Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

A weak cation-exchange monolith as stationary phase for the separation of peptide diastereomers by CEC

A CEC weak cation-exchange monolith has been prepared by in situ polymerization of acrylamide, methylenebisacrylamide and 4-acrylamidobutyric acid in a decanol-dimethylsulfoxide mixture as porogen. The columns were evaluated by SEM and characterized with regard to the separation of diastereomers and α/β-isomers of aspartyl peptides. Column preparation was reproducible as evidenced by comparison of the analyte retention times of several columns prepared simultaneously. Analyte separation was achieved using mobile phases consisting of acidic phosphate buffer and ACN. Under these conditions the peptides migrated due to their electrophoretic mobility but the EOF also contributed as driving force as a function of the pH of the mobile phase due to increasing dissociation of the carboxyl groups of the polymer. Raising the pH of the mobile phase also resulted in deprotonation of the peptides reducing analyte mobility. Due to these mechanisms each pair of diastereomeric peptides displayed the highest resolution at a different pH of the buffer component of the mobile phase. Comparing the weak-cation exchange monolith to an RP monolith and a strong cation-exchange monolith different elution order of some peptide diastereomers was observed, clearly illustrating that interactions with the stationary phase contribute to the CEC separations.     

Capillary electrochromatography-mass spectrometry of cationic surfactants

The CEC-MS of alkyltrimethylammonium (ATMA+) ions with chain lengths ranging from C1-C18 is optimized using an internally tapered column packed with mixed mode reversed phase/strong cation exchange stationary phase. A systematic study of the CEC separation parameters is conducted followed by evaluation of the ESI-MS sheath liquid and spray chamber settings. First, the optimization of CEC separation parameters are performed including the ACN concentration, triethylamine (TEA) content, buffer pH and ammonium acetate concentration. Using 90% v/v ACN with 0.04% v/v TEA as mobile phase, the separation of longer chain C6-C18-TMA+ surfactants could be achieved in 15 min. Lowering the ACN concentration to 70% v/v provided resolution of shorter chain C1, C2-TMA+ from C6-TMA+ although the total analysis time increased to 40 min. Furthermore, variation of both the ACN and TEA content as well as ionic strength has found to significantly influence the retention of longer chain surfactants as compared to shorter chains. The optimum CEC conditions are 70% v/v ACN, 0.04% v/v TEA, pH 3.0 and 15 mM ammonium acetate. Next, the optimization of the ESI-MS sheath liquid composition is conducted comparing methanol to isopropanol followed by the use of experimental design for analysis of spray chamber parameters. Overall, the developed CEC-ESI-MS method allows quantitative and sensitive monitoring of ATMA+ from < or =10 microg/mL down to 10 ng/mL. Utilizing the optimized CEC-ESI-MS protocol, the challenging analysis of commercial sample Arquad S-50 ATMA+ containing cis-trans unsaturated and saturated soyabean fatty acid derivatives is demonstrated.     

Comparison of zirconia- and silica-based reversed stationary phases for separation of enkephalins

In this study, the separation of biologically active peptides on two zirconia-based phases, polybutadiene (PBD)-ZrO2 and polystyrene (PS)-ZrO2, and a silica-based phase C18 was compared. Basic differences in interactions on both types of phases led to quite different selectivity. The retention characteristics were investigated in detail using a variety of organic modifiers, buffers, and temperatures. These parameters affected retention, separation efficiency, resolution and symmetry of peaks. Separation systems consisting of Discovery PBD-Zr column and mobile phase composed of a mixture of acetonitrile and phosphate buffer, pH 2.0 (45:55, v/v) at 70 degrees C and Discovery PS-Zr with acetonitrile and phosphate buffer, pH 3.5 in the same (v/v) ratio at 40 degrees C were suitable for a good resolution of enkephalin related peptides. Mobile phase composed of acetonitrile and phosphate buffer, pH 5.0 (22:78, v/v) was appropriate for separation of enkephalins on Supelcosil C18 stationary phase.     

Separation of peptide diastereomers using CEC and a hydrophobic monolithic column

A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. Using an acidic mobile phase, the peptides migrated due to their own electrophoretic mobility. Hydrophobic interactions with the stationary phase contributed to the separation. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase. Peak resolution increased with buffer pH and concentration. Di‐ and tripeptides composed only of L ‐configured amino acids migrated faster than peptides containing D ‐amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by either CZE or HPLC as well as the 24mer peptides tetracosactide and ¹⁶[D ‐Lys]‐tetracosactide could also be separated by CEC on the hydrophobic monolith.     

Incorporation of carbon nanotubes in a silica HPLC column to enhance the chromatographic separation of peptides: theoretical and practical aspects

The retention mechanism of a series of peptides on a single-wall carbon nanotube (SWCNT) stationary phase inside an HPLC column was investigated over a wide range of mobile phase compositions. While the similar size C18 column exhibited an efficiency of 11.5 μm, the SWCNT column increased the efficiency, i.e. 7.10 μm at a flow rate of 0.8 mL/min, and significantly affected the separation quality of the peptides. The values of enthalpy (ΔH) and entropy (ΔS(*)) of transfer of the peptides from the mobile to the SWCNT stationary phase were determined. The method studied each factor, i.e. ACN fraction x in the ACN/water mixture and column temperature. The changes in retention factor, ΔH and ΔS(*) as a function of the ACN fraction in the mobile phase were examined. These variations are explained using the organization of ACN in clusters in the ACN/water mixture and on the steric and electronic forces implied in the retention process. The information obtained in this work makes this SWCNT stationary phase useful for peptide research and demonstrated the role of ACN to improve the separation quality.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Enantioseparation of tryptophan and its unnatural derivatives by nano‐LC on CSP‐teicoplanin silica based

This work deals with the potentiality of nano liquid chromatography (Nano‐LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory‐assembled nano‐LC–UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.     

Enantioselective separation of thalidomide on an immobilized α1-acid glycoprotein chiral stationary phaseglycoprotein chiral stationary phase

Summary An easy and rapid enantioselective separation for assay of racemic thalidomide on an immobilized α₁-acid glycoprotein chiral stationary phase (GPA CSP) is described. The effects of tetrahydrofuran (THF) as organic modifier, buffer concentration to control the ionic strenth, and mobile phase pH were studied. These variations have consequences in terms of chromatographic retention (k), resolution (R _s), selectivity (α), and peak asymmetry (USP tailing factor). The main condition affecting chromatographic retention was mobile phase pH. At pH 4.5, no separation of thalidomide enantiomers was achieved whereas at pH 7.9 chiral separation was optimum. Peak tailing was directly related to changes in pH and to addition of THF as mobile phase modifier. Results also indicated that the resolution factor is THF concentration-dependent, and that the separation factor (α) is the best parameter for evaluating enantioselectivity. The best mobile phase was pH 7.0, 30 mM ammonium acetate containing 0.3% THF. Under these conditions validation including linearity, recovery, and precision was performed. The suitability of this method has been successfully proved in a limited in-vivo study after intravenous administration of thalidomide to a New Zealand male rabbit.     

Enantiomer separation of flavour and fragrance compounds by liquid chromatography using novel urea-covalent bonded methylated beta-cyclodextrins on silica

A novel methylated beta-cyclodextrin chiral stationary phase (CSP-ME), which was chemically immobilised onto porous silica via multiple urea-linkages was synthesised. The CSP-ME chiral stationary phase depicted good enantiomer separation abilities for some well-known flavour as well as fragrance compounds using high-performance liquid chromatography under reverse phase conditions. The optimum resolution for alpha-ionone, 3-methyl-alpha-ionone, flavanone, 5-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, naringenin and taxifolin was achieved using a mobile phase composition consisting of 1 wt.% triethylammonium acetate buffer (pH 4.68)-methanol. The effects of pH of triethylammonium acetate buffer and the methanol-acetonitrile content of the mobile phase composition on their retention time and resolution were examined to optimise the separation conditions.     

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-μm diol-silica particles. The CSP mixed with 5-μm amino silica particles (3:1) was packed into 75-μm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor R_s=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12–20 min).     

Preparation and enantiomer separation behavior of selectively methylated β-cyclodextrin-bonded stationary phases for high performance chromatography

Native and three selectively methylated β-cyclodextrin (β-CD)-bonded stationary phases without an unreacted spacer arm for liquid chromatography were prepared, where heptakis(2-O-methyl)-β-CD, heptakis(3-O-methyl)-β-CD and heptakis(2,3-di-O-methyl)-β-CD were used as the methylated β-CDs. The enantiomer separation abilities of the resulting β-CD stationary phases for 12 pairs of dansylamino acid enantiomers and six pairs of N-3,5-dinitrobenzoyl amino acid methyl esters as model solutes were investigated. The effects of pH and methanol content of the mobile phase on the retention and resolution were examined to optimize the mobile phase conditions. The optimum resolution for the dansylamino acids was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 4/6) on the β-CD stationary phase. Heptakis(3-O-methyl)- and heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases showed little enantiomer separation abilities for the dansylamino acids. The heptakis(2-O-methyl)-β-CD-bonded stationary phase exhibited no enantioselectivities for those solutes.

For the N-3,5-dinitrobenzoyl amino acid methyl esters, the optimum resolution was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 9/1) on a heptakis(2-O-methyl)-β-CD stationary phase. The heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases exhibited no enantioselectivities for the N-3,5-dinitrobenzoyl amino acid methyl esters. β-CD and heptakis(3-O-methyl)-β-CD-bonded stationary phases had no enantiomer separation abilities for those solutes except for the N-3,5-dinitrobenzoyl phenylalanine methyl ester.     

Sodium desoxycholate‐assisted capillary electrochromatography with methacrylate ester‐based monolithic column on fast separation and determination of coumarin analogs in Angelica dahurica extract

A rapid and sensitive CEC method with methacrylate ester‐based monolithic column has been developed for separation and determination of five coumarins (byakangelicin, oxypeucedanin hydrate, xanthotoxol, 5‐hydroxy‐8‐methoxypsoralen and bergapten) in Angelica dahurica extract. Surfactant sodium desoxycholate (SDC) was introduced into the mobile phase as the pseudostationary to dynamically increase the selectivity of analytes instead of increasing the hydrophobicity of stationary phase. In addition, other factors, pH of phosphate buffer, ACN content and applied voltage, for instance, have also an obvious effect on the resolution but little on the retention time. Satisfactory separation of these five coumarins was achieved within 6 min under a 30:70 v/v ACN–buffer containing 20 mM sodium dihydrogen phosphate (NaH₂PO₄) and 0.25 mM SDC at pH 2.51. The RSDs of intraday and interday for relative peak areas were less than 3.0% and 4.7%, respectively; and the recoveries were between 87.5% and 95.0%. The LODs were lower than 0.15 μg/mL and the LOQs were lower than 0.30 μg/mL, respectively, while calibration curves showed a good linearity (r² > 0.9979). Finally, five target coumarins from the crude extracts of A. dahurica were separated, purified, and concentrated by D‐101 macroporous resin, and were successfully separated and quantitatively determined within 6 min.     

Zwitterionic stationary phase with covalently bonded phosphorylcholine type polymer grafts and its applicability to separation of peptides in the hydrophilic interaction liquid chromatography mode

A novel phosphorylcholine type zwitterionic stationary phase was synthesized by graft polymerization of 2-methacryloyloxyethyl phosphorylcholine onto the surface of porous silica particles. The resulting material possesses both negatively charged phosphoric acid and positively charged quaternary ammonium groups, which renders it a low net charge over a wide pH range. The composition of the surface grafts were determined by elemental analysis and solid state NMR, and the surface charge (zeta-potential) in different buffer solutions were measured using photon correlation spectroscopy. Separation of several peptides was investigated on packed columns in the hydrophilic interaction liquid chromatography (HILIC) separation mode. It was shown that small peptides can be separated based on hydrophilic interaction and ionic interaction between the stationary phase and analyte. The organic solvent composition, the pH and the salt concentration of the eluent have strong effects on the retention time. Compared to native silica before grafting, the newly synthesized zwitterionic material gave more stable retention times for basic peptides over pH range 3-7 due to elimination of the dissociation of silanol groups.     

核苷与碱基的苯胺甲基键合硅胶固定相高效液相色谱分离

建立苯胺甲基键合硅胶固定相(PAMS)高效液相色谱分离核苷与碱基的方法；研究流动相有机溶剂浓度、磷酸缓冲液pH值、离子强度对核苷和碱基在该键合固定相上的色谱保留及分离选择性的影响，用磷酸缓冲液(pH＝4)为流动相快速分离了部分核苷与碱基。     

Analyses of preservatives by capillary electrochromatography using methacrylate ester-based monolithic columns

Five common food preservatives were analyzed by capillary electrochromatography, utilizing a methacrylate ester-based monolithic capillary as separation column. In order to optimize the separation of these preservatives, the effects of the pore size of the polymeric stationary phase, the pH and composition of the mobile phase on separation were examined. For all analytes, it was found that an increase in pore size caused a reduction in retention time. However, separation performances were greatly improved in monolithic columns with smaller pore sizes. The pH of the mobile phase had little influence on separation resolution, but a dramatic effect on the amount of sample that was needed to be electrokinetically injected into the monolithic column. In addition, the retention behaviors of these analytes were strongly influenced by the level of acetonitrile in the mobile phase. An optimal separation of the five preservatives was obtained within 7.0 min with a pH 3.0 mobile phase composed of phosphate buffer and acetonitrile 35:65 v/v. Finally, preservatives in real commercial products, including cold syrup, lotion, wine, and soy sauces, were successfully determined by the methacrylate ester-based polymeric monolithic column under this optimized condition.     

Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI‐MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high‐organic low‐aqueous mobile phases (i.e. under aqueous normal‐phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water‐rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.     

Reversed-phase liquid chromatographic separation of antiretroviral drugs on a monolithic column using ionic liquids as mobile phase additives

The use of 3-methylimidazolium cation-based ionic liquids (ILs) was evaluated as mobile phase additives for separation of antiretroviral drugs on a monolithic column by RP-HPLC. Separation of eight commonly used antiretroviral drugs was achieved on a Chromolith Flash, RP-18e column (25 × 4.6 mm, porous material) using water (pH 4.0 adjusted with acetic acid)/methanol v/v as a mobile phase containing ILs in a gradient elution mode. The effects of concentrations of ILs on retention, resolution and peak shape were studied and a regression equation correlating the interactions between stationary phase and the ILs was established. The retention of all the drugs was decreased notably by using 1-butyl-3-methylimidazolium tetrafluoroborate, while 1-ethyl-3-methylimidazolium methylsulfate reduced gradient drift drastically when compared to triethylamine.     

Titania-based stationary phase in separation of ondansetron and its related compounds

Improvements in stationary phase stability have been and remain a great task for research of new stationary phases. Metal oxide-based stationary phases appear as one of perspective alternatives to classical silica based stationary phases regarding to their similar effectiveness, different selectivity, different retention mechanism and mainly better chemical and thermal stability. In this study, the retention behaviour of ondansetron and its five pharmacopoeial impurities on TiO(2)-based reversed phase was investigated. The influence of buffer type, pH and concentration on retention was studied. Different types and amount of organic solvent in mobile phase were tested. The effect of temperature and flow rate on separation was investigated. The separation conditions were optimized and developed method validated. The retention parameters - retention time (t(R)), retention factor (k'), theoretical plate number (N), resolution between peaks due to nearby peaks (R(s)) and symmetry factor (A(s)) have been compared to parameters achieved on polybutadiene-coated zirconia column. The thermodynamic parameters of retention of analysed compounds - enthalpy, entropy and Gibbs free energy - were calculated and compared to those achieved on polybutadiene-coated zirconia column. This work proves similarity of retention behaviour of ondansetron and its five related compounds on zirconia-based and titania-based stationary phases and potential utilisation of polyethylene covered TiO(2)-based reversed stationary phase as an alternative to polybutadiene-coated ZrO(2) stationary phase in pharmaceutical analysis of ondansetron.