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.     

Chromatographic behaviour of selected steroids and their inclusion complexes with beta-cyclodextrin on octadecylsilica stationary phases with different carbon loads

Retention and separation studies of selected estrogens, progestogens and their inclusion complexes with beta-cyclodextrin were conducted using two C18 HPLC columns with different carbon loads. The difference in carbon load between investigated octadecylsilica packing materials was about 50%. The mobile phases were composed of a 30% v/v acetonitrile-water mixture without and with addition of beta-cyclodextrin at a concentration of 12 mM. The experimental data revealed that retention of the steroids was significantly reduced on the column with the lower carbon load. Moreover, it was found that this column offers better separation power and shorter analysis time at the temperatures studied. However, the calculated values of the retention factor ratios (k0(mMCD))/k(12mMCD)) of the steroids were similar for both columns investigated. This observation suggests that the stationary phase structure appears to have little effect on the formation of host-guest complexes if the complexation process is localised to the chromatographic mobile phase. From a practical point of view, when the mobile phase is modified with beta-cyclodextrin, the separation of the steroids is strongly influenced by temperature. The best chromatographic conditions were determined for the separation of multicomponent samples on the column with lower carbon load. A possible retention mechanism for components of interest in the presence of macrocyclic additives is discussed.     

Simple chamber for temperature-controlled planar chromatography

This article describes a construction of a simple developing device designed for temperature control of thin-layer chromatographic plates. The plates can be developed by the ascending technique under temperature gradient or non-gradient conditions. Saturated or unsaturated chamber conditions can be easily selected. The effects that give rise to pseudo-non-linear Van't Hoff plots, e.g. a temperature irregularity inside the chamber or heat evolving during solvent adsorption near the migrating front of the mobile phase are minimized. The preliminary temperature-retention studies show that the device is suitable for temperatures ranging from -20 to 60 degrees C. Using a binary mobile phase mixture (methanol-water, 70:30, v/v) the velocity of the mobile phase front on the HPTLC RP-18W plates at different temperatures was investigated. Under these conditions the retention profiles of four natural estrogens (estetrol, estriol, 17beta-estradiol and estrone) were examined. The application of the described device for temperature-retention studies is also discussed.     

Characterization of inclusion complexes of betamethasone-related steroids with cyclodextrins using high-performance liquid chromatography

HPLC was used to study the inclusion complexes formed between various beta- and gamma-cyclodextrins and a series of corticosteroids related to betamethasone. Apparent association constants were measured in acetonitrile-water for a set of 13 steroids. An increase in the stability of the steroid-cyclodextrin complex is observed at lower concentrations of acetonitrile. The effects of the nature of the halide at the 9-position, the location of a double bond within the C-ring, substitution at the 9- and 11-positions, and modification of the D-ring of the steroid backbone were studied. The 11- and 17-positions were found to be critically involved in the inclusion process. Larger apparent association constants were obtained with gamma-cyclodextrin (gamma-CD) than with beta-cyclodextrin (beta-CD) due to the increased diameter of the gamma-CD cavity. Van't Hoff plots were constructed to examine the thermodynamic properties of the inclusion process. Plots constructed using retention factors were found to be nonlinear when gamma-CD was present in the mobile phase. This is due to an increase in the strength of the inclusion complex as temperature decreases. Plots constructed using apparent association constants were linear, indicating that the mechanism of inclusion does not change over the range of temperatures studied (10 to 80 degrees C). Enthalpy-entropy compensation was observed for 11 of the 13 steroids studied. The usefulness of cyclodextrins to achieve the separation of steroids in HPLC is discussed and a practical application for the analysis of a steroid and three potential impurities is described.     

Investigations into the chromatographic behavior of a doxorubicin-peptide conjugate

HPLC impurity profile method development for a doxorubicin-heptapeptide conjugate included significant changes of the separation profile with diluent, eluent and pH. These separation variables were also temperature-dependent with a shift in retention from 35 to 45 degrees C. There was also a direct relationship of temperature with LC retention, and a pH minimum at 5.9. Atypical dependence of the impurity profile on diluent at a k' of 18 led to further investigation. A large change in retention by several minutes was a function of both the organic eluent composition and temperature between 15 and 30 degrees C. Several Van't Hoff temperature studies from 5 to 65 degrees C on several column types resulted in non-linear plots. Analysis of the molecular subunits suggested that the peptide portion of the analyte influenced the non-linear retention behavior. The stationary phase type was not a significant factor causing non-linearity. Circular dichroism-temperature studies indicated a notable transition in ellipticity for the amine regions (198-202 nm) that occurred between 39 and 44 degrees C. This transition temperature range coincided with the results of the Van't Hoff analysis, between 35 and 44 degrees C, to indicate that these effects were not primarily stationary phase induced.     

Chromatographic behavior of ion pair enantiomers of dansyl leucine cyclohexylammonium salt on a beta-cyclodextrin stationary phase and the effect of a competitive-binding mobile phase additive

The separation of dansyl leucine enantiomers on a beta-cyclodextrin stationary phase is significantly complicated by the association of the amino acid with its cyclohexylammonium counter ion, in a mobile phase of 80:20 (v/v) methanol-water. This produces very unusual chromatography, with two partially superimposed peaks observed for each enantiomer at lower column temperatures. The peak shape is attributed to the irreversible, oncolumn conversion of the ion pair (I) to the free, protonated (neutral) dansyl amino acid (II+H). Increasing the ionic strength of the mobile phase greatly improves the chromatography by transforming the solute species to enantiomers of II (the anionic, free amino acid). Van't Hoff plots are constructed for both species I and II (under different mobile phase conditions) to provide thermodynamic insight into the major enantioselective driving forces of separation. The chiral discrimination of the stationary phase is found to be primarily enthalpically driven for both solutes. Finally, 1-adamantanecarboxylic acid (ACA) is investigated as a solute-competitive mobile phase additive to intentionally block the hydrophobic cyclodextrin cavities on the stationary phase. By varying the concentration of ACA additive in the mobile phase, control over the retention and chiral recognition of the stationary phase is demonstrated.     

Role of the water as a surface tension modifier for the retention of a series of dansylaminoacids on a beta-CD stationary phase

In reversed phase liquid chromatography (RPLC), using a surface tension treatment, the retention and separation of a series of d,l-dansylaminoacids were investigated with native beta-cyclodextrin as a chiral stationary phase. The enantioselectivity thermodynamic parameters were determined from linear van't Hoff plots. An analysis of the experimental variations in the retention factor with different fractions of water in the mobile phase was performed. The number of water molecules, n, excluded from the solute beta-cyclodextrin cavity interface when the analyte transfer occurred, was determined. Using these n values, the relative degrees of compound inclusion were calculated and correlated to both the steric bulkiness of the solute and the thermodynamic data.     

朊病毒蛋白113-120序列多肽的反相液相色谱保留行为与构象稳定性的关系

朊病毒（Prion)蛋白是人和动物慢性中枢神经系统退化病的传染源,该蛋白的113－120序列被认为在其致病和传染机理中起着重要作用。以反相高效液相色谱为分析手段,研究了Prion蛋白113－120序列多肽的色谱保留行为。通过比较不同温度、不同流动相条件下该多肽色谱保留行为的变化,发现在乙腈溶液为上时,1nKw随温度的变化关系和Van′t Hoff曲线均比简单,说明该多肽在乙腈溶液中所采取的构象均较稳定,不易受温度的影响。以甲醇溶液为流动相时,具有游离末端的多肽的1nKw随温度变化关系和Van′t Hoff曲线比末端羧基和氨基分别被酰胺封闭的多肽要复杂,说明具有游离末端的多肽在甲醇溶液中所采取的构象相对较不稳定,易受环境的影响。这些结果进一步证明,113－120序列在Prion蛋白构象变化中可能起着重要作用。     

Unusual effect of column temperature on chromatographic enantioseparation of dihydropyrimidinone acid and methyl ester on amylose chiral stationary phase

This paper reports an unusual effect of column temperature on the separation of the enantiomers of dihydropyrimidinone (DHP) acid and its methyl ester on a derivatized amylose stationary phase by normal-phase liquid chromatography. The separation of the DHP acid enantiomers was investigated using both carbamate-derivatized amylose and cellulose stationary phases (Chiralpak AD and Chiralcel OD) with an ethanol-n-hexane (EtOH-n-Hex) mobile phase. On the amylose phase, the van 't Hoff plot of the retention factor of the S-(+)-DHP acid was observed to be non-linear while that of R-(-)-DHP acid was linear. Likewise, the van 't Hoff plot for DHP acid enantioselectivity was non-linear with a transition occurring at approximately 30 degrees C. Furthermore, the van 't Hoff plot for the DHP acid enantioselectivity factor for data taken when heating the column from 5 to 50 degrees C was not superimposable with the same plot prepared with data from the cooling process from 50 to 5 degrees C. This observation suggested that the stationary phase was undergoing a thermally induced irreversible conformational change that altered the separation mechanism between the heating and cooling cycles. Similar phenomena were observed for the separation of the enantiomers of the DHP ester probe compound. The conformational change of the AD phase was shown to depend on the polar component of the mobile phase. When 2-propanol (2-PrOH) was used as the modifier instead of EtOH, the van 't Hoff plots for DHP acid were linear and thermally reversible, suggesting that no such irreversible conformational change occurs with this modifier. Conversely, when the AD phase was pre-conditioned with a more polar methanol (MeOH) or water containing mobile phase, thermal irreversibility of DHP acid enantioselectivity was once again observed. Interestingly, when the stationary phase was changed to its cellulose analogue, the Chiralcel OD, all van 't Hoff plots for the retention and selectivity of DHP acid were thermally reversible for both EtOH-n-Hex and 2-PrOH-n-Hex mobile phases.     

Responses of enantioselective characteristics of imidazolinone herbicides and Chiralcel OJ column to temperature variations

Temperature affects not only the chromatographic characteristics of solute but may also alter the conformation of the stationary phase. However, temperature influences on enantioseparation of solute and conformation of chiral stationary phase (CSP) are seldom considered simultaneously. In this study, three temperature programs, a conventional heating procedure, a cyclic van't Hoff program, and a step-temperature program, were employed to evaluate temperature effects on enantioseparation of five imidazolinone herbicides on Chiralcel OJ column and the conformational state of the stationary phase. The van't Hoff plots of retention factor (k'), distribution constant (K) and separation factor (alpha) for imazapyr (1), imazapic (2), imazethapyr (3), and imazamox (4) were linear within 15-50 degrees C. Nonlinear van't Hoff plots of alpha were observed for imazaquin (5) with mobile phase of n-hexane (0.1% TFA)-2-propanol at 70/30 or 60/40 (v/v). The large molecular size of imazaquin (5) and van't Hoff plots of alpha were therefore more sensitive at detecting conformational changes of the stationary phase. Small but irreversible conformational changes occurred at 5-10 degrees C with the solvent ratio of 60/40. During the cyclic van't Hoff program, reversible conformational changes were observed at >or=15 degrees C. A switch was even visible at about 25 degrees C with the solvent ratio of 60/40 during the re-cooling cycle. The cyclic van't Hoff temperature program showed that using OJ column may yield satisfactory results at 15-50 degrees C but not at 相似文献   

Preparation and evaluation of nickelmesogen for micropacked gas chromatography

The preparation and mesomorphic properties of a substituted bis(dithiolene)nickel complex derived from 4, 4'-dimethoxybenzil are reported. The phase transition temperatures were based on data obtained by polarized light microscopy and differential scanning calorimetry. The mesogenic phase existed over the temperature range from 77 to 175 degrees C. A novel micropacked column (1.5 or 3 m x 1 mm i.d.) prepared from the slurry of bis[1,2-bis(4-n-undecyloxyphenyl)ethane-1,2-dithiolene] nickel(II) (5%, w/w), coated on Chromosorb W was applied for the separation of dialkyl sulfides. The non-linearity (discontinuity) of Van't Hoff plots suggests that the liquid crystal property existed even in the coated phase. Factors affecting the retention and the sample selectivity on the prepared column were examined by using a flame photometric detector (FPD). The separation might be based on the mechanism of ligand exchange, shape selectivity and polarity interaction besides the vapor pressure. LOD for the determination of dialkyl sulfides was below 1 ng for most of the analytes.     

阳离子交换色谱中色谱柱温度对碱金属与碱土金属离子保留的影响

研究了非抑制型阳离子交换色谱中色谱柱温度(25~50℃)对碱金属离子(Li+、Na+、K+、Rb+)和碱土金属离子(Mg2+、Ca2+、Sr2+)以及NH4+保留的影响。在Shim-pack IC-C1磺酸型阳离子交换柱上,以硝酸为流动相分离碱金属离子,以乙二胺或乙二胺-草酸(柠檬酸)为流动相分离碱土金属离子,随着色谱柱温度的升高,碱金属和碱土金属离子的保留时间均增长,其范特霍夫曲线具有良好的线性关系,斜率为负值,表明在此条件下碱金属和碱土金属离子的保留为吸热过程。在Shim-pack IC-SC1羧酸型阳离子交换柱上,以硫酸为流动相同时分离碱金属和碱土金属离子,随着色谱柱温度的升高,Mg2+、Ca2+的保留时间增长,而K+、Rb+的保留时间缩短,Li+、Na+、NH4+的保留时间基本不变。在此条件下,Mg2+、Ca2+、K+和Rb+的范特霍夫曲线具有良好的线性关系,其中Mg2+和Ca2+的曲线斜率为负值,K+和Rb+的曲线斜率为正值,表明Mg2+和Ca2+的保留表现为吸热过程,K+和Rb+的保留表现为放热过程。研究表明在不同固定相和流动相条件下,色谱柱温度对碱金属和碱土金属离子保留行为的影响不同。     

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.     

高效液相色谱使用两种类型的纤维素-三(对甲基苯甲酸酯)固定相手性拆分非洛地平的比较

以高效液相色谱手性固定相法对非洛地平(FEL)进行手性拆分。分别采用两种类型的纤维素-三(对甲基苯甲酸酯)手性柱Chiralcel OJ-R和Chiralcel OJ-H进行比较实验,以正己烷-异丙醇(90:10, v/v)为流动相,考察了流动相、柱温对保留及手性拆分的影响。实验显示,两柱拆分FEL的van’t Hoff图均发生了转折,在高温区域为焓驱动,在低温区域为熵驱动。两柱在温度升高时拆分FEL的分离度均提高,其中OJ-H的分离度优于OJ-R。两种手性柱对FEL具有相似的拆分机理。     

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.     

Characterisation of reversed-phase stationary phases for the liquid chromatographic analysis of basic pharmaceuticals by thermodynamic data

This paper describes the characterisation of reversed-phase liquid chromatography (RPLC) columns using thermodynamic measurements. Retention versus 1/T data were used to construct Van't Hoff plots. The slope of these plots indicates the standard enthalpy of transfer of the analyte from the mobile to the stationary phase. The standard entropy can be calculated from the intercept. Van't Hoff plots were linear for the investigated RPLC columns, meaning that for basic analytes over the temperature range studied no changes in the retention mechanism occurred. Enthalpies and entropies of transfer of basic analytes from the mobile to the stationary phase revealed information about the types of interaction of protonated and neutral compounds with the stationary phases. However, a clear view using the present set of basic compounds on how these thermodynamic data may explain the observed substantial differences in peak symmetry cannot be given. It is considered that addition of N,N-dimethyloctylamine (DMOA) to the eluent will results in a dynamically coating of the stationary phase. Addition of DMOA to the eluent resulted for protonated basic compounds in a reduction of both enthalpy and entropy. In practice, with DMOA in the eluent symmetrical peaks were obtained. It is assumed that this is due to blocking residual silanols and/or ion exclusion effects.     

Chromatographic study of terpene-β-cyclodextrin complexes on porous graphitic carbon stationary phase

Summary The effect of temperature on the retention of terpene derivatives on a porous graphitic carbon stationary phase has been investigated with methanol-water mobile phases containing β-cyclodextrin. Changes in Van't Hoff plots were evaluated in relation to the β-cyclodextrin concentration. Thermodynamic data were determined for both (i) solute transfer from the mobile phase to the PGC stationary phase and (ii) complexation of the solute with the β-CD. Enthalpyentropy compensation studies were performed for these two processes. Solute transfer was shown to be enthalpically driven and independent of terpene structure, β-cyclodextrin concentration, and mobile phase methanol content. The complexation reaction was shown to be entropically controlled and independent of terpene structure and mobile-phase composition. Comparison of the compensation temperatures of these two physicochemical processes led to the conclusion that each process had an similar influence on the global retention process.     

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.     

Determination of minor conformational changes of a doxorubicin-peptide conjugate under chromatographic conditions

Thermodynamic analysis of the reversed-phase retention behavior of a doxorubicin-peptide conjugate demonstrated that the degree of non-linearity observed in Van't Hoff plots was impacted by mobile phase acetonitrile content over the 25-38% acetonitrile (v/v) range tested. Small decreases in the non-polar surface area of the doxorubicin-peptide conjugate as a function of temperature were estimated from these data using linear solvent strength relationships, suggesting that the retention behavior may be the result of minor analyte conformational changes during the chromatographic experiment. This hypothesis was supported via circular dichroism (CD), Raman and 1H NMR spectroscopic studies of the doxorubicin-peptide conjugate in selected chromatographic mobile phase compositions. The CD and Raman data indicated small changes to the apparent analyte microenvironment as a function of temperature and bulk solvent environment, while 1H NMR studies specifically demonstrated the environmental sensitivity of protons on three non-polar peptide residues and the proximal aromatic region of the analyte. Together, these data suggest that minor changes to the conformational order of the essentially random structure of the doxorubicin-peptide conjugate are sufficient to impact chromatographic performance.     

Simultaneous separation of nonionic surfactants and polyethylene glycols by reversed phase high performance liquid chromatography

Summary The simultaneous separation of polyethylene glycol and its derivatives such as the lauryl alcohol and lauric acid ethoxylate oligomers was carried out by reversed phase high performance liquid chromatography. Branched fluorinated silica gel columns combined with evaporative light scattering detection were used for the characterization of nonionic surfactants. Lauryl alcohol ethoxylate oligomers were separated at 10°C with an isocratic eluent according to ethoxylate number and the retention time of the oligomers decreases with increasing ethoxylate number. The Van’t Hoff plots of retention factor of lauryl alcohol ethoxylate gave a complex cure, which is anomalous behavior for reversed phase high performance liquid chromatography. The anomalous Van’t Hoff plots were explained by a partial conformational change from polar to less polar conformers with increasing temperature. The most significant features for the analysis of the lauryl alcohol ethoxylate were the use of acetonitrile as mobile phase and operating temperature. The polyethylene glycol was separated according to ethoxylate number and the retention time of oligomers increased with increasing ethoxylate number. The Van’t Hoff plots of retention factor of polyethylene glycol had negative slopes. It was presumed that the polar conformation of the ethylene oxide chain decreased with increasing temperature. The lauryl alcohol ethoxylate and polyethylene glycol were separated simultaneously in gradient elution as a result of the conformational change of the ethylene oxide chain. As a practical example, lauric acid ethoxylate simultaneously separated into free polyethylene glycol, ethoxylate monolaurate and ethoxylate dilaurate in gradient elution.