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.     

十二烷基键合氧化锆填充电色谱柱的研究

以十二烷基键合氧化锆(C12-ZrO2)作为固定相,制备了填充毛细管电色谱(CEC)柱,较为系统地研究了流动相条件对电渗流的影响、填充CEC柱的稳定性、碱性与中性化合物的保留与流动相pH值和有机溶剂含量的关系。C12-ZrO2固定相填充CEC柱在pH3～11．7范围内具有极好的稳定性;利用磷酸盐与氧化锆表面之间较强的相互作用,能够有效解决传统硅胶键合烷基固定相在有机溶剂含量低的流动相条件下不稳定的问题;同时吸附磷酸盐的固定相表面使得在更宽的流动相pH值范围内CEC柱有足够的电渗流,进一步拓宽CEC的应用领域。     

Comparison of the performance of conventional microparticulates and monolithic reversed-phase columns for liquid chromatography separation of eleven pollutant phenols

The performance of isocratic separations of 11 pollutant phenols (PP) using monolithic (Chromolith RP-18e) and conventional reversed-phase 5 microm (Luna and Purospher C18) and 4 microm (Synergi C12) particulate size columns, selected from high purity silica materials, has been compared. The separations have been optimized based on a previously optimized separation in which a reversed-phase C18 Luna column and acetonitrile as organic modifier were used, allowing the separation of all phenols tested in 23 min. The optimization process was carried out for each column by studying the effect of the mobile phase (acetonitrile as organic modifier, pH, flow-rate) on phenols separation. Under the optimized separation conditions, all phenols were separated in less than 23 min for all columns tested. Asymmetry factors were further evaluated and used to estimate column efficiency using the Dorsey-Foley equation. The efficiency and asymmetry factors were lower for Chromolith than for Purospher and Luna columns respectively. The Chromolith column was finally selected, due to its lower flow resistance, analysis time and good efficiency and asymmetry factors. The PPs separation was achieved in 3 min. The asymmetry factors were in the range 0.9-1.5 using 50mM acetate buffer (pH = 5.25)-ACN (64:36, v/v) as mobile phase, T=45 degrees C and 4.0 ml min(-1) flow-rate.     

Reversed-phase retention thermodynamics of pure-water mobile phases at ambient and elevated temperature

The use of pure water at superheated temperatures, between 100 and 200 degrees C, as a mobile phase for reversed-phase separations is explored. The thermodynamics of the retention process at low temperature (15-55 degrees C) are compared to the thermodynamics at elevated temperature (125-175 degrees C). Significant differences in the enthalpy of the retention process are observed between the two temperature ranges. This is possibly due to changes in the hydrogen-bond network of the pure-water mobile phase, which would change the solvation, and therefore retention, of non-polar solutes. The change in thermodynamic values between the two temperature regions invalidates extrapolation of retention as a function of temperature between the two temperature regions for the prediction of room-temperature pure-water retention factors. The thermodynamic changes observed as the temperature is increased are similar to those seen when mobile phase composition is changed (by adding organic modifier) at constant temperature.     

Dependence of cyano bonded phase hydrolytic stability on ligand structure and solution pH

As part of our program to develop more stable cyano (CN) high-performance liquid chromatography (HPLC) column packings, we have evaluated hydrolytic stability as a function of ligand connectivity, chain length, and side group steric protection and the pH of the mobile phase. Three accelerated tests were used to evaluate stability: (1) A non-HPLC screening test measuring carbon loss in refluxing MeOH-100 mM KH2PO4 pH 4.5 (1:1, v/v) solution; (2) a continuous flow HPLC test measuring capacity factor maintenance in 1% trifluoroacetic acid in water (pH 1.02) at 80 degrees C; and (3) a continuous flow HPLC test measuring column efficiency maintenance in 50 mM triethylamine in water (pH 10.00) at 50 degrees C. The stability of the CN phases was found to be dependent on both ligand chemical structure and the pH of the test conditions. The starting screen test of intermediate pH was least able to differentiate the CN phases based on structure, because two different degradation mechanisms appear to offset each other (acid induced siloxane bond cleavage vs. base induced silica dissolution). A trifunctional and a sterically protected CN phase were notably stable under the acidic test conditions, but had poor stability under basic conditions. Conversely, chain extension afforded poor stability under acidic conditions, but did afford improved stability at higher pH. In total, the data indicate that good CN column stability can be achieved by using a trifunctional or a sterically protected phase in acidic mobile phases. However, as mobile phases of intermediate or higher pH are employed, shorter column lifetimes can be expected due to an accelerated dissolution of the underlying silica substrate. Materials were also compared chromatographically using a mixture of non-polar, polar, and basic analytes under reversed-phase conditions.     

High-performance liquid chromatographic stationary phases based on poly(methyloctylsiloxane) immobilized on silica. III. Stability evaluations

Several reversed-phase materials for high-performance liquid chromatography were obtained by deposition of poly(methyloctylsiloxane) (PMOS) on HPLC silica particles, followed by immobilization using different procedures. Each phase had characteristic physicochemical and chromatographic properties. The present work evaluates the stability of these phases with both neutral and basic mobile phases. All of the stationary phases were quite stable to neutral mobile phase, with less stability at higher pH. However, one thermally immobilized phase presented high stability even at an elevated temperature with a pH 10.0 mobile phase.     

System maps for retention of neutral organic compounds under isocratic conditions on a reversed-phase monolithic column

The solvation parameter model is used to create systems maps for the separation of neutral organic compounds on a Chromolith Performance RP-18e octadecylsiloxane-bonded silica-based monolithic column for water-acetonitrile and water-methanol mobile phase compositions from 10 to 70% (v/v) organic solvent. These results demonstrate that the retention properties of the monolithic column are similar to those of conventional octadecylsiloxane-bonded silica particle-packed columns. It is further shown that the selectivity for the monolithic column falls within the selectivity range for typical particle-packed columns at two mobile phase compositions for which a direct comparison is possible.     

High-performance liquid chromatographic stationary phases based on poly(dimethylsiloxane) immobilized on silica

This work describes the preparation and characterization of six stationary phases for high-performance liquid chromatography (HPLC) obtained by deposition of poly(dimethylsiloxane) (PDMS) in HPLC silica particles, followed by immobilization using different processes (thermal treatments, thermal treatment + microwave irradiation, self-immobilization + gamma irradiation and self-immobilization + microwave irradiation). The chromatographic parameters of all the phases were evaluated with a mixture of test compounds having varied natures (acid, basic and neutral). The stability of one of these phases was evaluated in both a neutral mobile phase and a higher pH mobile phase used at an elevated temperature, with promising results.     

万古霉素键合手性固定相高效液相色谱法直接分离泰妥拉唑对映体

利用反相高效液相色谱法在大环抗生素类手性固定相万古霉素键合手性固定相(Chirobiotic V)上直接分离了泰妥拉唑对映体。考察了缓冲溶液的种类、浓度和pH值,有机改性剂的种类和浓度,柱长和柱温等对手性分离的影响。优化后的色谱条件为:Chirobiotic V色谱柱(150 mm×4.6 mm,5 μm),流动相为0.02 mol/L 醋酸铵缓冲液(pH 6.0)-四氢呋喃(体积比为93∶7),流速为0.5 mL/min,柱温为20 ℃,检测波长为306 nm。在此条件下泰妥拉唑对映体达到了基线分离,分离度达1.68;对映体保留时间的相对标准偏差分别为0.48%和0.49%(n=6),峰面积的相对标准偏差分别为0.45%和0.55%(n=6)。所建立的手性分离方法具有简便快速及重复性好等优点。     

Chromatographic behavior of epirubicin and its analogues on high-purity silica in hydrophilic interaction chromatography

Hydrophilic interaction chromatography has been applied for the separation of epirubicin and its analogues using high-purity silica column with aqueous-organic mobile phase. Parameters affecting the chromatographic behavior of the solutes such as organic modifier, buffer pH, ionic strength and sample size, have been investigated. Of utmost importance for successful separation of these analogues is the choice of organic modifier, since it impacts both the solvent selectivity and the ionization of silica silanols as well as buffer solution, and consequently the retention behavior of solutes. Acetonitrile was shown to offer superior separation of these analogues to methanol, isopropanol or tetrahydrofuran. Results of the effects of organic modifier, buffer pH and ion strength indicate that the retention mechanism is a mixed-mode of adsorption and ion exchange. In addition, an irreversible adsorption of these compounds was found on silica in the weakly acidic or neutral mobile phases, and the effect of various factors on irreversible adsorption was also preliminarily discussed. More significantly, these basic compounds have exhibited peaks with a slanted front and a sharp tail, a typical overloading peak profile belonging to the behavior of competitive anti-Langmuir isotherm by increasing the sample size at the experimental conditions.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

硅胶电色谱分离机理的研究

对硅胶电色谱柱的性能进行了考察,发现在水／有机溶剂流动相条件下,几乎不存在气泡问题,流动相的组成在有机溶剂浓度、电解质浓度、ＰＨ值等方面可以在较大范围变化,选用５种典型样品,对硅胶电色谱的分离机理进行了系统研究,发现有反相分离机理、正相吸附机理、离子交换机理以及电泳机理参与作用。同时考察了有机溶剂浓度、电解质浓度、ＰＨ等对分离的影响。此外,还首次提出了一种全新的电色谱模式－动态改性硅胶电色谱。     

Analysis of polymer additives in high-temperature liquid chromatography

A high-temperature liquid chromatographic technique is employed for the separation of commercially available polymer additives to enhance the resolution and speed. Separation efficiencies and elution behaviors for seven phthalate plasticizers and five antioxidants are evaluated at elevated column temperatures and with a thermal gradient. Diamondbond C18 (octadecylsilica), Zirchrom PS (zirconia-based polystyrene), and Zirchrom PBD (zirconia-based polybutadiene) columns are selected for the study because of their thermal stability. The temperature programming is controlled with a column oven in conjunction with an independent mobile phase preheater and a post-column effluent cooling assembly. Van't Hoff plots show that the reverse-phase liquid chromatography mechanism is maintained over a wide range of column temperatures. A 1% increase of acetonitrile in the mobile phase is estimated to have a comparable effect as a 7-7.5 degrees C column temperature increase on the retention time changes.     

Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

An ultra acid stable reversed stationary phase

We report a new reversed phase liquid chromatography (RPLC) phase with remarkable acid stability using dimethyl(ethylphenylchloromethyl)chlorosilane (1), oligomeric polystyrene (PS), and octylbenzene (C8). This phase Si-1-PS-C8 was prepared using silica modification processes and Friedel-Crafts alkylation chemistry. Under highly aggressive mobile phase conditions, Si-1-PS-C8 exhibited remarkable stability as evinced by only minimal reduction in retention factor (k') after 1400 column volumes at pH = 0.5 and 150 degrees C. The peak shapes for a variety of basic solutes were symmetric using Si-1-PS-C8. Evidence for a highly cross-linked coating of the silica particles was observed using scanning electron microscopy. The remarkable stability of this phase is unparalleled as compared to all other RPLC phases reported to date.     

Separation of B6 vitamers with micellar liquid chromatography using UV and electrochemical detection

Separation of six vitamers of vitamin B6 was performed by RP-HPLC using micellar mobile phase, UV and electrochemical detection. Effect of temperature, type and amount of organic modifier in mobile phase on efficiency and asymmetry factor showed that, the appropriate conditions were temperature of 35 degrees C and 3.0-5.0% (v/v) 1-butanol in mobile phase. Variations of selectivity factor versus 1-butanol concentration, pH of mobile phase, and SDS concentration was investigated and the following optimized conditions were selected for the separation: 3.0% (v/v) 1-butanol, pH=5.5 and 65 mM SDS in mobile phase. Electrochemical behavior of vitamers in optimized mobile phase was investigated using cyclic voltammetry, and potential of +1.2 V versus Ag/AgCl(Sat.) was chose as working potential. Finally, separation of B6 vitamers using UV detection at 254 nm and electrochemical detection at +1.2 V was compared.     

替考拉宁键合手性固定相高效液相色谱法直接拆分泮托拉唑钠对映体

以替考拉宁为手性选择剂制备了大环抗生素类手性固定相替考拉宁键合手性固定相(T-CSP),建立了T-CSP反相液相色谱直接拆分泮托拉唑钠对映体的方法。考察了流动相中有机改性剂的种类和比例、柱温以及流动相流速对拆分泮托拉唑钠对映体的影响。研究发现,用甲醇作有机改性剂比乙腈更有利于对映体的分离;在研究的温度范围内,随着柱温的升高,对映体的保留时间缩短,同时分离因子和分离度降低;在一定范围内降低流速有利于对映体的分离。采用T-CSP色谱柱(150 mm×4.6 mm i.d.,5 μm),以甲醇-水(体积比为35∶65)为流动相,在流速0.6 mL/min、检测波长290 nm、柱温20 ℃的条件下,泮托拉唑钠对映体获得了近于基线的分离,所建立的方法具有简便快速及重复性好等优点。     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Comparison of the performance of different reversed-phase columns for liquid chromatography separation of 11 pollutant phenols

A systematic optimization of the HPLC separation of a mixture containing 11 pollutant phenols (PPs) using a Hypersil ODS (250 mm x 4.6 mm, 5 microm) column and UV-DAD detection has been carried out. The binary mobile phases used were obtained by mixing 50 mM phosphate (pH = 3.0) and methanol, ACN, or THF as organic modifiers. After selecting ACN as an organic modifier, the effects of pH and temperature on PPs separation were studied. A mobile phase of 50 mM acetate (pH = 5.0)-ACN (60:40 v/v) at 50 degrees C allowed the separation of 11 phenols but not to baseline in 17 min. To improve the performance of this separation, the following RP columns were tested: Luna C18 (2), Purospher C18, Synergi C12, Synergi Fusion C18, Gemini C18, Luna Cyano, Lichrospher C8, and Envirosep-PP (polymeric). In all the cases, the performance (analysis time, retention, selectivity, resolution, asymmetry factors, and efficiency) was evaluated. A further reoptimization of the mobile phase was carried out for all the columns by studying the ACN content and pH, with the aim of improving the above-mentioned separations and selecting the most suitable one for PPs analysis.     

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

Summary Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantios-electivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantioselectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.