Retention mechanisms of imidazoline and piperazine-related compounds in non-aqueous hydrophilic interaction and supercritical fluid chromatography based on chemometric design and analysis.

The experimental design methodology based on central composite design of experiments was applied to compare the retention mechanisms in supercritical fluid chromatography (SFC) and non-aqueous hydrophilic interaction liquid chromatography (NA-HILIC). The selected set consists of 26 compounds that belong to imidazoline and serotonin receptor ligands. The different chemometric tools (multiple linear regression, principal component analysis, parallel factor analysis) were used to examine the retention, as well as to identify the most significant retention mechanisms. The retention mechanism was investigated on two different stationary phases (diol, and mixed-mode diol). In NA-HILIC, the mobile phase contains acetonitrile as a main component, and methanolic solution of ammonium formate (+ 0.1% of formic acid) as a modifier. The same mobile phase modifier was used in SFC, with a difference in the main component of the mobile phase which was CO2. The retention behaviour differs significantly between HILIC and SFC conditions. The retention pattern in HILIC mode was more partition-like, while in SFC the solute-sorbent interactions allowed retention. The retention mechanism between mixed-mode diol and the diol phases varies depending on the applied chromatographic mode, e.g., in HILIC the type of stationary phase significantly affects the elution order, while in SFC this was not the case. The HILIC retention behaviour was influenced by the number of tertiary amines-aliphatic, and N atom-centred fragments in tested compounds. On the other hand, the number of pyrrole and pyridine rings in the structure of the compound showed correlation with their SFC retention, simultaneously increasing the molecular weight and rapid elution of larger compounds. It was found that temperature surprisingly plays a major role in SFC mode. The increase in temperature reduces the relative contribution of enthalpy factors to total retention, so the separation in SFC was more entropy-controlled. For further pharmaceutical research and optimization, the SFC would be considered more beneficial compared to HILIC since it gives good selectivity in separation of chosen impurities.

Multicomponent Ugi reaction as a tool for fast and easy preparation of stationary phases for hydrophilic interaction liquid chromatography. Part I: The influence of attachment and spacing of the functional ligand obtained via the Ugi reaction.

A number of novel stationary phases for hydrophilic interaction liquid chromatography (HILIC) containing both amides and hydroxyl groups in the functional ligand were obtained by the multicomponent Ugi reaction on the aminated silica surface. The ligands were either attached directly to the substrate surface or spaced by 1,4-butanediol diglycidyl ether (1,4-BDDGE) as a linker. Chromatographic parameters of the obtained stationary phases were evaluated using Tanaka test and model mixtures of sugars, amino acids, and water-soluble vitamins. The adsorbent obtained by direct substrate modification by the Ugi reaction showed the increase of hydrophilicity according to the Tanaka test and high selectivity toward all the analytes with separation efficiency of up to 40000 N/m. The separation of 9 sugars in 12 min, 7 amino acids in less than 18 min, and 7 vitamins in less than 10 min with only 10 cm long column was provided. The effect of introducing 1,4-BDDGE as a linker between the substrate surface and the ligand formed via the Ugi reaction was demonstrated and discussed.

Manipulating selectivity of covalently-bonded hyperbranched anion exchangers toward organic acids. Part IV: General algorithm based on the variation of external part of the functional layer.

Novel anion exchangers with mono- and dialkanolamines in the external part of covalently-bonded hyperbranched functional layer were synthesized. Comparison of the chromatographic properties of the prepared stationary phases in suppressed ion chromatography (IC) mode using hydroxide eluent allowed us to evaluate the effect of the number of substituents at nitrogen atom in alkanolamine on selectivity of anion exchangers toward organic acids. Obtained anion exchangers were also examined together with previously described hyperbranched stationary phases with different mono- and diamines in the external part of the layer for evaluating the influence of various parameters on their selectivity. Effects of hydrophilicity, functionality of amine, and the number of substituents at nitrogen atom of amine used in the last modification cycle were established independently from each other, which provided the possibility to tailor selectivity toward organic acids when preparing anion exchanger for solving particular analytical task. Predominance of hydrophilicity as a key factor affecting the separation of weakly retained organic acids over other studied parameters was demonstrated.

Quantification of inorganic anions and organic acids in apple and orange juices using novel covalently-bonded hyperbranched anion exchanger with improved selectivity.

Chromatographic analysis of orange and apple juices is provided using novel covalently-bonded poly(styrene-divinylbenzene)-based (PS-DVB) hyperbranched anion exchanger for suppressed ion chromatography (IC) with improved selectivity toward inorganic anions and organic acids. The obtained stationary phase provides baseline resolution of weakly retained organic acids such as glycolate, acetate, lactate, and formate, which are not separated to baseline with modern commercially available anion exchangers. The proposed method is validated with respect to linearity, recovery, limits of detection, and intra-day and inter-day precision.

A novel strategy for isolation and determination of sugars and sugar alcohols from conifers.

The ultrasound-assisted extraction method for isolation of 17 sugars and sugar alcohols from conifers with a subsequent hydrophilic interaction liquid chromatography-tandem mass spectrometry method for their determination is proposed. The optimization of extraction parameters was carried out using Taguchi - L9 (34) orthogonal array experimental design for the following parameters-a methanol concentration in the extraction solution, an extraction time, a type of plant sample and an extraction temperature. The optimal ultrasound-assisted extraction conditions were-MeOH concentration - 30% (water - 70%), extraction time - 30 min, type of plant sample - II (grinded leaves 2-4 mm long), extraction temperature - 60 °C. Pure water and acetonitrile were used as eluents in gradient elution mode to separate the analytes. Direct determination of multiple sugars and sugar alcohols was carried out using a mass spectrometric detector operated in a multiple reaction monitoring mode, providing detection limits in the range between 0.1 and 20 ng/mL and good analytical characteristics of the method without derivatization. The developed approach was validated by multiple successive extraction method applied to test its performance on a series of 10 samples, i.e. 2 samples per each of 5 genera: Abies, Larix, Picea, Pinus (Pinaceae) and Juniperus (Cupressaceae), widely distributed in the boreal conifer forests of Eurasia. The novel strategy can be used for profiling of sugars and sugar alcohols in a wide range of plant species.

Novel polymer-based anion-exchangers with covalently-bonded functional layers of quaternized polyethyleneimine for ion chromatography.

For the first time novel pellicular anion-exchangers with functional layers of quaternized branched polyethyleneimine (PEI) covalently bonded to the surface of aminated poly(styrene-divinylbenzene) (PS-DVB) with 1,4-butanediol diglycidyl ether (1,4-BDDGE) as a linker are obtained and studied. The proposed method of synthesis includes alkylation of PS-DVB substrate containing secondary aminogroups with 1,4-BDDGE followed by amination with branched PEI containing primary, secondary, and tertiary amino groups. Quaternization of amino groups of PEI required for increasing ion-exchange capacity of the stationary phase is provided with two epoxides - glycidol and 1,4-BDDGE, which results in the considerable variations of ion-exchange selectivity. Chromatographic properties of the obtained anion-exchangers are evaluated using various model mixtures of anions (F-, HCOO-, CH3COO-, C2H5COO-, Cl-, BrO3-, NO2-, Br-, NO3-, ClO3-, S2O32-, C2O42-, CrO42-, SO42-, PO43-, I-, SCN-) in hydroxide and carbonate/bicarbonte eluents. All anion-exchangers show better selectivity toward the anions of weakly retained organic acids (acetate, formate, propionate) as compared with previously reported chemically derivatized anion-exchangers with monomeric functional sites in the ion-exchange layer. In terms of separation abilities, novel anion-exchangers with covalently-bonded PEI are not inferior to some commercial columns with grafted polymeric anion-exchange layer. Anion-exchanger quaternized with more hydrophilic glycidol packed in 10-cm long column enables the separation of 16 anions including weakly retained and highly retained polarizable ones within 26 min using gradient elution. Column efficiencies for both anion-exchangers with quaternized PEI are up to 32000 N/m and 37000 N/m in case of hydroxide and carbonate eluent, respectively, which is comparable with the values demonstrated by commercially available columns of related chemistry.

Covalently-bonded hyperbranched poly(styrene-divinylbenzene)-based anion exchangers for ion chromatography.

A number of covalently-bonded hyperbranched poly(styrene-divinylbenzene)-based (PS-DVB) anion exchangers having functional ion exchange layers of different branching degrees are prepared and investigated. The attachment of the hyperbranched functional layers to the substrate surface is realized via anchor secondary amino groups inserted into the polymeric backbone by means of acylation with acetic anhydride followed by reductive amination with methylamine. Further modification of the obtained secondary amino groups is provided by repeating the steps of alkylation with 1,4-butanediol diglycidyl ether (1,4-BDDGE) and amination of the terminal epoxide rings with methylamine (MA). The variation of the number of cycles including modification with 1,4-BDDGE and MA results in selectivity alterations for the obtained anion exchangers. Chromatographic parameters of the obtained stationary phases are evaluated using the model mixtures of anions (F-, HCOO-, Cl-, EtCOO-, BrO3-, NO2-, Br-, NO3-, SO42-, PO43-) with hydroxide and carbonate/bicarbonate eluents. The anion exchangers show the increase of NO2-/EtCOO- and NO2-/BrO3- selectivity and the decrease of EtCOO-/Cl- selectivity with increasing the number of modification cycles. In case of anion exchanger obtained after three modification cycles, the calculated values of column efficiencies for polarizable NO2- and Br- are up to 18,000 and 16,000N/m, respectively.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry methylphosponic and alkyl methylphosphonic acids determination in environmental samples after pre-column derivatization with p-bromophenacyl bromide.

Once exposed to the environment organophosphate nerve agents readily degrade by rapid hydrolysis to the corresponding alkyl methylphosphonic acids which do not exist in nature. These alkyl methylphosphonic acids are finally slowly hydrolyzed to methylphosphonic acid. Methylphosphonic acid is the most stable hydrolysis product of organophosphate nerve agents, persisting in environment for a long time. A highly sensitive method of methylphosphonic acid and alkyl methylphosphonic acids detection in dust and ground mixed samples has been developed and validated. The fact that alkyl methylphosphonic acids unlike methylphosphonic acid did not react with p-bromophenacyl bromide under chosen conditions was discovered. This allowed simultaneous chromatographic separation and mass spectrometric detection of derivatized methylphosphonic acid and underivatized alkyl methylphosphonic acids using HILIC-MS/MS method. Very simple sample pretreatment with high recoveries for each analyte was developed. Methylphosphonic acid pre-column derivate and alkyl methylphosphonic acids were detected using tandem mass spectrometry with electrospray ionization after hydrophilic interaction liquid chromatography separation. The developed approach allows achieving ultra-low detection limits: 200 pg mL(-1) for methylphosphonic acid, 70 pg mL(-1) for ethyl methylphosphonic acid, 8 pg mL(-1) for i-propyl methylphosphonic acid, 8 pg mL(-1) for i-butyl methylphosphonic acid, 5 pg mL(-1) for pinacolyl methylphosphonic acid in the extracts of dust and ground mixed samples. This approach was successfully applied to the dust and ground mixed samples from decommissioned plant for the production of chemical weapons.

Preparation and chromatographic performance of polymer-based anion exchangers for ion chromatography: A review.

In the last decade the developments in the field of ion chromatography (IC) were aimed at increasing the efficiency, sensitivity and rapidity of analysis, as well as on improving separation selectivity. Since selectivity and efficiency to the large extent depend on the surface chemistry of the stationary phase, the development of novel anion exchangers remains one of the priority tasks in modern IC. The exact chemistry of commercially available resins is not known and not many literature data devoted to the procedures of preparing anion exchangers for IC have become available in the last 10-15 years. However, the knowledge about the surface chemistry of anion exchangers can provide understanding of the trends in selectivity and efficiency changes, as well as help with the choice of the stationary phase type suitable for solving a particular analytical task. The current review is devoted to the methods of preparing anion exchangers based on polystyrene-divinylbenzene (PS-DVB) and ethylvinylbenzene-divinylbenzene (EVB-DVB) for IC of inorganic and small organic anions and is aimed at demonstrating the improvement of their performance over the years, which was brought by the development of the new types of stationary phase architecture.

Anion exchangers with branched functional ion exchange layers of different hydrophilicity for ion chromatography.

Novel polystyrene-divinylbenzene (PS-DVB) based anion exchangers differing from each other in the structure of the branched functional ion exchange layer are prepared to investigate the role of linker and functional site on ion exchange selectivity. The proposed method of synthesis includes the obtaining of aminated PS-DVB particles by means of their acylation with following reductive amination with methylamine. Further modification of the obtained secondary aminogroups is provided by the alkylation with either 1,4-butanediol diglycidyl ether (1,4-BDDGE) or resorcinol diglycidyl ether (RDGE), which form the linkers of different hydrophobicity, and amination of terminal epoxide rings with trimethylamine (TMA), dimethylethanolamine (DMEA), methyldiethanolamine (MDEA) or triethanolamine (TEA). The variation of the structure and hydrophobicity of the linker and terminal quaternary ammonium sites in the functional layer allows the alteration of selectivity and separation efficiency of the obtained adsorbents. The ion exchange selectivity and separation efficiency of the anion exchangers are evaluated using the model mixtures of anions (F(-), HCOO(-), Cl(-), NO2(-), Br(-), NO3(-), HPO4(2-) and SO4(2-)) in potassium hydroxide eluents. The adsorbents show the decrease of selectivity with increasing the hydrophilicity of the terminal functional site. The anion exchangers having more flexible and hydrophilic 1,4-BDDGE linker provide smaller separation factors for most of the analytes as compared with RDGE-containing adsorbents with the same terminal ion exchange sites, but are characterized with higher column efficiencies and better peak symmetry for polarizable anions. In case of 1,4-BDDGE-modified anion exchangers of the particle size of 3.3µm functionalized with DMEA and MDEA the calculated values of column efficiencies for polarizable NO3(-) and Br(-) are up to 49,000 and 53,000N/m, respectively, which is almost twice higher than the values obtained for the RDGE-containing analogues.

Preparation and characterisation of anion exchangers with dihydroxy-containing alkyl substitutes in the quaternary ammonium functional groups.

Novel poly(styrene-divinylbenzene) (PS-DVB) based anion exchangers having one and two (2,3-dihydroxypropyl) substitutes in the quaternary ammonium functional groups are prepared and characterised by ion chromatography (IC). The introduction of bulky and hydrophilic substitutes to the anion-exchange groups allows the elimination of non-ionic interactions between the polarisable anions and the aromatic rings from the matrix and the improvement of separation selectivity as compared with the traditional trimethylammonium (TMA) functionalised ion exchangers. The synthesis of the ion exchangers includes acylation of PS-DVB particles with acetic anhydride followed by reductive amination either with methylamine hydrochloride or dimethylamine and further alkylation with oxiranes under varied conditions. The ion exchange selectivity and separation efficiency of nine adsorbents having different structure of bonded groups, ion exchange capacity (9-98µequiv.g(-1)) or particle size is studied for model mixture of inorganic anions (F(-), Cl(-), NO2(-), Br(-), NO3(-), HPO4(2-) and SO4(2-)) using carbonate and hydroxide eluents. The adsorbents with more hydrophilic substitutes provided superior columns efficiencies and better peak symmetry as compared with analogues having hydrophobic functional groups. The calculated values of column efficiencies for polarisable NO3(-) and HPO4(2-) are 18,500 and 29,000N/m, respectively, for anion-exchanger, having N-methyl-N',N″-di-(2,3-dihydroxypropyl)ammonium groups which is significantly higher than 1800 and 12,000N/m obtained for these anions with anion exchanger bearing TMA functional groups.

Determination of underivatized glucosamine in human plasma by high-performance liquid chromatography with electrochemical detection: application to pharmacokinetic study.

A simple, rapid and sensitive high-performance liquid chromatography method with electrochemical detection was developed for the determination of glucosamine in human plasma. Plasma samples were analyzed after a simple two-step procedure of protein precipitation with subsequent dilution. The chromatographic separation was performed on a Carbopack column (3 mm x 150 mm) with a mobile phase consisting of water and 200 mM sodium hydroxide. Detection was performed electrochemically in a pulsed voltammetry mode. The limit of detection was 2.0 ng/ml, inter- and intra-day precision were less than 10%. The method was successfully applied to the investigation of the pharmacokinetics of glucosamine in healthy man volunteers.

Chiral high-performance liquid chromatography analysis of alpha-amino acid mixtures using a novel SH reagent--N-R-mandelyl-L-cysteine and traditional enantiomeric thiols for precolumn derivatization.

Several chiral thiols, i.e. traditionally used enantiomerically pure SH reagents and novel N-R-mandelyl-L-cysteine (R-NMC) containing additional chiral center, have been applied as co-reagents in precolumn derivatization with o-phthalaldehyde for enantiomeric HPLC analysis of individual alpha-amino acids and their mixtures. The R-NMC-derived isoindoles as well as adducts with other thiols have a characteristic absorption maximum at 340 nm, and are highly fluorescent allowing detection of 10 microg/l of an amino acid. Investigated 19 amino acids were analyzed separately and in a mixture by a gradient HPLC after precolumn derivatization. The chromatographic behavior of formed isoindoles substantially differs for each of the thiols used for modification. In contrast to traditional enantiomeric thiols application of diastereomeric R-NMC provides higher resolution for alpha-amino acid enantiomers, with L,D-elution order (except for Arg). Combined use of R-NMC and other thiol enlarges the possibilities of this method, allowing accurate chiral analysis of complex amino acid mixtures.

Bionanocompositional chitosan-silica sorbent for liquid chromatography.

Preparation of hybrid nanocompositional chitosan/silica sorbent was carried out. It was shown that formation of gel in sol-gel process of hydrolytic polycondensation of tetraethoxysilane (TEOS) with including of chitosan consists of two stages. Suppression of crystallization of chitosan in obtained two-phase system and changes in IR spectra are evidenced of interactions between molecules of chitosan and silanol groups of silica network. The resulting hybrid chitosan/silica sorbent was tested by high-performance liquid chromatography (HPLC).

Optical photothermal detection in HPLC.

A mode-mismatched parallel dual-beam thermal lens spectrometer with a far-field single-channel detector system was used as a detector in HPLC. An expert estimation of the measurement results was applied to optimize the optical-scheme configuration of the spectrometer to achieve the longest linear calibration range and highest repeatability under chromatographic flow conditions. Chelates with 4-(2-pyridylazo)resorcinol were separated and determined with the limits of detection of n x 10(-8)- n x 10(-7) mol L(-1); the relative standard deviation of measurements was 46%. Xylenol Orange, 4-(2-thiazolylazo)resorcinol, and dithizone were studied as post-column reagents in thermal lens detection in ion chromatography. The limits of detection were n x 10(-8)- n x 10(-7) mol L(-1); the linear calibration ranges were about three orders; the relative standard deviation of measurements was 3-7%. A combined photothermal-refractometric detector for HPLC based on a polarization interferometer is proposed. Metal complexes as 4-(pyridylazo)resorcinol chelates (limits of detection of n x 10(-8)- n x 10(-7) mol L(-1)) and sugars (limits of detection of 10-20 ng L(-1)) were investigated as model substances. Obtained results were compared with results for traditional detectors, which show that photothermal detection has higher sensitivity than photometric and other absorption detectors.

Application of polyelectrolyte complexes as novel pseudo-stationary phases in MEKC.

Polyacrylic acid (PAA) and polymethacrylic acid (PMAA) with carboxyl groups partially blocked by dodecyltrimethylammonium bromide (DTAB) and tetrabutylammonium bromide (TBAB) were tested as new pseudo-stationary phases in micellar electrokinetic chromatography (MEKC). The separation of was examined using PAA and PMAA. Excellent resolution of the substituted phenols and derivatized amino acids was demonstrated using additives of PAA-DTAB polyelectrolyte complex in the running phosphate buffer. It was found that the capacity factors were proportional to the concentration of the complex PAA/DTAB. Critical micelle concentration was effectively zero. It was found that the migration times and efficiency of separation of phenols and derivatives of amino acids depended on the type of polymers and alkyltrimethylammonium salts used.

Separation and enantioseparation of derivatized amino acids and biogenic amines by high-performance liquid chromatography with reversed and chiral stationary phases.

This study demonstrates that an amino-beta-cyclodextrin-bonded phase column exhibits enantioselectivity for various amino acid derivatives. Mixtures of methanol, acetonitrile, tetrahydrofuran or dioxan and triethylamine buffers (pH 4.0-7.0) were used as mobile phases. The effect of the mobile phase on the resolution process was studied by varying the mobile phase composition (type and percentage of organic modifiers, pH, and ionic strength of the buffer solution). The 1-octanol-water partition coefficients are calculated and tabulated for 16 derivatized amino acids. The chromatographic data for 42 pairs of derivatized amino acids resolved on the amino-beta-cyclodextrin-bonded phase are summarised. The separation of adrenaline, noradrenaline and amphetamine on a novel vancomycin stationary phase is demonstrated.

Separation selectivity of anionic metal complexes of N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid in ion and ion electrokinetic chromatography.

The complexes of Fe(III), Co(III), Mn(III), Al(III), Cu(II), Ni(II), Cd(II) and Zn(II) with N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) were separated by ion exchange in different modes: ion chromatography (IC) and ion electrokinetic chromatography (IEKC). In column IC these complexes were separated on an IonPac AS4a anion-exchange column (Dionex, USA). Parameters of the background electrolyte that were examined in IEKC mode include polymer, competing ion concentration and pH. The use of poly(diallyldimethylammonium chloride) (PDADMACl) as a modifier in IEKC provides separation selectivity only slightly different from that observed in IC on the IonPac AS4a column. Optimal separation conditions were found to be: 0.1 mM HBED, 50 mM PDADMAOH, 10 mM Na2 B4 O7, pH adjusted to 10 with acetic acid. The use of an aromatic ligand allowed a 10-fold decrease in detection limits of metal ions in comparison with previously studied EDTA. A separation efficiency up to 400,000 theoretical plates was demonstrated for IEKC.

Potential of ethylenediaminedi(o-hydroxyphenylacetic acid) and N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid for the determination of metal ions by capillary electrophoresis.

Two aromatic polyaminocarboxylate ligands, ethylenediaminedi(o-hydroxyphenylacetic acid) (EDDHA) and N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED), were applied for the separation of transition and heavy metal ions by the ion-exchange variant of electrokinetic chromatography. EDDHA structure contains two chiral carbon centers. It makes it impossible to use the commercially available ligand. All the studied metal ions showed two peaks, which correspond to meso and rac forms of the ligand. The separation of metal-HBED chelates was performed using poly(diallyldimethylammonium) polycations in mixed acetate-hydroxide form. Simultaneous separation of nine single- and nine double-charged HBED chelates, including In(III), Ga(III), Co(II)-(III) and Mn(II)-(III) pairs demonstrated the efficiency of 40,000-400,000 theoretical plates. The separation of Co(III), Fe(III) complexes with different arrangements of donor groups and oxidation of Co(II), Mn(H), Fe(II) ions in reaction with HBED have been discussed.

Ion-chromatographic selectivity of polyelectrolyte sorbents based on some aliphatic and aromatic ionenes.

An aliphatic ionene with hydroxyl group (2HP-8 ionene), mixed aliphatic-aromatic ionenes (3-X and 6-X ionenes), aromatic ionene (Ph-X ionene), and viologen (Dp-X ionene) - polymers with quaternary nitrogen atoms in the main chain served as modifiers in synthesising polyelectrolyte sorbents for ion chromatography. The selectivity of produced and several previously prepared anion exchangers was compared with those of aliphatic ionenes. It was found that aromatic ionenes having a rigid structure of polymer chains are similar to their aliphatic analogues with shorter chains with a high charge density. Polyelectrolyte sorbents based on aromatic ionenes show higher selectivity to aromatic acids (e.g., 1-naphthalenesulfonic acid) as compared with aliphatic ionenes due to specific pi-pi interactions.