology developed can be easily used for relative quantification of each isomer within a mixture and can be applied for semi-preparative purification of each one. The evaporative light scattering detector allows the detection of these non UV-visible absorbing molecules.     

Chiral supercritical fluid chromatography on porous graphitic carbon using commercial dimethyl beta-cyclodextrins as mobile phase additive

Using dimethylated-beta-cyclodextrin mixtures (MeCD) as chiral selectors in CO2-polar modifier mobile phase and porous graphitic carbon as solid-phase, chiral supercritical (or subcritical) fluid chromatography was performed. The adsorbed quantity of MeCD onto the porous graphitic carbon (Hypercarb) was measured for various chiral selector concentrations using the breakthrough method with evaporative light scattering detector. The effects of MeCD concentration in the mobile phase, the nature of the polar modifier, the outlet pressure, the column temperature and the nature of the commercial MeCD mixture on the retention and the enantioselectivities were studied. For a given solute, the enantioselectivity is greatly dependent on the commercial MeCD mixture used. The retention mechanism was also studied. From the data, we find that the dominant mechanism for the chiral discrimination is the diastereoisomeric complexation in the mobile phase.     

Evaluation of reversed phase columns designed for polar compounds and porous graphitic carbon in "trapping" and separating neurotransmitters

Quantification of neurotransmitters as biologically active analytes in neurological samples is of high interest for studying their effect on multiple targets. This work is part of a strategy involving two-dimensional liquid chromatography (2D LC) system with mass spectrometry (MS) detection. The concept of the on-line LC system is the coupling of reversed phase liquid chromatography (RPLC, the second separation dimension) to ion-exchange chromatography (IEC, the first dimension). Our objective in this study is to find the appropriate second dimension column, ensuring that samples of neurotransmitters are refocused and separated on it. Silica-based columns designed specifically to retain polar compounds were tested in LC conditions and compared with results obtained with a porous graphitic carbon (PGC, Hypercarb) column. These polar embedded, polar endcapped, and high-density alkyl chain columns successfully separated analytes in question using mobile phase systems with high percentage of water, or even pure water. Only Hypercarb column provided efficient retention of the most polar neurotransmitters and could be used for trapping and preconcentrating the compounds without rapid breakthrough.     

Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography--electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases

Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC-MS on Hypercarb is presented.     

A novel porous carbon and its perspectives for the solid phase extraction of volatile organic compounds

A porous carbon sorbent prepared by controlled pyrolysis of saccharose in a matrix of silica gel was tested for the preconcentration of a variety of compounds differing in polarity and volatility (hydrocarbons, halocarbons, oxygenated compounds) in simulated and real air samples. The trapped components were desorbed off-line by liquid-(carbon disulphide and carbon disulphide with 1% methanol) and on-line by thermo-desorption in combination with HRCGC. Adsorption-desorption properties of the sorbent were evaluated according to the determined recovery values, whereby physicochemical interactions of both the sorbent and solute were taken into consideration. With oxygenated compounds recovery data indicate the presence of active sites on the surface of the sorbent.Dedicated to Professor Dr. Dr. h.c. mult. J. F. K. Huber on the occasion of his 70th birthday