Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions

Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns.     

Thermally-treated clay as a stationary phase in liquid chromatography

Spray-dried, spherical synthetic hectorite particles have been thermally-treated at 500 degrees C for 16 h and used as adsorbent materials in reversed-phase liquid chromatography. The retention of a 22 mono and disubstituted aromatic compounds was evaluated to study the retention mechanisms on the clay mineral. The retention of solutes on the thermally-treated clays was markedly different than that measured on octadecylsilica (ODS) columns under identical conditions, but remarkably similar to retention characteristics of the same solutes on porous graphitic carbon columns. The clay columns exhibit an enhanced selectivity over the ODS column in separation of nitroaromatic positional isomers. Under identical mobile phase compositions, a selectivity, alpha, of 7.15 between ortho- and para-dinitrobenzene isomers was measured on the clay column compared to a alpha of 1.04 on the ODS column.     

Influence of column characteristics on retention and selectivity in RP-HPLC

Summary Eight commercially available reversed-phase (RP) columns of widely different characteristics were evaluated and compared using the linear solvation energy relationships (LSER). Retention factors of 32 solutes of different types were determined under isocratic conditions using an acetonitrile-water (30∶70) mobile phase. Stationary phase properties were compared by the fitting coefficients of the LSER-based regression equations which are characteristic of the individual stationary phases and represent the extent of various molecular interactions contributing to the retention process. The good agreement between the calculated and measured logk values for different type of compounds support the adequacy and applicability of the LSER model to describe chromatographic retention. Characterization of column performance for the separation of various type of compounds was established by the determination of the different selectivity factors representing hydrophobic selectivity, polar selectivity and specific selectivity.     

Effect of temperature on the chromatographic retention of ionizable compounds. II. Acetonitrile-water mobile phases

The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upon changes in column temperature has been theoretically and experimentally studied. The study focuses on examining the temperature dependence of the retention of various solutes at eluent pH close to their corresponding pKa values, and on the indirect role exerted by the buffer ionization equilibria on retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffer solutions dissolved in 30% (v/v) acetonitrile as eluent at five temperatures in the range from 25 to 50 degrees C were carefully measured. Six buffer solutions were prepared from judiciously chosen conjugated pairs of different chemical nature. Their pKa values in this acetonitrile-water composition and within the range of 15-50 degrees C were determined potentiometrically. These compounds exhibit very different standard ionization enthalpies within this temperature range. Thus, whenever they are used to control mobile phase pH, the column temperature determines their final pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are evaluated. This study demonstrates the significant role that the selected buffer would have on retention and selectivity in RPLC at temperatures higher than 25 degrees C, particularly for solutes that coelute.     

Polybutadiene-coated zirconia packing materials in solvating gas chromatography using carbon dioxide as mobile phase

Summary In this paper, practical considerations of column efficiency, separation speed, thermal stability, and column polarity of capillary columns packed with polybutadiene-coated zirconia were investigated under solvating gas chromatography (SGC) conditions using carbon dioxide as mobile phase. When compared with results obtained from conventional porous octadecyl obtained from conventional porous octadecyl bonded silica (ODS) particles, PBD-zirconia particles produced greater change in mobile phase linear velocity with pressure than conventional ODS particles under the same conditions. The maximum plate number per second (N_t) obtained with a 30 cm PBD-zirconia column was approximately 1.5 times higher than that obtained with an ODS column at 100 °C. Therefore, the PBD-zirconia phase is more suitable for fast separations than conventional ODS particles in SGC. Maximum plate numbers per meter of 76,900 and 63,300 were obtained using a 57 cm×250 μm i.d. fused silica capillary column packed with 3 μm PBD-zirconia at 50 °C and 100 °C, respectively. The PBD-zirconia phase was stable at temperatures up to 320 °C under SGC conditions using carbon dioxide as mobile phase. Polarizable aromatic compounds and low molecular weight ketones and aldehydes were eluted with symmetrical peaks from a 10 cm column packed with 3 μm PBD-zirconia. Zirconia phases with greater inertness are required for the analysis of more polar compounds by SGC.     

HPLC Separation of Different Groups of Small Polar Compounds on a Novel Amide-Embedded Stationary Phase

Retention behaviors of an amide-embedded silica base stationary phase, which was recently developed by our group, were studied by using six different groups of small polar compounds including phenolic compounds, substituted anilines, chlorinated herbicides, Sudan dyes and some nucleotides and nucleosides in HPLC. The chromatographic behaviors of the prepared stationary phase for these analytes were compared with those of a commercially available reversed-phase column ACE C18 under same conditions. Among the six groups of analytes studied, the amide-silica stationary phase showed enhanced selectivity towards phenolic compounds, substituted anilines, Sudan dyes and herbicides under reversed-phase conditions and satisfactory selectivity towards nucleosides and nucleotides which could not be separated with ACE C18 column under HILIC conditions. Experimental data provided some evidence that functional groups on the stationary phases might have certain degrees of influence on selectivity possibly through secondary interactions with the model compounds. The retentions of the moderately polar compounds such as phenolic acids, anilines and herbicides on the stationary phase are higher than highly polar compounds such as nucleotides and nucleosides due to both the hydrophobic and hydrophilic interactions between the stationary phase and analytes. The quantitative determination of Sudan dyes (I, II, III, and IV) in red chilli peppers was performed. Many red chilli peppers were screened and three of them contained Sudans dyes.     

HPLC Separation of Different Groups of Small Polar Compounds on a Novel Amide-Embedded Stationary Phase

Retention behaviors of an amide-embedded silica base stationary phase, which was recently developed by our group, were studied by using six different groups of small polar compounds including phenolic compounds, substituted anilines, chlorinated herbicides, Sudan dyes and some nucleotides and nucleosides in HPLC. The chromatographic behaviors of the prepared stationary phase for these analytes were compared with those of a commercially available reversed-phase column ACE C18 under same conditions. Among the six groups of analytes studied, the amide-silica stationary phase showed enhanced selectivity towards phenolic compounds, substituted anilines, Sudan dyes and herbicides under reversed-phase conditions and satisfactory selectivity towards nucleosides and nucleotides which could not be separated with ACE C18 column under HILIC conditions. Experimental data provided some evidence that functional groups on the stationary phases might have certain degrees of influence on selectivity possibly through secondary interactions with the model compounds. The retentions of the moderately polar compounds such as phenolic acids, anilines and herbicides on the stationary phase are higher than highly polar compounds such as nucleotides and nucleosides due to both the hydrophobic and hydrophilic interactions between the stationary phase and analytes. The quantitative determination of Sudan dyes (I, II, III, and IV) in red chilli peppers was performed. Many red chilli peppers were screened and three of them contained Sudans dyes.

     

Fast separations of chiral β-blockers on a cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia monolithic column by capillary electrochromatography

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) is an excellent chiral selector for enantioseparation of a wide variety of chiral compounds. The monolithic chiral columns are becoming popular in liquid chromatography and capillary electrochromatography. In this work, we present the fast separation of chiral β-blockers on a CDMPC-modified zirconia monolithic column by capillary electrochromatography (CEC). The porous zirconia monolithic capillary column was prepared by using the sol-gel technology and then zirconia surface modified with CDMPC. The enantioseparations were performed in reversed-phase (RP) eluents of a phosphate solution (pH 4.4) modified with acetonitrile or alcohol. The enantioseparations of a set of eight chiral β-blockers were achieved in less than one minute. Influences of the applied voltage, column temperature, concentration of acetonitrile and the type of alcohol as the organic modifier in the mobile phase, and sample injection time on enantioseparation were investigated. CEC separations at the applied voltage of 10 kV and 15 °C in the ACN-modified mobile phase provided the best resolutions for the analytes studied. Run-to-run and day-to-day repeatabilities of the column in the RP-CEC separation were less than 1 and 2%, respectively.     

溶质在动态改性氧化锆液相色谱柱上的保留行为

分别采用硬脂酸、环糊精和十二烷基磺酸钠动态改性自制的ZrO2微球,研究了流动相中甲醇和改性剂浓度对苯酚及苯甲酸的衍生物、苯胺衍生物及芳香烃类化合物的色谱保留行为的影响。中性及碱性化合物的保留时间较短,色谱峰对称;酸性化合物保留时间较长,色谱峰拖尾较严重。改性氧化锆表现出反相色谱性能。     

Determination of atropine in biological specimens by high-performance liquid chromatography

A sensitive and selective method for the determination of atropine in biological specimens has been developed. Samples alkalinized with sodium hydroxide were extracted with dichloromethane, and the organic phase was evaporated in a water-bath at 50 degrees C for ca. 10 min. The residue was dissolved in the mobile phase and injected into a reversed-phase column (TSK gel ODS-120A). The retention time for atropine could be varied by changing either the acetonitrile-water ratio in the mobile phase or the pH of the mobile phase. Acetonitrile-water (2:8, v/v) containing 6 mM phosphoric acid was used as mobile phase. Samples of 200 microliters or less were injected into the chromatography and measured at 215 nm. The recoveries of atropine added to drug-free specimens were satisfactory with coefficients of variation of 4% or less. Ninety-two compounds tested did not interfere with the assay of atropine. The method has been applied for monitoring atropine concentrations in cases of organophosphate and drug poisoning.     

Utilising retention correlation for the separation of oligostyrenes by coupled-column liquid chromatography

The separation of n = 2-5 n-butyloligostyrenes has been illustrated by reversed-phase reversed-phase (RP-RP) coupled-column liquid chromatography. The coupled-column separation has been achieved by use of a C18 column with methanol as the mobile phase followed by a DiamondBond C18 column with acetonitrile (ACN) mobile phase. The DiamondBond C18 is a hybrid carbon clad zirconia (CCZ)-C18 stationary phase. Unlike a C18-carbon clad zirconia two-dimensional liquid chromatographic system, which is orthogonal, the C18 and DiamondBond C18 columns combination exhibit correlations based upon the molecular weight of n-butyloligostyrenes. Using an alternative strategy to two-dimensional liquid chromatography, the molecular weight dependence displayed by both the C18 column and DiamondBond C18 has been used to increase throughput or decrease analysis time in the analysis of the n-butyloligostyrenes. However, this is at the expense of a portion of the two-dimensional peak capacity displayed by the C18-carbon clad zirconia system.     

High temperature normal phase liquid chromatography of aromatic hydrocarbons on bare zirconia

The normal phase HPLC behavior of a bare zirconia column was studied at temperatures up to 200 °C using a hexane mobile phase. The use of elevated column temperatures significantly decreased the retention of twenty five aromatic model compounds according to the van't Hoff equation (>30-fold decrease for some compounds). Large improvements in peak shape, efficiency (>2.2-fold), aromatic group-type selectivity, and column re-equilibration times (>5-fold) were obtained at elevated temperatures. The thermal decomposition of two polar nitrogen compounds (indole and carbazole) was observed in a hexane/dichloromethane mobile phase at temperatures greater than 100 °C. The first order decomposition of carbazole was studied in further detail.     

Separation of steroids using temperature-dependent inclusion chromatography

The influence of temperature on retention and separation of estrogens, progesterone derivatives and beta-cyclodextrin in reversed-phase high-performance liquid chromatography has been studied. Steroids were detected using direct UV detection at 240 and 280 nm. Detection of beta-cyclodextrin was achieved using a post-column indirect photometric method. Chromatographic experiments were performed using an acetonitrile-water mobile phase (30%, v/v) and a wide range of column temperatures from 0 to 80 degrees C with 20 degrees C steps. Linear Van't Hoff plots were observed for steroids and beta-cyclodextrin when an unmodified binary mobile phase was applied. The retention of steroids was strongly influenced by temperature when the mobile phase was modified with beta-cyclodextrin at a concentration of 12 mM. Particularly, for 17beta-estradiol and 20alpha-hydroxyprogesterone a strong deviation from the linear Van't Hoff plots and a remarkable affinity for beta-cyclodextrin was observed. Polynomial regression calculations were performed to fit the set of experimental data points. Using third-order polynomial equations, minimum separation factor values (alphamin) were calculated for temperatures from -10 to + 100 degrees C with 1 degrees C steps. The best chromatographic conditions for separation of multicomponent samples were chosen. A possible retention mechanism for solutes in the presence of macrocyclic additives is discussed. The results presented describe the role of temperature in high-performance liquid chromatography systems in which the mobile phase is modified with an inclusion agent.     

高效液相色谱-串联质谱法结合多糖衍生物手性固定相拆分氰戊菊酯

建立了以多糖衍生物为手性固定相的高效液相色谱-串联质谱(HPLC-MS/MS)直接拆分氰戊菊酯对映体的方法。在反相液相色谱条件下,考察了手性固定相的种类、流动相组成、柱温、流速对氰戊菊酯4个立体异构体分离的影响。同时,利用热力学方法对氰戊菊酯的立体异构体与固定相之间的色谱保留和分离的热力学机理进行了探讨。结果表明:采用Lux Cellulose-3(纤维素-三(4-甲基苯甲酸酯))手性色谱柱,在以流动相为乙腈-水(5 mmol/L甲酸铵)=(55:45,V:V)流速0.4 mL/min,柱温30℃的条件下,可在14 mins内实现氰戊菊酯4个立体异构体的基线分离。拓展了HPLC-MS/MS在菊酯类手性农药对映体分离及检测上的应用。     

Retention mechanism of peptides on a stationary phase embedded with a quaternary ammonium group: A liquid chromatography study

We investigated the mechanisms involved in the retention of various peptides on a stationary phase embedded with a quaternary ammonium group (BS C23), by high-performance liquid chromatography. This was compared with peptide retention on a conventional reversed-phase C18 (RP C18) column under isocratic conditions, to understand better the various mechanisms involved. Chromatographic characterization of the two stationary phases with “model” compounds showed that BS C23 is less hydrophobic than RP C18 and induces electrostatic interaction (attraction or repulsion) with ionized compounds. If reversed-phase partitioning was the predominant retention phenomenon, for both stationary phases, the retention mechanisms in BS C23 provided different selectivity to that of RP C18. Electrostatic attraction or repulsion was clearly observed between peptides and the permanent positively charged group embedded in BS C23 depending on the pH. For most of the peptides, a weak anion-exchange mechanism was observed on the quaternary ammonium-embedded stationary phase if mobile phases at neutral pH and low ionic strengths were employed.     

Fast enantioseparations of basic analytes by high-performance liquid chromatography using cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia stationary phases

In this work, we study the influence of the mobile phase and column temperature on the enantioresolution of basic compounds on microparticulate porous zirconia coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC). The chiral analytes are amino compounds, including a number of beta-blockers. Analytes are eluted with hexane-alcohol mobile phases. We investigated the effect of alcohol (type and concentration), basic eluent additives, and column temperature on the parameters that control resolution (column efficiency, retention and selectivity). Conditions for achieving an adequate separation in the least time have been determined for numerous racemic mixtures. For most solutes, baseline resolution of the enantiomeric pair was achieved in less than 1 min; 12 of 13 pairs were separated in less than 2 min.     

Combined effect of solvent content, temperature and pH on the chromatographic behaviour of ionisable compounds

The organic solvent content and the pH in the mobile phase are the usual main factors in reversed-phase liquid chromatographic separations, owing to their strong effects on retention and/or selectivity. Temperature is often neglected. However, even in cases where the impact of this factor on selectivity is minor, the reduction in analysis time is still an interesting reason to consider it. In addition, ionisable compounds may exhibit selectivity changes, owing to the interaction of organic solvent and/or temperature with pH. The separation of ionisable compounds (nine diuretics: bendroflumethiazide, benzthiazide, bumetanide, chlorthalidone, furosemide, piretanide, probenecid, trichloromethiazide and xipamide, and two beta-blockers: oxprenolol and propranolol) exhibiting different acid-base behaviour was studied. The compounds were tested in a Zorbax SB C18 column under a wide range of conditions: 25-45% (v/v) acetonitrile, pH 3-7 and 20-50 degrees C. Models considering two factors (organic solvent/pH and temperature/pH), and three factors (organic solvent/temperature/pH) were developed from a previously reported equation, which considers the polarity contributions of solute, stationary and mobile phases. This allowed a comprehensive method to predict the retention of the 11 compounds, the modification of their acid-base behaviour (i.e. determination of protonation constants and shifts of the retention versus pH curves), and the selectivity changes within the studied factor ranges.     

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

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

Reversed-phase separations on cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase

Summary The chiral separation of eight racemic compounds has been investigated on a cellulose tris(3,5-dimethylphenylcarbamate) chiral column under reversed-phase conditions. The discrimination mechanism under reversed-phase conditions is discussed. Addition of acid to the mobile phase is necessary for resolution of acidic racemic compounds. The presence of ion-pair reagent in the mobile phase is a key factor in the resolution of basic racemic compounds. Retention of the racemates is also affected by addition of acid or salt. The anion in buffer not only interacts with the racemete, but also with the polysaccharide derivative on the silica gel surface.     

A study of the effects of column porosity on gradient separations of proteins

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample elution. Hydrodynamic properties, efficiency and gradient elution of proteins were investigated on five commercial C18 columns with wide-pore totally porous particles, with superficially porous layer particles, non-porous particles and a silica-based monolithic bed. The efficiency in the terms of reduced plate height is higher for low-molecular ethylbenzene than for proteins, but depends on the character of the pores in the individual columns tested. The superficially porous Poroshell and the non-porous Micra columns provide the best efficiency for proteins at high mobile phase flow rates, probably because of similar pore architecture in the stationary phase. The Zorbax column with similar pore architecture as the Poroshell active layer, i.e. narrow pore distribution of wider pores shows better efficiency than the packed column with narrow pores and broad pore distribution. The monolithic column shows lower efficiency for proteins at high flow rates, but it performs better than the broad-pore distribution totally porous particulate columns. Different pore architecture affects also the retention and selectivity for proteins on the individual columns. The retention times on all columns can be predicted using the model for reversed-phase gradient elution developed originally for low-molecular compounds. Consideration of the limited pore volume accessible to the biopolymers has negligible effect on the prediction of retention on the columns packed with non-porous or superficially porous particles, but improves the accuracy of the predicted data for the totally porous columns with broad pore distribution.