Separation of α‐ from β‐arylalanines by nickel nitrilotriacetate chromatography

A method is described to separate α‐ from β‐arylalanines by ligand exchange chromatography on a nickel nitrilotriacetate agarose column with UV monitoring of the effluent. Separate mixtures containing an α‐ and β‐arylalanine pair (1 mg of each) were individually loaded onto the nickel resin pre‐equilibrated with the mobile phase at room temperature, and the amino acids were eluted from the column with a gradient from pH 12.0–8.0. The β‐arylalanines eluted first, followed by the α‐isomers. The four α/β‐amino acid pairs tested were well separated with baseline resolution. An aliquot of each fraction was chemically treated to derivatize the amino acids to their N‐acyl methyl ester analogs, and their identities were confirmed by GC/MS analysis. The sample recovery was quantitative (>98%), and the column matrix was very resilient, as demonstrated by consistent separation of the solutes after ～100 preparative cycles.     

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH₂-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH₂-100 column. TSK-gel NH₂ column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.

     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.

     

Hydrophilic interaction liquid chromatography in the separation of a moderately lipophilic drug from its highly polar metabolites--the cardioprotectant dexrazoxane as a model case

This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers.     

Retention Mechanism Studies of Selected Amino Acids and Vitamin B6 on HILIC Columns with Evaporative Light Scattering Detection

The goal of the study was to investigate separation mechanism of selected “essential” amino acids (leucine, isoleucine, threonine, tryptophan, proline, and glycine) and vitamin B6 in hydrophilic interaction liquid chromatography (HILIC) with the evaporative light scattering detection. Chromatographic measurements were made on three different HILIC columns: amide-silica (TSK-gel Amide-80), amino-silica (TSK-gel NH₂-100), and cross-linked diol (Luna HILIC). The retention behaviour of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–90 % (v/v) acetonitrile. The compounds studied were separated under isocratic and gradient conditions. The best results of tested biologically active compounds separation were obtained on the TSK-gel NH₂-100 column. TSK-gel NH₂ column showed mixed HILIC–ion-exchange mechanism, the highest separation efficiency and better selectivity and resolution for tested analytes than the other studied column, especially at concentration of water in mobile phase lower than 30 % (v/v). Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes.     

Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography‐mass spectroscopy from green alga Chlorella kessleri

A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of heterotrophically cultivated green freshwater alga Chlorella kessleri and their identification as picolinyl esters by means of liquid chromatography‐mass spectrometry with atmospheric pressure chemical ionization (LC‐MS with APCI). The method is based on the use of preparative reversed phase HPLC of hundred‐milligram amounts and their subsequent identification by microbore APCI LC‐MS. A combination of these two techniques was used to identify unusual VLCFAs up to C₄₇, both saturated and monounsaturated, with two positional isomers (ω‐9 and ω‐26).     

Preparation of a Novel Amino-Phosphate Zwitterionic Stationary Phase for Hydrophilic Interaction Chromatography

A new stationary phase which contains both negatively charged phosphate groups and positively charged amino groups was successfully synthesized by modification of amino-functionalized silica particles with trichlorophosphine oxide (POCl₃) for hydrophilic interaction chromatography (HILIC). The composition of the surface grafts was determined by Fourier transform infrared spectroscopy and elemental analysis. Various parameters, such as column temperature, water content, pH values and ionic strength of the mobile phase were investigated to study the retention mechanism. The results demonstrated that the stationary phase involved a complex retention process including surface adsorption, partitioning and electrostatic interactions. Under optimized conditions, the separation of nucleobases and nucleosides, water-soluble vitamins, organic acids on the novel stationary phase could be achieved in the HILIC mode.     

The performance of 1‐ethyl‐3‐methylimidazolium tetrafluoroborate ionic liquid as mobile phase additive in HPLC‐based lipophilicity assessment

Ionic liquids have been widely used as green alternative mobile phase additives to shield the residuals silanols groups and modify the stationary/mobile phase HPLC systems. The present study aimed to evaluate the performance of the ionic liquid 1‐ethyl‐3‐methylimidazolium tetrafluoroborate ([EMIM][BF4]) in producing extrapolated logk_w indices suitable to substitute for octanol–water logP or logD values. The effect of [EMIM][BF4] was investigated for a set of basic and neutral drugs using two different columns, BDS and ABZ⁺. [EMIM][BF4] was added simply alone or in combination with n‐octanol and was compared with the conventional masking agent n‐decylamine. [EMIM][BF4] reduced the retention by suppressing silanophilic interactions, althoug to a lower extent than n‐decylamine. Addition of n‐octanol further decreased the retention by shielding silanol sites on BDS and/or interacting with polar groups through hydrogen bonding on ABZ⁺. Logk_w/logD_7.4 relationships proved moderate compared with those derived upon addition of n‐decylamine. They were considerably improved upon the introduction of protonated fraction F⁺ in the correlation, reflecting ion pair formation between the chaotropic anion [BF4]^‐ and the protonated basic compounds. In this aspect, the ionic liquid [EMIM][BF4], although efficient as a masking agent, cannot be recommended as mobile phase additive to reproduce octanol–water partitioning. Copyright © 2010 John Wiley & Sons, Ltd.     

Dual Retention Mechanism in Two-Dimensional LC Separations of Barbiturates,Sulfonamides, Nucleic Bases and Nucleosides on Polymethacrylate Zwitterionic Monolithic Micro-Columns

In-house prepared zwitterionic polymethacrylate micro-columns using in situ polymerization of N,N-dimethyl-N-metacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MEDSA) functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) cross-linker provided excellent stability and reproducibility of preparation and separation efficiency of 60,000–70,000 theoretical plates m⁻¹ for small molecules under isocratic conditions. The column showed a dual retention mechanism, reversed-phase (RP) in highly aqueous mobile phases and aqueous normal-phase (HILIC) in acetonitrile-rich mobile phases. This property can be used to obtain complementary separation and combined information on the sample from repeated injections of a sample on a single column, in different mobile phases characteristic for the HILIC and for the RP modes, which is in fact a form of offline two-dimensional chromatography on a single column. The dual retention mechanism has been observed with a variety of columns, however, often with impractically narrow retention range in one of the two modes. To take full advantage from the combined single-column RP–HILIC experiments, the column should provide a sufficiently broad mobile phase interval both in the RP and in the HILIC mode. The BIGDMA-MEDSA micro-columns proved suitable earlier for the combined RP–HILIC separations of some phenolic compounds and flavonoids. In the present work, we investigated the effects of the mobile phase composition on the retention of a variety of polar compounds over full retention range of buffered aqueous acetonitrile mobile phases, to find potentially useful HILIC and RP retention ranges for barbiturates, sulfonamides, nucleosides and nucleic bases. In the HILIC mode, proton donor–acceptor interactions show a major effect on retention and selectivity of separation, whereas the size of the non-polar hydrocarbon part of the sample molecule is the most important factor in the water-rich mobile phases. The sample structure strongly affects the composition of aqueous–organic mobile phases at which the transition between the two retention modes occurs. Of the investigated sample types, barbiturates show better separation under reversed-phase conditions, whereas nucleosides and nucleic bases in the HILIC mode. Aromatic carboxylic acids and sulfonamides can be separated either in the reversed phase or under HILIC conditions, the two separation modes showing complementary selectivity of separation.

     

Effect of analyte lipophilicity on the resolution of α‐ and β‐amino acids on liquid chromatographic ligand exchange chiral stationary phases

Separation of the two enantiomers of racemic α‐ and β‐amino acids on two ligand exchange chiral stationary phases (CSPs) prepared previously by covalently bonding sodium N‐((S)‐1‐hydroxymethy‐3‐methylbutyl)‐N‐undecylaminoacetate or sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate on silica gel was studied with variation of the organic modifier (methanol) concentration in the aqueous mobile phase. In particular, the variation of retention factors with changing organic modifier concentration in the aqueous mobile phase was found to be strongly dependent on both the analyte lipophilicity and the stationary phase lipophilicity. In general, the retention factors of relatively lipophilic analytes on relatively lipophilic CSPs tend to increase with increasing organic modifier concentration in the aqueous mobile phases while those of less lipophilic or hydrophilic analytes tend to increase. However, only highly lipophilic analytes show decreasing retention factors with increasing organic modifier concentration in the aqueous mobile phase on less lipophilic CSPs. The contrasting retention behaviors on the two CSPs were rationalized by the balance of the two competing interactions, viz. hydrophilic interaction of analytes with polar aqueous mobile phase and the lipophilic interaction of analytes with the stationary phase.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of α‐amino acids and proton pump inhibitors on a ligand exchange chiral stationary phase

A new ligand exchange chiral stationary phase (new CSP) containing residual silanol group‐protecting n‐octyl groups on the silica surface was prepared by treating a ligand exchange CSP (original CSP) based on sodium N‐[(R)‐2‐hydroxy‐1‐phenylethyl]‐N‐undecylaminoacetate bonded to silica gel with excess n‐octyltriethoxysilane. The new and original CSPs containing an identical amount of chiral selector were applied to the resolution of α‐amino acids and proton pump inhibitors (PPIs) including omeprazole, pantoprazole, lansoprazole, and rabeprazole. The separation factors (α) and resolutions (R_S) were greater on the new CSP than on the original CSP except for the resolution of asparagine. The trends of the retention factors (k₁) for the resolution of α‐amino acids on the new and original CSPs with the variation of the organic modifier content in aqueous mobile phase were opposite to those for the resolution of PPIs. Removal of the nonenantioselective interactions between the residual silanol groups and the analytes and the improved lipophilicity of the new CSP were proposed to be responsible for the improved chiral recognition ability of the new CSP and the different retention behaviors of the enantiomers between the new and original CSPs.     

Evaluation of various chromatographic approaches for the retention of hydrophilic compounds and MS compatibility

The goal of this study was to compare the performance of three separation techniques for the analysis of 57 hydrophilic compounds. RPLC, hydrophilic interaction liquid chromatography (HILIC) and subcritical fluid chromatography (SFC) were tested. The comparison was based on the retention, selectivity, peak shape (asymmetry and peak width) and MS sensitivity. As expected, RPLC had some obvious limitations for such classes of compounds, and on average the %ACN required to elute these hydrophilic substances was 4, 7, and 11% ACN at pH 3, 6, and 9, respectively. However, a hybrid polar‐embedded C₁₈ phase with an appropriate mobile phase could represent a viable strategy for hydrophilic basic compounds with log D greater than –2 on average. HILIC and SFC were found to be more appropriate for analyzing a large majority of these hydrophilic analytes (～60 and 70% of compounds eluted during the gradient in HILIC and SFC), while maintaining good MS sensitivity. Finally, this work demonstrated the complementarity of the three analytical techniques and showed that the selection of a suitable strategy should mostly be based on physicochemical properties of the analytes (pK_a, log D, H‐bonding capability, etc.).     

Preparation and application of novel zwitterionic monolithic column for hydrophilic interaction chromatography

A novel zwitterionic hydrophilic porous monolithic stationary phase was prepared based on the thermal‐initiated copolymerization of N,N‐dimethyl‐N‐(3‐methacryl‐amidopropyl)‐N‐(3‐(sulfopropyl)ammonium betaine and ethylene glycol dimethacrylate. A typical hydrophilic separation mechanism was observed at a highly organic mobile phase (ACN >60%) on this optimized zwitterionic hydrophilic interaction chromatography (HILIC) monolithic stationary phase. Good permeability, stability, and column efficiency were observed on the final monolithic column. Additionally, a weak electrostatic interaction for charged analytes was confirmed in analysis of six benzoic acids by studying the influence of mobile phase pH and salt concentration on their retention behaviors on the obtained zwitterionic HILIC monolithic column. The optimized zwitterionic HILIC monolith exhibited good selectivity for a range of polar test analytes.     

Synthesis of polyoxazolines using glycerol carbonate derivative and end chains functionalization via carbonate and isocyanate routes

We report the cationic ring‐opening polymerization of 2‐methyl‐2‐oxazoline (MOx) using bio‐based initiator (GCTs). The functional initiator GCTs was prepared by tosylation of the corresponding alcohol: glycerol carbonate (GC). The termination stage of the polymerization was achieved in presence of KOH and the telechelic polyoxazoline carrying five‐membered cyclic carbonate and oxazolium end groups (GC‐POx^ium) was converted to ((HO)₂‐POx‐OH) carrying α‐diol and ω‐hydroxyl groups. End‐functionalized polyoxazolines (HO)₂‐POx‐OH with M_n ranging from 4200 to 8400 g mol^?1 were synthesized. According to GPC results, the polymerizations of MOx using GCTs and other initiator coming from 1,2‐isopropylidene‐glycerol (Solk‐Ts) were compared. On the basis of FTIR and NMR spectroscopies, the chemical modification of end chains of polyoxazolines was investigated by two alternative synthetic routes. The isocyanate route is a postpolymerization urethanization. The nucleophilic reactivity of the α‐diol and ω‐hydroxyl groups of (HO)₂‐POx‐OH was studied with functional isocyanate (TESPI). In the carbonate route, the electrophilic reactivity of α‐ and ω‐end groups of GC‐POx^ium were explored with amine. It was demonstrated that during the termination stage of the polymerization in presence of allylamine both urethane linker in α‐end chain was synthesized and the ω‐oxazolium group was converted into terminal amine. The carbonate route is an alternative to synthesize urethane without isocyanate. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 4027–4035, 2010     

糖类化合物亲水作用色谱保留行为评价

以糖类化合物为研究对象,系统评价了其在亲水模式下的色谱保留行为。分别考察了流动相、固定相和缓冲盐等对糖类化合物保留的影响,建立了糖类化合物在亲水模式下的保留方程。结果表明,糖类化合物随着流动相中乙腈比例的降低,保留时间减小;随着缓冲盐浓度的增加,保留时间增加;同时,糖类化合物的保留行为还会受到有机溶剂种类和固定相类型的影响;其保留行为可使用顶替吸附-液相相互作用模型定量描述。将该模型进一步用于实际样品中糖类化合物保留行为的预测,获得了较好的实验结果,预测保留时间与实测保留时间的相对误差小于0.3%。对糖类化合物亲水模式下的保留行为进行了系统的评价和定量描述,该研究结果将有助于糖类化合物亲水作用色谱分离方法的发展。     

Investigating the Effect of Chromatographic Conditions on Retention of Organic Acids in Hydrophilic Interaction Chromatography Using a Design of Experiment

Small organic acids have shown significant retention on various stationary phases, such as amide, amino, aspartamide, silica and sulfobetaine phase commonly used in hydrophilic interaction chromatography (HILIC). This study investigated the effect of chromatographic conditions on the retention behavior of organic acids in HILIC using the tool of design of experiment (DOE). The results of the DOE study indicated that both the content of organic solvent (i.e., acetonitrile) and salt concentration in the mobile phase had significant effects on the retention of organic acids. Higher content of organic solvent in the mobile phase led to a significant increase in retention on all types of stationary phases. Increasing salt concentration also resulted in a moderate increase in retention; however, the effect of salt concentration varied with the type of stationary phase. The study also revealed that column temperature had less impact on retention than organic solvent content and salt concentration in HILIC.     

The acetonitrile shortage: Is reversed HILIC with water an alternative for the analysis of highly polar ionizable solutes?

In hydrophilic interaction chromatography (HILIC), best results are obtained with high concentrations of ACN. In the framework of green chromatography and the present shortage and very high price of this hazardous solvent, reversing the stationary phase to apolar and the mobile phase to aqueous can be of interest for several applications. The features of the aqueous RP technique called per aqueous LC (PALC) are illustrated with the analysis of catecholamines, nucleobases, acids, and amino acids. The ca. three-fold higher viscosity of water compared to ACN has consequences on the shape of the Van Deemter plot. For dopamine (N = 26.450 on a 25 cm×4.6 mm id, 5 μm bare silica column), a reduced plate height of 1.9 at an u_opt of 0.3 mm/s was calculated. The plate number, however, strongly depends on pH and ionic strength. As in RP separations, retention is shortened by adding an organic modifier. In the framework of green chromatography, the biodegradable ethanol was used. On the other hand, retention increased by lengthening the carbon chain of ion-pair reagents supporting the RP mechanism as well.     

Determination of the n‐octanol/water partition coefficients of weakly ionizable basic compounds by reversed‐phase high‐performance liquid chromatography with neutral model compounds

A strategy to utilize neutral model compounds for lipophilicity measurement of ionizable basic compounds by reversed‐phase high‐performance liquid chromatography is proposed in this paper. The applicability of the novel protocol was justified by theoretical derivation. Meanwhile, the linear relationships between logarithm of apparent n‐octanol/water partition coefficients (logK_ow′′) and logarithm of retention factors corresponding to the 100% aqueous fraction of mobile phase (logk_w) were established for a basic training set, a neutral training set and a mixed training set of these two. As proved in theory, the good linearity and external validation results indicated that the logK_ow′′–logk_w relationships obtained from a neutral model training set were always reliable regardless of mobile phase pH. Afterwards, the above relationships were adopted to determine the logK_ow of harmaline, a weakly dissociable alkaloid. As far as we know, this is the first report on experimental logK_ow data for harmaline (logK_ow = 2.28 ± 0.08). Introducing neutral compounds into a basic model training set or using neutral model compounds alone is recommended to measure the lipophilicity of weakly ionizable basic compounds especially those with high hydrophobicity for the advantages of more suitable model compound choices and convenient mobile phase pH control.     

Retention prediction and hydrophobicity estimation of weak acidic compounds by reversed-phase liquid chromatography using acetic and perchloric acids as ion suppressors

Simple acids are usually applied to suppress the ionization of weakly ionizable acidic analytes in reversed-phase liquid chromatography. The purpose of this study is to investigate the retention behavior of various weak acidic compounds (monoprotic, diprotic, triprotic, and tetraprotic acids) using acetic or perchloric acid as ion suppressor in a binary hydroorganic mobile phase. The apparent n-octanol–water partition coefficient (K _ow ″) was proposed to calibrate the n-octanol–water partition coefficient (K _ow) of weak acidic compound. LogK _ow ″ was found to have a better linear correlation with logk _w, the logarithm of the retention factor obtained by extrapolating to neat aqueous fraction of the mobile phase, for all weakly ionizable acidic compounds. This straightforward relationship offers a potential medium for direct measurement of K _ow data of weak acidic analytes and can be used to predict retention behavior of these compounds in the ion suppression reversed-phase liquid chromatographic mode.     

Zirconia—A stationary phase capable of the separation of polar markers of myocardial metabolism in hydrophilic interaction chromatography

Creatine, phosphocreatine, and adenine nucleotides are highly polar markers of myocardial metabolism that are poorly retained on RP silica sorbents. Zirconia represents an alternative material to silica with high promise to be used in hydrophilic interaction chromatography (HILIC). This study describes a first systematic investigation of the ability of ZrO₂ to separate creatine, phosphocreatine, adenosine 5′‐monophosphate, adenosine 5′‐diphosphate, and adenosine 5′‐triphosphate and compares the results with those obtained on TiO₂. All analytes showed a HILIC‐like retention pattern when mobile phases of different strengths were tested. Stronger retention and better column performance were achieved in organic‐rich mobile phases as compared to aqueous conditions, where poor retention and insufficient column performance were observed. The effect of mobile phase pH and ionic strength was evaluated as well. The analysis of myocardial tissue demonstrated that all compounds were separated in a relevant biological material and thus proved ZrO₂ as a promising phase for HILIC of biological samples that deserves further investigation.