HPLC of Steroids in Non-aqueous Mobile Phase at Subambient Temperature

Abstract

Increased resolution of steroid mixtures was found in high performance liquid chromatography of steroids at subambient temperatures. With aqueous mobile phase using a reversed phase column it was not possible to decrease temperatures below ?10°C due to increased viscosity. This report describes further increase in resolution at ?50°C using a non-aqueous mobile phase for the separation of steroids. Retention times were shorter several fold while resolution was improved.     

Ligand-exchange chromatography of amino acid enantiomers and its application to the biotransformation of DL-aspartic acid byPseudomonas dacunhae

Summary The separation of the D and L enantiomers of eighteen essential α amino acids has been investigated by ligand-exchange chromatography (LEC). The effect of column temperature on the retention times and resolution of individual amino acid enantiomers has been studied by varying the temperature from 25 to 50 °C for a mobile phase containing Cu²⁺ ions. By use of a temperature of 50 °C and Zn²⁺ in the mobile phase, eight of the eighteen amino acid enantiomers can be resolved sufficiently well for practical application. Only phenylalamine, tyrosine, and tryptophan can be separated by use of Ni²⁺ as complexation metal at 50 °C. LEC has been used to monitor the decarboxylation of racemic DL-aspartic acid byPseudomonas dacunhae. Analysis of DL amino acid enantiomers in different media was performed at column temperatures of 30 and 50°C by addition of 0.125 mM Cu²⁺ to the aqueous mobile phase. It was found that the analytical performance is most dependent on the identity of the metal used for complexation; the concentration of the metal was of secondary importance and the column temperature less important still.     

Liquid chromatographic separation and thermodynamic investigation of stereoisomers of darunavir on Chiralpak AD‐Hcolumn

Liquid chromatographic separation of stereoisomers of darunavir on Chiralpak AD‐H, a column containing the stationary phase coated with amylose tris(3,5‐dimethylphenylcarbamate) as a chiral selector, was studied under normal‐phase conditions at different temperatures between 20 and 50°C. The effect of quality and quantity of different polar organic modifiers viz: methanol, ethanol, 1‐propanol, and 2‐propanol in the mobile phase as well as column temperature on retention, separation, and resolution was investigated and optimized. The optimum separation was accomplished using a mobile phase composed of n‐hexane/ethanol/diethyl amine (80:20:0.1 v/v/v) at 40°C. Apparent thermodynamic parameters ΔH⁰ and ΔS* were derived from the Van't Hoff plots (lnk′ versus 1/T) and used to explain the strength of interactions between the stereoisomers and amylose tris(3,5‐dimethylphenylcarbamate) coated chiral stationary phase.     

Effect of Temperature and Mobile Phase Composition on RP-HPLC Separation of Cephalosporins

Abstract

The effects of mobile phase composition and column temperature on resolution in reversed-phase high performance liquid chromatography (RP-HPLC) were used to separate the following cephalosporins: cefonicid, cefaclor, cephazolin, cefodizime, cephaloridine, cephamandole and cephalotin, from a single sample. The capacity factor (k') was described as a function of temperature and mobile phase composition. Semi-empirically estimated values of k' were determined using a small number of experimental data for different temperatures between 20°C and 60°C, and mobile phase compositions (acetate buffer/isopropanol). The capacity factor of each cephalosporin was observed to decrease with increasing temperature and the volume fraction (V) of isopropanol in the mobile phase; linear relationships were obtained for plots of In k' versus 1/T and log k' versus log V. The method developed, while simple, reveals the optimal isocratic elution conditions for column temperature and eluent concentration for the complete separation and rapid analysis of the cephalosporins studied.     

Isocratic separation of ginsenosides by high-performance liquid chromatography on a diol column at subambient temperatures

An improved high-performance liquid chromatographic method for separation of a number of ginsenosides has been developed. The influence of temperature (from 0 to 25°C) on the retention and separation of the ginsenosides was studied by applying a binary mobile phase (acetonitrile/water, 82:18 v/v) and a diol column (LiChrospher 100 Diol). The column temperature is one of the more important parameters for the retention and separation of the components investigated. Selected thermodynamic parameters, including changes of enthalpy (ΔH°) and entropy (ΔS°), were estimated from linear van’t Hoff plots, and possible retention mechanisms were discussed. Moreover, the best separation conditions were selected based on optimization criteria including maximum retention time (t _{R max}), minimum resolution (R _{s min}), and relative resolution product (r). Temperature regions close to 14°C offered the highest selectivity and almost equal distribution of the ginsenosides peaks across the chromatogram. Under such isocratic conditions, excellent separation of chromatographic standards and selected ginseng samples was achieved in less than 16 min.     

Reversed-phase high-performance liquid chromatographic separation of mono- and divinyl chlorophyll forms using pyridine-containing mobile phases and a polymeric octadecylsilica column

Summary The separation of chlorophyll forms was studied employing a wide bore polymeric octadecylsilica column and pyridine containing mobile phases, giving consideration to considering the influence of mobile phase composition and column temperature on the resolution of monovinyl forms from their divinyl analogues. A method involving gradient elution and operating at 15°C is proposed for the separation of several polar and non-polar mono- and divinyl chlorophylls from etiolated tissues of higher plants and from marine phytoplankton. The advantages of pyridine as a mobile phase additive in the reversed-phase liquid chromatography of chlorophylls are discussed.     

High Temperature Liquid Chromatography of Tocol-Derivatives on Polybutadiene-Coated Zirconia Stationary Phases

High temperature reversed-phase high performance liquid chromatography is evaluated as a new approach for the separation of vitamin E isomers (α-, γ-, δ-tocopherol, and α-, γ-, δ-tocotrienol) and α-tocopherol acetate. The separations of each analyte were examined by varying the eluent composition and column temperature. Using a polybutadiene-coated zirconia column at temperatures of 80 °C–140 °C, complete separations were achieved within 10–20 min using organic modifier (acetonitrile) in the range of 40–50%. Irregularity of the Van’t Hoff analysis was noted over the temperature studied. The plot tends to deviate from linearity at higher operating temperatures (140 °C and 150 °C) with linear deviation errors of 12.7% and 41.6%, respectively, for the mobile phase examined.     

Temperature effects in Microcolumn Size Exclusion Chromatography

Abstract

Effects of column temperature on the column efficiency, retention time and stability of analytes were studied in microcolumn size-exclusion chromatography. Larger theoretical plates were achieved at column temperatures betwen 70 and 100 °C. In the constant-flow mode the retention time of analytes decreased with increasing column temperature, which was due mainly to thermal expansion of the mobile phase. When the column temperature was around or higher than the critical temperature of the mobile phase, the retention times of the analytes observed under supercritical pressure conditions still indicated dominancy of the size-exclusion mechanism, while another retention mechanism was involved under subcritical pressure conditions. In the case of the analysis of saccharides, the column temperature should be lower than 100 °C because oligosaccharides were decomposed at higher temperatures.     

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.     

The Study of Separation and Thermodynamics of Adsorption of the Enantiomers of Ethofumasate on a Modified Cellulose

Abstract

Cellulose and cellulose derivatives are biopolymers that are often used as stationary phases for the separation of enantiomers. Describing the mechanism of such separations is a difficult task due to the complexity of these phases. In the present study, direct enantiomeric resolution of ethofumesate has been achieved, using hexane as the mobile phase with various alcoholic modifiers on cellulose tri(3,5‐dimethylphenylcarbamate) chiral stationary phase (CDMPC CSP). The influence of the mobile phase composition and the column temperature on the chiral separation was studied. It was found that at a constant temperature and within a certain range of alcohol modifier concentration, the conformation of the polymeric phase, and the selective adsorption sites were not affected by alcohol modifier concentration. The type and the concentration of the alcoholic modifiers influenced the retention factor and the separation factor. Ethofumesate gained the best enantioseparation using sec‐butanol as alcoholic modifier at 25°C with α‐value 1.70. And the separation factor decreased with the increase of the column temperature. The van't Hoff plots were linear (R ²>0.96) for ethofumesate from 25°C to 50°C. That showed the enantioselective interactions do not change over the temperature range studied. Furthermore the values of ΔH° and ΔS° were both negative, which indicated an enthalpy‐driven separation. And the possible chiral recognition mechanism of the analyte and CDMPC was discussed. It was found that hydrogen bonding plays an important role on enantioseparation of CDMPC CSP. The inclusion and fitness of solute shape in the chiral cavity significantly contributed to the enantioseparation of solute.     

Rapid Supercritical Fluid Chromatography Method for Separation of Chlorthalidone Enantiomers

Supercritical fluid chromatography employing chiral stationary phases is a popular separation technique to perform enantioselective separations. The main advantages of supercritical fluid chromatography are low analysis time, low consumption of organic modifiers, and therefore lower costs and higher environmental friendliness. A novel method for the separation of chlorthalidone enantiomers, widely used diuretic drug, is reported that clearly demonstrates the advantages of supercritical fluid chromatography. The effects of the amount and type of organic modifiers, temperature, and back pressure on enantioselectivity and resolution of the enantiomers were evaluated. The baseline separation was achieved in less than 2.5 min in the optimized system composed of Chiralpak AD column, mobile phase CO₂/MeOH 50/50 (v/v), temperature 40°C, a flow rate of 4.0 mL/min, and 120 bar back pressure. Moreover, enantiomers of chlorthalidone were determined in two commercially available pharmaceuticals. The proposed method may be easily transferred to a semi-preparative scale.     

HTLC: A Viable Approach to Fast LC of Biogenic Amines in Food with Legacy Equipment

Nowadays, there is a demand for fast liquid chromatography (LC) of biogenic amines in food with the equipment available in the average laboratory. To this end, high-temperature liquid chromatography (HTLC) is presented in this study as a viable option, working on a conventional LC platform at moderately high temperatures up to 80 °C. The LC platform was adapted by including an appropriate length of stainless-steel microbore tubing as a preheater. Biogenic amines as benzoyl derivatives showed good thermostability on a thermally rugged column (150?×?4.6 mm, 5 µm). As a model application the HTLC conditions were developed for wine separation. The separation selectivity changed considerably with temperature in the range 36–84 °C, and baseline resolution was obtained only at temperatures well above ambient. At 73 °C oven temperature, the system allowed a flow rate as high as 3 mL min^?1 with the backpressure not exceeding 195 bar (2800 psi). The separation took less than 5 min, comparably fast to documented fast LC separations, and typically at least three to five times faster than a conventional separation.     

An efficient and inexpensive refrigerated LC system for H/D exchange mass spectrometry

Loss of deuterium label during the LC step in amide hydrogen/deuterium exchange mass spectrometry (H/D-MS) is minimized by maintaining an acidic mobile phase pH and low temperature (pH 2.5, 0 °C). Here we detail the construction and performance of a low-cost, thermoelectrically refrigerated enclosure to house high-performance liquid chromatography (HPLC) components and cool mobile phases. Small volume heat exchangers rapidly decrease mobile phase temperature and keep the temperature stable to ±0.2 °C. Using a superficially porous reversed-phase column, we obtained excellent chromatographic performance in the separation of peptides with a median peak width of 4.4 s. Average deuterium recovery was 80.2% with an average relative precision of 0.91%.     

Chromatographic behavior of small organic compounds in low‐temperature high‐performance liquid chromatography using liquid carbon dioxide as the mobile phase

Low‐temperature high‐performance liquid chromatography, in which a loop injector, column, and detection cell were refrigerated at –35ºC, using liquid carbon dioxide as the mobile phase was developed. Small organic compounds (polyaromatic hydrocarbons, alkylbenzenes, and quinones) were separated by low‐temperature high‐performance liquid chromatography at temperatures from –35 to –5ºC. The combination of liquid carbon dioxide mobile phase with an octadecyl‐silica (C₁₈) column provided reversed phase mode separation, and a bare silica‐gel column resulted in normal phase mode separation. In both the cases, nonlinear behavior at approximately –15ºC was found in the relationship between the temperature and the retention factors of the analytes (van't Hoff plots). In contrast to general trends in high‐performance liquid chromatography, the decrease in temperature enhanced the separation efficiency of both the columns.     

Effect of Column Type and Experimental Parameters on the HPLC Separation of Dipeptides

Abstract

The separation of underivatized phenylalanine dipeptides by high performance liquid chromatography using a beta-cyclodextrin bonded silica and a reversed-phase C-18 column was evaluated. Parameters such as per cent organic modifier, pH, buffer type, and temperature were shown to have various effects on the dipeptides separation. The results revealed that the separation of dipeptides was possible using the beta-cyclodextrin column. Changing the percent methanol in the mobile phase from 15 to 10 percent improved the separation only slightly. Changing the pH of the mobile phase affected the peak shape and retention times depending on the amino acid in the dipeptide. It was found that pH changes affected there tention times of Phe-Asp, Phe-Clu, and Phe-Trp more than the other dipeptides analyzed. Two buffer systems were evalusted, triethylammonium acetate (O.l%, pH 4.0) and ammonium acetate (0.01 M, pH 4.5); both systems gave comparableseparations. The effect of temperature on the Separation of the phenylalanine dipeptides was also studied. The only observed effects in varying the temperature from ambient t o 57°C were the decrease in retention times and the peaks were shirperat higher than lower temperatures.     

Direct observation of morphological differences as a function of reaction temperature in model systems for polyurethane foams

A series of polyurea urethanes was isothermally synthesized from toluene diisocyanate (TDI), water, and trifunctional poly(propylene oxide) in the temperature range of 50–150°C. Morphologies of the samples vary significantly as a function of reaction temperature. In this system, phase separation competes with polymerization and crosslinking. Both transmission electron microscopy and atomic force microscopy have shown a network type of structure for the 50°C samples, while the 150°C samples appear to be homogeneous. Infrared analysis shows that samples prepared at 150°C possess a morphology that is less strongly hydrogen bonded and has a broader distribution of hydrogen-bonded states compared to those prepared at lower temperatures. From this combination of techniques, it can be inferred that phase separation occurs faster than crosslinking at low temperatures; consequently, a phase-separated morphology forms. In contrast, crosslinking occurs faster than phase separation at higher reaction temperatures. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 3065–3077, 1998     

HPLC Method for Chiral Separation and Quantification of Antidepressant Citalopram and Its Precursor Citadiol

HPLC method enabling chiral separation and determination of citalopram (CIT), a widely used antidepressant, and its synthetic precursor citadiol in one analysis was developed and validated. Moreover, supercritical fluid chromatography was also tested and was proved to be less effective for this separation purpose. The optimized HPLC system was composed of Chiralcel OD-H column and n-hexane/propane-2-ol/triethylamine 96/4/0.1 (v/v/v) as mobile phase, column temperature 25 °C, flow rate 1.0 mL min^?1, UV detection at 250 nm. The effects of amount of propane-2-ol, triethylamine addition, and temperature on enantioselectivity and resolution of the enantiomers were evaluated. The method was found to be suitable for determination of the enantiomeric purity of CIT in bulk drugs. Enantiomers of CIT were determined in two commercially available pharmaceuticals.     

纤维素衍生物手性固定相高效液相色谱法分离盐酸川丁特罗对映体

以纤维素三-(3,5-二甲基苯基氨基甲酸酯)为手性固定相(Lux Cellulose-1),建立了在正相色谱条件下直接分离盐酸川丁特罗对映体的高效液相色谱法。考察了乙醇、异丙醇等有机改性剂,三氟乙酸、二乙胺等流动相添加剂和柱温对对映体分离的影响。结果显示,酸性和碱性添加剂对对映体分离的影响最为显著: 添加二乙胺时两对映体无分离趋势;添加三氟乙酸时对映体保留强,且分离趋势明显;而同时添加三氟乙酸和二乙胺则两对映体分离显著改善,分离度可达4.0。优化后的色谱条件: 色谱柱为Lux Cellulose-1手性柱(250 mm×4.6 mm, 5 μm),流动相为正庚烷-乙醇-三氟乙酸-二乙胺(88:12:0.3:0.05, v/v/v/v),流速为1.0 mL/min,紫外检测波长为246 nm,柱温为25 ℃。该方法简便,快速,可用于左旋盐酸川丁特罗原料中右旋异构体杂质的检查。     

Chiral ligand-exchange high-performance liquid chromatography with copper (II)-L-phenylalanine complexes for separation of 3,4-dimethoxy-α-methylphenylalanine racemes

L-3, 4-dimethoxy-α-methylphenylalanine (L-DMMD) is an important intermediate for the synthesis of 3-hydroxy-α-methyl-L-tyrosine (L-methyldopa). This paper describes an efficient, accurate, and low-priced method of high-performance liquid chromatography (HPLC) using chiral mobile phase and conventional C₁₈ column to separate L-DMMD from its enantiomers. The effects of ligands, copper salts, organic modifiers, pHs of mobile phase, and temperatures on the retention factors (k') and selectivity (α) were evaluated to achieve optimal separation performance. Then, thermal analysis of the optimal separation conditions was investigated as well. It was confirmed that the optimal mobile phase was composed of 20 % (v/v) methanol, 8 mM L-phenylalanine (L-Phe), and 4 mM cupric sulfate in water of pH 3.2, and the column temperature was set at 20 °C. Baseline separation of two enantiomers could be obtained through the conventional C₁₈ column with a resolution (R) of 3.18 in less than 18 min. Thermodynamic data (??H and ??S) obtained by Van't Hoff plots revealed the chiral separation was an enthalpy-controlled process. To the best of our knowledge, this is the first report regarding the enantioseparation of DMMD by chiral ligand-exchange HPLC.     

Gas Chromatographic Analysis of Cholesterol Oxidation Products on a Thermostable Medium Polarity Capillary Column

The performance of a fused silica column coated with 50% phenyl-/50% dimethyl-polysiloxane, characterized by a high thermal stability, was evaluated for the gas chromatographic determination of cholesterol oxidation products. A silylated mixture of eighteen oxysterol standards (0.1 mg/ml of each compound) was injected into the gas chromatographic system. The temperature was programmed from 230 to 260°C at 2.5°/min and then to 290°C at 1°/min; the injector and detector temperatures were set at 325°C, and the gas linear velocity was 22.8 cm/s. The column gave a fast (15 min) and satisfactory resolution of the main cholesterol oxides. Good separation of the hydroxy from the epoxy derivatives was achieved.The suitability of the method for the determination of oxysterols in food matrices was successfully tested on a saponified lipid extract from egg yolk powder, previously enriched and purified by NH₂ solid phase extraction.