Function Of Column Temperature Research Articles

Introducing Multifactorial Peak Crossover in Analytical and Preparative Chromatography via Computer-Assisted Modeling.

Modern pharmaceutical processes can often lead to multicomponent mixtures of closely related species that are difficult to resolve under chromatographic conditions, and even worse in preparative scale settings. Despite recent improvements in column technology and instrumentation, there remains an urgent need for creating innovative approaches that address challenging coelutions of critical pair and poor chromatographic productivity of purification methods. Herein, we overcome these challenges by introducing a simple and practical technique named multifactorial peak crossover (MPC) via computer-assisted chromatographic modeling. The approach outlined here focuses on mapping the separation landscape of pharmaceutical mixtures to quickly identify spaces of peak coelution crossings which enables one to conveniently switch the elution order of target analytes. Diverse examples of MPC diagrams as a function of column temperature, mobile phase gradient or a multifactorial combination in reversed phase and ion exchange chromatography (RPLC and IEC) modes are generated using ACD Laboratories/LC Simulator software and corroborated with experimental data match (overall retention time differences of less than 1%). This powerful MPC technique allows us to gain massive productivity increases (shorter cycle time and higher sample loading) for purification of pharmaceuticals by selectively switching the elution order of target components away from undesired tailing peaks and coelution spaces. MPC chromatography dramatically reduces the time spent developing productive analytical and preparative scale separations. In addition, we illustrate how this new MPC concept can be used to gain substantial improvements of the signal-to-noise ratio, enabling straightforward ppb detection of low-level target components with direct impact in the quantitation of metabolites and potential genotoxic impurities (PGIs). These innovations are of paramount importance in order to facilitate efficient isolation, characterization, and quantitation of drug substances in the development of new medicines.

A System Map for the Ionic Liquid Stationary Phase 1,9-Di(3-vinylimidazolium)nonane Bis(trifluoromethylsulfonyl)imide

The solvation parameter model is used to characterize the retention properties of the ionic liquid stationary phase 1,9-di(3-vinylimidazolium)nonane bis(trifluoromethylsulfonyl)imide (SLB-IL100) over the temperature range 60–200 °C. The results are presented in the form of a system map comprising a continuous plot of the system constants of the solvation parameter model determined at 20 °C intervals as a function of column temperature. The ionic liquid stationary phase is a cohesive, polar solvent with significant hydrogen-bonding capability. It differs from polar siloxane-based and poly(ethylene glycol) stationary phases by being a moderate hydrogen-bond acid and is shown to possess separation properties that are not duplicated in a comprehensive database of system constants for commercially available open-tubular columns.

HPLC study of the host–guest complexation between fluorescent glutathione derivatives and β-cyclodextrin

RP-HPLC and the van’t Hoff law were used to study the association in which β-cyclodextrin forms inclusion complexes with aminothiol–phthaldialdehyde derivatives prepared from either glutathione (GSH) or γ-glutamylcysteine (γ-glucys) and either naphthalene-2,3-dicarboxaldehyde (NDA) or o-phthaldialdehyde (OPA). Elution was carried out at pH 8.5, the derivatization pH which gave the highest fluorescence signal during batch experiments. The variation of the retention factor (k) was monitored as a function of column temperature (10–35 °C) and β-cyclodextrin concentration (0–5 mM) in the mobile phase. Apparent binding constants, enthalpy and entropy were calculated from van’t Hoff plots for the complexation reaction. These data lay the groundwork for the improvement of high throughput GSH quantification methods using fluorimetry in biological and vegetal samples.

Evaluation and prediction of the shape of gas chromatographic peaks

The evaluation and prediction of the shape of asymmetric gas chromatographic peaks is important as the knowledge of the amount of tailing permits to foresee the resolution between closely eluting peaks and to select the best analytical conditions for an efficient and rapid separation. A model function was tested in order to approximate the true peak shape obtained on non-polar column by injecting different compounds. The trend of the parameters involved in the used equation has been investigated as a function of column temperature and inlet pressure. The reproduction of the symmetrical or asymmetrical shape of gas chromatographic peaks is satisfactory and the method also permits to predict the shape of peaks obtained in different conditions of temperature and pressure.

Kinetic analysis and subambient temperature on-line on-column derivatization of an active aldehyde

The chromatographic analysis of aldehydes under typical reversed-phase conditions may be a challenging task due to an equilibrium process leading to the formation of a gem diol species regardless of acidic or basic conditions. Initially, a reversed-phase HPLC gradient elution was developed to determine the amount of an acetylenic aldehyde in a reaction mixture. Significant fronting was observed under acidic and basic conditions even at −5 °C. In order to circumvent this problem, a reversed-phase HPLC gradient method on a C 18 column at subambient temperature was developed using diethylamine as a mobile phase additive for on-line on-column derivatization of the aldehyde moiety. The on-line on-column reaction rate for the derivatization of the aldehyde with diethylamine was determined as a function of column temperature. An Arrhenius plot was constructed and the activation energy was calculated. The chromatographic behavior of the derivatized acetylenic aldehyde and products formed in-situ in the chromatographic system were studied at various temperatures ranging from −10 to 60 °C. It was found that the reaction products could be controlled by adjusting the column temperature. Different reaction pathways were identified as a function of temperature. The products and the reaction pathways were characterized by NMR, LC–MS and UV spectra.

Effect of column temperature on the behaviour of some angiotensin converting enzyme inhibitors during high-performance liquid chromatographic analysis

The chromatographic behaviour of the ACE inhibitors lisinopril, enalapril and its two degradation products, enalaprilat (hydrolytic degradation product) and diketopiperazine (DKP) (cyclization degradation product) was studied as a function of column temperature and pH of the mobile phase. The rate of isomerization (which influences the peak shape or even peak splitting during chromatographic analysis) increases with temperature. The shape of the chromatographic peak for enalapril, enalaprilat and lisinopril is also pH dependent. At high temperature (80°C) and low pH (pH=2) all studied compounds appear on the chromatogram as a narrow chromatographic peak. Chromatographic peaks become broader or they split by lowering the column temperature. Enalapril appears at 6°C on the chromatogram in two peaks which belong to its cis- and trans-rotation isomers. Separation of the rotamers was confirmed by NMR spectroscopy.

Retention study on silica gel in high-temperature liquid chromatography (HTLC)

Retention volumes (V N) of different kinds of test compound have been measured on silica gel as a function of column temperature (T) in the range 20 to 210°C.n-Heptane and mixtures ofn-heptane and polar modifiers were used as mobile phases. With nC7 and nC7-1,4-dioxane mixtures, there was a good linear relationship between InV N and 1/T for column temperatures above 100 °C. When more polar modifiers such as butanol, ethanol and acetic acid were used, ‘N’-shaped curves were obtained for the plots of InV N against 1/T. Possible explanations of this retention behavior have been proposed on the basis of related studies and common physical chemistry relationships. Column equilibration time after alteration of water content or column temperature is greatly reduced at elevated temperatures. This will benefit the application of LSC on silica gel.

Conformer Interconversion in the LC Analysis of Triostin A and Its Under-N-Methylated Synthetic Analogue

Multiple peak formation and interconversion in the liquid chromatographic (LC) analysis of triostin A and its under-N-methylated synthetic analogue, [N-MeCys3,N-MeCys7]-TANDEM (MCTANDEM), are investigated as a function of column temperature. Slow interconversion between chromatographic peaks, ascribed to the presence of the n- and p-solution conformers of the peptides, is exhibited in the normal-phase elution profiles of triostin A and in the reversed-phase elution profiles of MCTANDEM. A chromatographic model is developed to estimate the kinetics of conformer interconversion. Reversed-phase LC analysis of the n- and p-conformers of MCTANDEM yields a value of 0.01/s for the apparent interconversion rate constant (kn-p) at 25 degrees C, with a corresponding activation energy of 16 kcal/mol. Normal-phase LC analysis of the n- and p-conformer interconversion of triostin A dissolved in chloroform results in a value of 0.04/s for kn-p at 25 degrees C, with a corresponding activation energy of 18 kcal/mol. For triostin A, normal-phase LC findings as a function of column temperature are compared with 1H nuclear magnetic resonance (NMR) line-width measurements between 80 degrees C and 140 degrees C for the n- and p-conformers, which yield an activation energy of 19 kcal/mol and an extrapolated value of 0.02/s at 25 degrees C for kn-p in deuteriochloroform.

Effect of temperature on the separation of DNA restriction fragments in capillary gel electrophoresis

Two different separation modes were used in high-performance capillary gel electrophoresis to study the effect of temperature on the separation of DNA restriction fragments, isoelectrostatic (use of constant applied electric field) and isorheic (use of constant current). In both instances, the migration properties and resolution of the DNA molecules were studied as a function of column temperature between 20 and 50°C. In the isoelectrostatic separation mode, the migration time and resolution decrease as temperature increases. In the isorheic separation mode, increasing the column temperature results in a maximum migration time for all of the DNA fragments and shows a maximum resolution for the lower molecular weight fragments (<200 kilodalton).

Prediction of thermodynamic retention behaviour in reversed-phase liquid chromatography with aqueous eluents and various concentrations of methanol by using the solubility concept model

The retention of a series of non-polar solutes in reversed-phase liquid chromatography was studied as a function of column temperature and mobile phase composition using methanol—water mixtures. The enthalpy change on transferring a solute from a stationary phase to a mobile phase was predicted from physicochemical parameters of the solute on elution with methanol—water between 40:60 and 100:0 by using the solubility concept model.

Resolution of triglycerides in capillary SFC as a function of column temperature

Resolution of triglycerides in capillary SFC as a function of column temperature

Mobile phase effects in reversed-phase chromatography IV. Retention by n-alkylbenzenes as a function of column temperature and the nature and concentration of organic co-solvent

Mobile phase effects in reversed-phase chromatography IV. Retention by n-alkylbenzenes as a function of column temperature and the nature and concentration of organic co-solvent