Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined. The choice of modifier depended on the nature of the stationary phase. The more polar solvents were better suited for stationary phases with higher percentage of free silanol groups. Isomeric species were eluted according to chain length, number of double bonds, and the position of the unsaturated acyl chain in the glycerol molecule. Retention increases in the order 20:5 < 22:6 < 18:3 < 20:4 < 18:2. Within each isomeric pair, the species with unsaturated acyl chain occupying either the sn-1- or the 3-position were retained preferentially. Complete simultaneous regioselective separation of 10 isomeric triacylglycerols in a single isocratic run on a single ODS column was demonstrated.     

High temperature fast chromatography of proteins using a silica-based stationary phase with greatly enhanced low pH stability

Reversed-phase liquid chromatography (RPLC) is very widely used for the separation and characterization of proteins and peptides. A novel type of highly stable silica-based stationary phase has been developed for protein separations. A dense monolayer of dimethyl-(chloromethyl)phenylethyl)-chlorosilane (DM-CMPES) on the surface of silica is "hyper-crosslinked" with a polyfunctional aromatic crosslinker through Friedel-Crafts chemistry resulting in stationary phases with extraordinary stability in acidic media. Elemental analysis data confirm the high degree of cross-linking among the surface groups. The hyper-crosslinked phases are extremely stable under highly acidic mobile phase conditions even at a temperature as high as 150 degrees C. A wide-pore (300 A) material made in this way is used here to separate proteins by a reversed-phase mechanism and compared to a commercially available "sterically protected" C18 phase. For small molecules, including neutral and basic compounds, these crosslinked phases give comparable peak shape and efficiency to the commercial phase. Our results show that no pore blockage takes place as commonly afflicts polymer coated phases. In consequence, protein separations on the new phases are acceptable. Using strong ion-pairing reagents, such as HPF6, improves the separation efficiency. Compared to the commercial phases, these new phases can be used at lower pHs and much higher temperatures thereby enabling much faster separations which is the primary focus of this work. Better efficiency for proteins was obtained at high temperature. However, at conventional linear velocities the instability of proteins at high temperature becomes a problem which establishes an upper temperature limit. Uses of a narrowbore column and high flow rates both solves this problem by reducing the time that proteins spend on the hot column and, of course, speeds up the separation of the protein mixture. Finally, an ultrafast gradient (<1 min) protein separation was obtained by utilizing the high temperature and thus high linear velocities afforded by the extreme stability of these new phases. The phases are stable even after 50h of exposure to 0.1% TFA at 120 degrees C. This paper is dedicated to the memory of Csaba Horvath whose work in high temperature HPLC inspired the development of the stationary phases described here.     

Effect of stationary phase hydrophobicity and mobile phase composition on the separation of carboxylic acids in ion chromatography

In a previous work, we studied the retention behavior of monovalent and divalent carboxylic acids on a highly cross-linked polystryene-divinylbenzene anion-exchange column (IonPac AS4A-SC) using a carbonate-based buffer, and a retention model was applied to the chromatographic data obtained. In this work we characterized the retention of carboxylates (formic, acetic, propionic, lactic, pyruvic, oxalic, malonic, succinic, fumaric, maleic, tartaric, glutaric, adipic, malic, mucic, trans-beta-hydromuconic, trans,trans-muconic acids) on a column with higher hydrophilicity (IonPac AS11) according to analyte and stationary phase properties, using previously investigated eluent compositions and comparing the retention data obtained. Moreover, the effect of organic modifiers (CH3OH and CH3CN) in the eluent on the retention factors was also evaluated. The chromatographic data obtained on the IonPac AS11 column were fitted by the retention model and allowed one to obtain and to compare ion-specific selectivity constants (parameters of the model) with the ones obtained with the previous column.     

Temperature-responsive stationary phase utilizing a polymer of proline derivative for hydrophobic interaction chromatography using an aqueous mobile phase

A new method of chromatography is proposed, utilizing a thermo-responsive polymer carrying an amino acid ester residue for the stationary phase of high-performance liquid chromatography (HPLC). We have been investigating the new concept of chromatography, a temperature-responsive chromatography, using temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)-modified surface for HPLC with a constant aqueous media as the mobile phase. In this study, we designed and synthesized thermo-responsive poly(acryloyl-L-proline methyl ester) and its copolymer with N-isopropylacrylamide (NIPAAm). Homopolymers of acryloyl-L-proline methyl ester and copolymer were prepared by the reaction of radical telomerization. These polymers underwent a reversible phase transition from water-soluble forms into aggregates by changing the temperature, similar to PNIPAAm. The surface properties and functions of stationary phases modified with poly(acryloyl-L-proline methyl ester) were controlled by the external temperature. In the chromatographic system, we separated steroids and amino acids with a variety of hydrophobicities using a sole aqueous mobile phase. In contrast to a PNIPAAm-modified surface, a poly(acryloyl-L-proline methyl ester)-modified surface showed a greater affinity for hydrophobic amino acids.     

Retention properties of acetone-water mobile phases on a biphenylsiloxane-bonded silica stationary phase in reversed-phase liquid chromatography

The solvation parameter model system constants and retention factors were used to interpret retention properties of 39 calibration compounds on a biphenylsiloxane-bonded stationary phase (Kinetex biphenyl) for acetone-water binary mobile phase systems containing 30–70% v/v. Variation in system constants, phase ratios, and retention factors of acetone-water binary mobile phases systems were compared with more commonly used acetonitrile and methanol mobile phase systems. Retention properties of acetone mobile phases on a Kinetex biphenyl column were more similar to that of acetonitrile than methanol mobile phases except with respect to selectivity equivalency. Importantly, selectivity differences arising between acetone and acetonitrile systems (the lower hydrogen-bond basicity of acetone-water mobile phases and differences in hydrogen-bond acidity, cavity formation and dispersion interactions) could be exploited in reversed-phase liquid chromatography method development on a Kinetex biphenyl stationary phase.     

Chiral separation of selected proline derivatives using a polysaccharide type stationary phase by high-performance liquid chromatography

Proline derivatives, such as Boc-proline, Boc-2-methylproline, Boc-2-methylproline benzyl ester and Boc-2-methyl-4-hydroxy-proline benzyl ester, have been widely used as a building block leading to a variety of pharmaceutical compounds. Therefore, there is a wide interest in the chiral separation of these compounds. High-performance liquid chromatography (HPLC) methods were developed using a Chiralpak AD-H column to separate enantiomers of these proline derivatives. The effect of mobile phase composition and column temperature was studied. For the proline derivatives studied in this work, good resolution was achieved using a mobile phase composition of hexane, ethanol and 0.1% TFA. For prolines containing carboxyl or hydroxy group, resolution was changed dramatically corresponding to changes as little as 1% of ethanol in the mobile phase, suggesting that the dominant chiral recognition is from hydrogen bonding interactions. On the other hand, for prolines containing a benzyl ester instead of hydroxy group next to the chiral center, resolution was not affected as significantly with the changes of ethanol content in the mobile phase, indicating a different leading chiral recognition mechanism, such as inclusion, steric effect, or possible pi-pi interaction. Linearity, precision and limit of detection were also measured for Boc-2-methylproline and Boc-2-methylproline benzyl ester.     

Polyethylene glycol diacrylate-based supermacroporous monolithic cryogel as high-performance liquid chromatography stationary phase for protein and polymeric nanoparticle separation

A supermacroporous monolithic cryogel was directly prepared by in situ cryo-copolymerization in a stainless steel cartridge (70mmx5.0mm I.D.) using methacrylic acid (MAA) as functional monomer and polyethylene glycol diacrylate (PEGDA) as crosslinker. The highly crosslinked (90%, molar ratio) poly(MAA-PEGDA) cryogel had more uniform supermacropores with a mean diameter of 25microm compared to the poly(acrylamide)-based cryogels. The viability of poly(MAA-PEGDA) cryogel as a medium was demonstrated for separations of lysozyme from chicken egg white (CEW) and water-soluble poly(N-isopropylacrylamide-co-3-(dimethylamino) propyl methacrylamide) (NIPAM-DMAPMA) nanoparticles from its crude reaction solution. The dynamic binding capacities of lysozyme and the polymeric nanoparticles were 4.51x10(-3)micromol/ml and 33.4microg/ml, respectively. The lysozyme recovered from the above separations had a purity of more than 85%, and retained 90% of its enzymatic activity.     

直链淀粉手性固定相高效液相色谱法拆分比索洛尔对映体

采用直链淀粉手性固定相高效液相色谱法在正相条件下直接拆分了比索洛尔对映异构体。分别以异丙醇、乙醇为有机改性剂,考察了流动相的组成与配比、流速及柱温等因素对比索洛尔对映体分离的影响。确定了比索洛尔对映体的最佳拆分条件:流动相正己烷-乙醇-二乙胺(体积比为88∶12∶0.1),流速0.6 mL/min,检测波长270 nm,柱温20 ℃。该方法可快捷、简便地拆分比索洛尔对映体。     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for anionic analytes and mobile phase components on polystyrene polymers using an alkaline mobile phase

Summary Retention models for anionic analytes and mobile phase compounds in reversed-phase ion-pair chromatographic systems have been studied. The solid phase was a polystyrene-divinylbenzene copolymer. The analytes were mono- and divalent anions monitored by indirect detection technique. The mobile phase was highly alkaline and contained a detectable anion, sulfanilic acid.Expressions for the ion-pair retention of solutes and mobile phase components have been developed assuming Langmuir distribution of ion-pairs to a solid phase with one kind of binding site. The validity of the expressions and the origin of system peaks have been confirmed by determination of distribution constants using linear regression.     

α-磺酸脂肪酸聚乙二醇酯混合物的柱色谱分离

依据液固色谱的分离原理,在50 cm长、200～260目硅胶G柱上、常压下,对α-磺酸脂肪酸聚乙二醇酯混合物[1]进行液固色谱分析.通过改变样品混合物处理条件及流动相极性,得到分离产品.α-磺酸脂肪酸聚乙二醇(6000)单、双酯的收率分别为56%和21%.由红外光谱对组分结构进行认证.     

Effect of amine mobile phase additives on chiral subcritical fluid chromatography using polysaccharide stationary phases

Increased retention and selectivity in the subcritical fluid chromatography (SFC) of various amine compounds on polysaccharide chiral stationary phases (CSP) was observed upon incorporation of cyclic amines into the modifier. The retention increases are most pronounced with 2-propanol and are almost absent when methanol is used as modifier. This suggests that the effect may arise from a restriction to the modifier access to the binding site required to effect elution. The effect of the amine additives in SFC does not remain after their removal from the mobile phase. Findings were applied to the development of a 5 min separation of amphetamine and methamphetamine enantiomers.     

Chiral separation using cyclodextrins as mobile phase additives in open-tubular liquid chromatography with a pseudophase coating

The chiral separation of various analytes (dichlorprop, mecoprop, ibuprofen, and ketoprofen) was demonstrated with different cyclodextrins as mobile phase additives in open-tubular liquid chromatography using a stationary pseudophase semipermanent coating. The stable coating was prepared by a successive multiple ionic layer approach using poly(diallyldimethylammonium chloride), polystyrene sulfonate, and didodecyldimethyl ammonium bromide. Increasing concentrations (0–0.2 mM) of various native and derivatized cyclodextrins in 25 mM sodium tetraborate (pH 9.2) were investigated. Chiral separation was achieved for the four test analytes using 0.05–0.1 mM β-cyclodextrin (resolution between 1.11 and 1.34), γ-cyclodextrin (resolution between 0.78 and 1.27), carboxymethyl-β-cyclodextrin (resolution between 1.64 and 2.59), and 2-hydroxypropyl-β-cyclodextrin (resolution between 0.71 and 1.76) with the highest resolutions obtained with 0.1 mM carboxymethyl-β-cyclodextrin. %RSD values were <10%. This is the first demonstration of chiral open-tubular liquid chromatography using achiral chromatographic coatings and cyclodextrins as mobile phase additives.     

CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with a strong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of 75 microm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, and temperature on peptides' resolution was evaluated. Baseline separation of the studied IOPs was achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were performed in the short side mode where the stationary phase was packed for 7 cm only. The selection of the experimental parameters strongly influenced the retention time, resolution, and retention factor. An acidic pH was selected in order to positively charge the analyzed peptides, the pI's of which are about 3 in water buffer solutions. A good selectivity and resolution was achieved at pH <2.8; at higher pH the three parameters decreased due to reduced or even zero charge of peptides. The increase in the ionic strength of the buffer present in the mobile phase caused a decrease in retention factor for all the studied compounds due to the decreased interaction between analytes and stationary phase. Raising the ACN concentration in the mobile phase in the range 40-80% v/v caused an increase in both retention factor, retention time, and resolution due to the hydrophilic interactions of IOPs with free silanols and sulfonic groups of the stationary phase.     

Optimisation of the chlorthalidone chiral separation by capillary electrochromatography using an achiral stationary phase and cyclodextrin in the mobile phase

The separation of chlorthalidone enantiomers in capillary electrochromatography on an achiral stationary phase when adding a chiral selector, hydroxypropyl-β-cyclodextrin, to the mobile phase, was optimised. The goal was to investigate the feasibility of modelling retention times and resolution when during the optimisation procedure regular replacement of columns is required due to their fragility. Therefore, it is essential that the packing procedure delivers reproducible columns. The optimisation of an existing chlorthalidone separation was chosen as case study. The influence of two factors, chiral selector concentration and organic modifier content, on the responses was modelled. The experiments performed prior to modelling were defined by a central composite design. Results on different columns, obtained under identical experimental conditions, were found comparable and thus modelling was possible in situations where several columns were required to complete a design. A second-order polynomial model was built for both responses. Optimal separations were also predicted using Derringer’s desirability functions. The optimum was found at 33 mM cyclodextrin and 16% (v/v) acetonitrile on two types of columns (with different packing times) leading to a strong reduction in analysis time for an equally good separation compared to the initial conditions. Measured and predicted responses were found comparable, indicating that acceptable models were obtained.     

Ultra-fast HPLC separation of common anions using a monolithic stationary phase

In this work, a monolithic column was used to perform ultrafast separations of common inorganic anions in as little as 15 seconds. Separations were performed using ion-interaction chromatography with tetrabutylammonium-phthalate as the ion-interaction reagent and were monitored using either direct conductivity or indirect absorbance detection. Detection limits for direct conductivity were in the low ppm range, whereas those for indirect absorbance detection were up to an order of magnitude higher. The reproducibility was 0.4% and 2% RSD for retention time and peak area, respectively, for a one minute separation, and 2.8% and 3-15%, respectively, for the 15 second separation. The proposed method was validated versus standard ion chromatography with suppressed conductivity detection for the analysis of an industrial water sample.     

Retention in gas-liquid chromatography with a polyethylene oxide stationary phase: molecular simulation and experiment

Configurational-bias Monte Carlo simulations in the isobaric-isothermal Gibbs ensemble were carried out to investigate the partitioning of normal alkanes, primary and secondary alcohols, symmetric alkyl ethers and arenes between a helium vapor phase and a polyethylene oxide stationary phase (M(W)=382 g mol(-1)). The united-atom version of the transferable potentials for phase equilibria force field was used to model all solutes, polyethylene oxide and helium. The Gibbs free energies of transfer and Kovats retention indices of the solutes were calculated directly from the partition constants at two different temperatures, 353 and 393 K. Chromatographic experiments on a Carbowax 20M retentive phase were performed for the same set of solutes and temperatures ranging from 333 to 413 K. The predicted retention indices for alcohols, ethers and arenes are overestimated by about 120, 70 and 20 retention index units, respectively, pointing to an overestimation of the first-order electrostatic interactions in the model system. Molecular-level analysis shows that hydrogen-bonding and dipole-dipole interactions lead to orientational ordering for the alcohol and ether analytes, whereas the weaker dipole-quadrupole interactions for the arene solutes are not sufficient to induce orientational ordering. The retention indices of alcohols and ethers decrease with increasing temperature because of the large entropic cost of hydrogen-bonding and orientational ordering. In contrast, the retention indices for arenes increase with increasing temperature because the entropic cost of cavity formation is smaller for arenes than for comparable alkanes.