Separation characteristics of alkylated guanines in high-performance liquid chromatography

The retention behavior of thirteen alkylated guanines on normal-phase silica gel and amino columns and on reversed-phase ODS and phenyl columns was studied. The larger the alkyl substituent at the same position of guanine the weaker was the retention in the normal-phase chromatographic system and the greater the retention during reversed-phase chromatography. O6-Derivatives possess the lowest polarity in each set of isomers. An amino column was found to be of highest efficiency in terms of separation of the set of ethylguanine isomers and of benzylguanines studied. A phenyl column provided the best resolution of methylated guanines.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

Liquid chromatographic profiles of individual compounds of technical toxaphene

The elution orders of 20 hexa- to nonachlorobornanes and five hexa- to octachlorocamphenes were studied with normal-phase silica and amino phase HPLC, reversed-phase HPLC, as well as gel-permeation chromatography (GPC). Twenty-one compounds of technical toxaphene (CTTs) are commercially available and four were isolated from environmental samples. Structure-activity relationships and chromatographic properties were deduced from the data sets derived on these LC systems. The retention on silica (low-resolution LC and HPLC) increased with the polarity of the CTTs. The elution order of CTTs on amino normal-phase HPLC was, for the most part, the same as on silica normal-phase HPLC. The degree of chlorination determined the elution order of CTTs on C18 RP-HPLC. CTTs eluted from medium-pressure GPC with decreasing molecular size. Chlorobornanes with dichloro substituents on the six-membered ring eluted after the chloroboranes without geminal chlorine atoms on secondary carbons, indicating that these congeners are larger. Altogether, the results increase the knowledge of complex substance class and may serve as a tool in order to gain further standard components.     

Mechanism and prediction of retention of oligomers in normal-phase and reversed-phase HPLC

Summary The simultaneous dependence of the retention in oligomeric series on the number of repeat structural units and on the mobile phase composition may be described by very similar equations for reversed-phase and for normal-phase systems.In reversed-phase systems, the separation selectivity of the individual oligomers is determined mainly by the size and by the polarity of the repeat structural unit, but the influence of a bulky and polar structural residue may also become important so that even reversed order of elution may be observed for oligomeric series with the same oligomeric units but significantly different end groups. For example, oligoethylene glycols are eluted in the order of increasing size of the oligomers, whereas ethoxylated nonylphenols are eluted in the order of decreasing size.In normal-phase systems, the separation selectivity in oligomeric series depends on the adsorption energy and on the adsorbed area of the oligomeric unit. If the oligomeric unit is small, the concentration of the polar solvent in the binary organic mobile phase has only a minor effect on retention and selectivity, which may be controlled by taking account of the nature of the adsorbent and of the polar solvent or by varying the proportion of two polar solvents in a ternary mobile phase.     

Matrix effects and selectivity issues in LC-MS-MS

Summary Chlorobenzenes, triazine and phenylurea herbicides were separated by normal micellar electrokinetic chromatography (MEKC) and by micellar electrokinetic chromatography with reversed flow (RF-MEKC) in running buffers containing organic solvents. The relationship between the two techniques is similar to that between reversed-phase and normal-phase HPLC. Using RF-MEKC, the separation of lipophilic compounds is often improved compared to normal MEKC. The migration in MEKC and in RF-MEKC was characterised by lipophilic and polar indices. The experimental values of the lipophilic indices of the compounds tested in the two techniques were close to the indices in reversed-phase HPLC (RP-HPLC). This enables the use of the indices determined in RP-HPLC for predicting the effects of changing composition of the running buffers on migration times in MEKC and in RF-MEKC. Presented at Balaton Symposium '01 on High-Performance Separation Methods, siófok, Hungary, September 2–4, 2001     

Thermodynamic investigations on shape selective separation behaviors of poly(4-vinylpyridine)-grafted silica for polycyclic aromatic hydrocarbons in both normal-phase and reversed-phase high-performance liquid chromatography

With the successful implementation of poly(4-vinylpyridine)-grafted silica prepared by grafting-from approach (GF-VP(n)) as a stationary phase for the separation of polycyclic aromatic hydrocarbons (PAHs) in normal-phase HPLC, this paper describes the chromatographic retention behaviors of PAHs with GF-VP(n) in reversed-phase HPLC. Significantly higher retention factor along with enhanced shape selectivity were observed with GF-VP(n). Thermodynamic study on the retention behaviors of PAHs with GF-VP(n) in normal-phase and reversed-phase HPLC revealed that retention of PAHs was exothermic in both phases. Furthermore, higher entropic contribution was observed in reversed-phase HPLC compared to normal-phase HPLC.     

氨基甲酸酯功能化离子液体高效液相色谱固定相的制备与初步评价

合成一种了氨基酸衍生物：4,4′-二苯亚甲基桥联-二[2-(1-咪唑基)-3-苯基丙醇氨基甲酸酯](ImPh-Carb),并将其键合到硅胶上制备了一种新的氨基甲酸酯功能化的离子液体HPLC固定相（ImPh-Carb-Silica）。 利用1H NMR、13C NMR、MS和FTIR对ImPh-Carb进行了表征;通过FTIR和元素分析对ImPh-Carb-Silica固定相进行了表征,根据N含量计算得到ImPh-Carb-Silica的键合量为0.19 mmol/g。 以5种芳烃、5种酚类化合物和4种有机磷农药为分析物,分别在正相和反相色谱模式下对固定相的色谱分离性能进行了评价,同时考察了流动相的变化与溶质保留因子lg k之间的关系。 结果表明,该固定相与溶质分子间存在多重作用力,如疏水、氢键、π-π和偶极-诱导偶极作用等,使其能同时在正相和反相色谱模式下使用;在正相色谱条件下固定相对酚类化合物和有机磷农药表现出较好的分离选择性。     

Prediction of peptide retention times in normal-phase liquid chromatography with only a single gradient run.

Previous studies of peptide separation by normal-phase liquid chromatography have shown a linear relationship between the logarithm of the capacity factor and the logarithm of the volume fraction of modifier in the mobile phase. This permitted the use of a model to predict isocratic and gradient retention times based on data obtained by two initial gradient runs. In the present study, chromatographic behavior of 25 peptides in normal-phase liquid chromatography with isocratic elution have been studied and a linear relationship between the slope (S) and intercept [log k(0)] was obtained. This relationship was combined with the algorithm of prediction reported in the previous paper. The prediction of peptide retention times with only a single experimental gradient retention data was investigated.     

Poly(vinylpyrrolidone)-coated silica: A versatile,polar stationary phase for HPLC

Summary Poly(vinylpyrrolidone) (PVP) is immobilized on both, small- and large-pore silicas by thermal treatment, γ-radiation, or peroxide initiated polymerization. The hydrolytic stability of such a highly polar stationary phase significantly exceeds that of a comparable phase prepared by the chemical reaction of silica with a pyrrolidone ethyl dimethylchlorosilane silanization reagent. The properties of the different PVP-silicas are evaluated by elemental analysis, spectroscopy, and chromatography. Columns of PVP-silica packings can be used in several modes: a) under normal-phase conditions as a polar bonded stationary phase, b) under reversed-phase conditions, for the separation of organic proton-donor and hydrogen-bonding compounds, c) for the aqueous size exclusion chromatography of proteins, and d) with salt gradients for the hydrophobic interaction chromatography of proteins. The minimum observable reduced plate-height of PVP-silica columns is about 3. Double-layer polymer coating experiments using PVP-silica covered with poly(methyloctadecylsiloxane) have been performed to study diffusion and shielding effects of different polymer layers in the stationary phase. Depending on separation conditions, one or the other polymer governs the retention process. A mixed selectivity was observed in a reversed phase mode with acidic eluents.     

Hypercrosslinked polystyrene as a novel type of high-performance liquid chromatography column packing material. Mechanisms of retention

An experimental material, Chromalite 5HGN (Purolite, UK), that represents hypercrosslinked polystyrene as a new type of neutral stationary phase for HPLC was examined. The material contains no functional groups, but is compatible with any kind of nonpolar and highly polar mobile phase, and even with water. It is chemically resistant and thermally stable. When using aqueous organic mobile phases, Chromalite 5HGN works similar to standard C18 reversed-phase packings, but is characterized by much greater hydrophobicity and, sometimes, unusual selectivity. When using nonpolar mobile phases, i.e. under "quasi normal-phase" conditions, the retention is mostly governed by the interactions between pi-electronic systems of the adsorbent and adsorbate. Adding highly polar, even hydrophilic solvents into the mobile phase, leads to a shift of retention times toward the "reversed-phase" kind of chromatography, which gives an additional possibility in fine tuning the column selectivity.     

Calculation of amino acid hydrophilicity indices for retention of peptides on amide, diol and silica columns in normal-phase liquid chromatography

Summary A set of hydrophilicity parameters in a normal-phase liquid chromatography of peptides is presented in order to clarify the contribution of individual amino acid residues to peptide retention and to predict retention times. The retention of 100 peptides was studied using normal-phase liquid chromatography on amide, diol and silica columns. An acetonitrile-water mixed solution containing 0.2% trifluoroacetic acid +0.2% triethylamine was used as the mobile phase in a linear gradient elution system. The contribution of each residue upon retention was calculated by linear multiple regression analysis. This paper described the contribution values as “hydrophilicity retention coefficients”. Using these hydrophilicity retention coefficients, retention times could be predicted for peptides of known amino acid content and sequence. A set of hydrophilicity retention coefficients on each column was successfully explained by contributions to the degree of retention.     

Reversed-phase ion-pairing liquid chromatography/ion trap mass spectrometry for the analysis of negatively charged,derivatized glycans

The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.     

Recent developments in adsorption liquid chromatography (NP-HPLC) A review

The development of normal phase chromatography mainly in the past ten years is summarized. At first, the difference between normal-phase and reversed-phase chromatography is briefly discussed. According to Snyders theory of retention, some theoretical considerations are added. Most of the stationary phases synthesized in the past years and their field of use in NP-LC are given. They are sorted by linkage to silica and by their polar substituents. The effects resulting from variation of phases and eluents are discussed. Examples for group separation and other applications of NP-LC are given. Received: 10 December 1997 / Revised: 24 February 1998 / Accepted: 26 February 1998     

Recent developments in adsorption liquid chromatography (NP-HPLC) A review

The development of normal phase chromatography mainly in the past ten years is summarized. At first, the difference between normal-phase and reversed-phase chromatography is briefly discussed. According to Snyders theory of retention, some theoretical considerations are added. Most of the stationary phases synthesized in the past years and their field of use in NP-LC are given. They are sorted by linkage to silica and by their polar substituents. The effects resulting from variation of phases and eluents are discussed. Examples for group separation and other applications of NP-LC are given. Received: 10 December 1997 / Revised: 24 February 1998 / Accepted: 26 February 1998     

烷基酚聚氧乙烯醚在不同液相色谱分离模式中的分离研究

考察了烷基酚聚氧乙烯醚在反相色谱、正相键合色谱、硅胶吸附色谱、体积排阻色谱4种不同液相色谱分离模式中的分离效果,分别采用Kromasil C_(18)(250 mm× 4.6 mm,5 μm)、Agilent ZORBAX NH2(250 mm× 4.6 mm,5 μm)、Waters Spherisorb S3W(150 mm×2.0 mm,3 μm)和Shodex MSpak GF-310 2D(150 mm×2.0 mm,5 μm)色谱柱,以225 nm为紫外检测波长,对不同液相色谱分离模式的流动相组成、梯度洗脱条件、柱温、流速等进行了优化,并对烷基酚聚氧乙烯醚在不同液相色谱分离模式中的保留机理进行了初步探讨.结果表明,正相键合色谱实现了烷基酚聚氧乙烯醚的最佳分离;硅胶吸附色谱和体积排阻色谱的分离效果较正相键合色谱稍差.     

Limitation of predictive 2-D liquid chromatography in reducing the database search space in shotgun proteomics: in silico studies

A two-dimensional (2-D) liquid chromatography (LC) separation of complex peptide mixtures that combines a normal phase utilizing hydrophilic interactions and a reversed phase offers reportedly the highest level of 2-D LC orthogonality by providing an even spread of peptides across multiple LC fractions. Matching experimental peptide retention times to those predicted by empirical models describing chromatographic separation in each LC dimension leads to a significant reduction in a database search space. In this work, we calculated the retention times of tryptic peptides separated in the C18 reversed phase at different separation conditions (pH 2 and pH 10) and in TSK gel Amide-80 normal phase. We show that retention times calculated for different 2-D LC separation schemes utilizing these phases start to correlate once the mass range of peptides under analysis becomes progressively narrow. This effect is explained by high degree of correlation between retention coefficients in the considered phases.     

Chromatographic retention of adamantane derivatives in high-performance liquid chromatography

The retention characteristics of some adamantane derivatives under conditions of normal-phase and reversed-phase high-performance liquid chromatography were examined. The values of the retention factor and relative retention factor for chromatographing with various eluents were obtained. The effects of the nature and elution strength of the mobile phase on the retention characteristic of adamantane derivatives were studied.     

Evaluation of Inclusion Selectivity of (2-hydroxy-3-methacryloyloxypropyl β-cyclodextrin-co-N-vinylpyrrolidone) Copolymers Coated Silica Stationary Phase by High Performance Liquid Chromatography

A comparative study of the retention behaviour of disubstituted benzene derivatives on (2-hydroxy-3-methacryloyloxypropyl -cyclodextrin-co-N-vinylpyrrolidone) copolymers physically adsorbed onto porous silca gel was achieved. Under reversed-phase mode, the separation process is based on an inclusion complex formation, and the inclusion selectivity is strongly influenced by hydrogen bonds and/or steric effects. The influence of cyclodextrin derivatives used as mobile phase additives for the conventional reversed-phase high-performance liquid chromatography was also examined with the aim to confirm the formation of inclusion complexes between CD derivatives and test molecules. Under normal-phase mode, it was found that the retention and separation processes are mainly controlled by hydrophilic interactions between the polar substituents of the solute and the hydroxyl groups of the cyclodextrin units. Finally, the enantiomer separation ability was briefly demonstrated.     

Retention mechanism of poly(ethylene oxide) in reversed-phase and normal-phase liquid chromatography

The retention behavior of low- and high-molecular-mass poly(ethylene oxide) (PEO) in reversed-phase (RP) and normal-phase (NP) liquid chromatography was investigated. In RPLC using a C18 bonded silica stationary phase and an acetonitrile-water mixture mobile phase, the sorption process of PEO to the stationary phase showed deltaH(o) > 0 and deltaS(o) > 0. Therefore, PEO retention in RPLC separation is an energetically unfavorable, entropy-driven process, which results in an increase of PEO retention as the temperature increases. In addition, at the enthalpy-entropy compensation point the elution volume of PEO was very different from the column void volume. These observations are quite different from the RPLC retention behavior of many organic polymers. The peculiar retention behavior of PEO in RPLC separation can be understood in terms of the hydrophobic interaction of this class of typical amphiphilic compounds with the non-polar stationary phase, on the one hand, and with the aqueous mobile phase, on the other. The entropy gain due to the release of the solvated water molecules from the PEO chain and the stationary phase is believed to be responsible for the entropy-driven separation process. On the other hand, in NPLC using an amino-bonded silica stationary phase and an acetonitrile-water mixture mobile phase, PEO showed normal enthalpy-driven retention behavior: deltaH(o) < 0 and deltaS(o) < 0, with the retention decreasing with increasing temperature and PEO eluting near the column void volume at the enthalpy-entropy compensation point. Therefore, high-resolution temperature gradient NPLC separation of high-molecular-mass PEO samples can be achieved with relative ease. The molecular mass distribution of high-molecular-mass PEO was found to be much narrower than that measured by size-exclusion chromatography.     

The Application of Perdeuterated Polycyclic Aromatic Hydrocarbons (PAH) as Internal Standards for the Liquid Chromatographic Determination of PAH in a Petroleum Crude Oil and Other Complex Mixtures

Abstract

A sequential liquid chromatographic (LC) procedure for the determination of polycyclic aromatic hydrocarbons (PAH) in a petroleum crude oil and other complex mixtures is described. The procedure includes normal-phase LC on an aminosilane column to isolate fractions containing isomeric PAH and reversed-phase LC on a polymeric C₁₈ column to separate the individual PAH isomers. Appropriate perdeuterated PAH are added to the sample so that each isomeric fraction will contain one internal standard. The perdeuterated PAH are excellent internal standards for this sequential LC procedure. Perdeuterated PAH have normal-phase and reversed-phase LC retention characteristics similar to those of the parent PAH. In the normal-phase LC separation, the perdeuterated PAH elute in the same fraction as the parent PAH. In the reversed-phase LC separation, the perdeuterated PAH elute first and are generally resolved from the parent PAH. The optimized spectrofluorometric detection of each PAH analyte is accomplished by programming appropriate sets of excitation and emission wavelengths to correspond with the elation time of each analyte on the polymeric C₁₈ column. The analytical results obtained from this procedure for the analysis of a shale oil sample [Standard Reference Material (SRM) 1580] and a petroleum crude oil (SRM 1582) are compared to values obtained by gas chromatography - mass spectrometry.