Open-tubular liquid chromatography under turbulent and secondary flow conditions

Summary Liquid chromatography in open tubes at flow rates around 0.1 cm · s^–1 and less can generate an enormous number of theoretical plates but suffers from long analysis times. The Golay and van Deemter equations are also obeyed with high accuracy.If straight tubes of circular cross section and very smooth inner surface are used, secondary flow effects can be neglected. Thus the plate height should depend only on the flow velocity and the number of theoretical plates should decrease with increase in linear flow, whereas the number of plates per unit time should be constant.Under turbulent conditions, however, the plate height seems to be more or less independent of linear flow over a wide range. Increasing the flow to several hundred cm · s^–1 causes, for example, 0.2 mm ID capillaries to show the reverse effect and a slight decrease in plate height is found. Alkali treated and silanized capillaries below 100 m i. d. deviate from the Golay prediction before turbulence occurs and a relative decrease in the plate height and thus higher efficiency is found possibly caused be secondary flow effects in the coiled tube or the roughness of the inner walls. These effects allow an increase in the efficiency of given open-tubular systems simply by increasing the solvent velocity drastically. Although plate heights are in the range mm to cm, efficiencies, in theoretical plates per second, similar to classic HPLC, are found. Therefore the quite interesting possibility of overcoming the well-known restrictions of classical HPLC to achieve high efficiency liquid chromatograms exists.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Sorption of catechins under conditions of reverse-phase high-efficiency liquid chromatography

The physico-chemical principles of catechin sorption from various polar solvents onto silica gel modified with octadecyl groups were studied. Thermodynamic characteristics of the sorption were calculated, and the applicability of different models of retention was demonstrated for catechins under the conditions of reverse-phase high-efficiency liquid chromatography.     

A study of benzobisoxazole and benzobisthiazole compounds and polymers under hydrolytic conditions

The stability of benzobisoxazole and benzobisthiazole compounds and polymers under hydrolytic conditions was studied. 2,6-Bis(4-tert-butylphenyl)benzo[1,2-d;4,5-d′]bisoxazole (1) dissolved in acetonitrile containing sulfuric acid and water at 80°C is stable. A suspension of 2,6-bis[4-(2-benzoxazoyl)phenyl]benzo[1,2-d;5,4-d′]bisoxazole (2) in 0.2 N H₂SO₄ or 0.2 N NaOH solution at 100°C for 21 days is stable. The intrinsic viscosity of a poly(p-phenylene)benzobisoxazole (PBO) fiber sample soaked in 0.2 N H₂SO₄, water with 1 wt % polyphosphoric acid (PPA), or 0.2 N NaOH remained the same. Under very severe hydrolytic conditions such as dissolution of compound 2 or PBO in PPA or methanesulfonic acid with residual water followed by coagulation in water, benzobisoxazole underwent bond cleavage to generate carboxylic acid and o-aminophenol functional groups. This is in contrast to an earlier hypothesis that the decrease in intrinsic viscosity under these conditions was due to chain association. Poly(p-phenylene)benzobisthiazole (PBT) also underwent bond cleavage under these very severe conditions, which are unlikely to be encountered in normal applications. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 2637–2643, 1999     

Towards early detection of the hydrolytic degradation of poly(bisphenol A)carbonate by hyphenated liquid chromatography and comprehensive two-dimensional liquid chromatography

The hydrolytic degradation of poly(bisphenol A)carbonate (PC) has been characterized by various liquid chromatography techniques. Size exclusion chromatography (SEC) showed a significant decrease in molecular mass as a result of hydrolytic degradation, while ‘liquid chromatography at critical conditions’ (LC-CC) was very successful for observing differences in functionality due to degradation, i.e. the formation of OH end-groups. To characterize and identify the observed differences semi on-line coupling of liquid chromatography to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Fourier transform infrared spectroscopy (FT-IR) has proved to be very useful.Comprehensive two-dimensional liquid chromatography (2D-LC) was also applied to study the hydrolytic degradation of poly(bisphenol A)carbonate. LC-CC × SEC showed that the formation of poly(bisphenol A)carbonate with OH end-groups occurred over the whole molecular mass range. This information could not be obtained with the separate liquid chromatographic techniques, thereby illustrating the usefulness of 2D-LC.The main degradation processes of poly(bisphenol A)carbonate under the applied hydrolysis conditions turned out to be disappearance of cyclic PC oligomers, chain scission of PC and (subsequent) formation of OH end-groups. FT-IR, SEC and LC-CC have been used to follow the hydrolytic degradation with time. LC-CC proved to be the most promising technique to detect the degradation of poly(bisphenol A)carbonate at an early stage.     

Purification of E. coli 30S ribosomal proteins by high-performance liquid chromatography under non-denaturing conditions

High-performance ion-exchange chromatography was applied to the separation of proteins from the 30S ribosomal subunit under non-denaturing conditions. It was shown that a single chromatographic step only allows the purification of nine proteins. To increase the number of separated proteins, a prefractionation step was added that depends on the physical characteristics of the proteins to be purified. Sixteen out of 21 proteins could be purified by using prefractionation (gel permeation and lithium chloride salt washing). This method is well suited to preparing fresh samples on demand for optical studies owing to the simplicity of the buffers used and the amounts of proteins recovered in the eluted peaks (0.05-0.1 mg/ml).     

High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography

Simvastatin (SS) is an effective cholesterol-lowering medicine, and is hydrolyzed to simvastatin acid (SSA) after oral administration. Due to SS and SSA inter-conversion and its pH and temperature dependence, SS and SSA quantitation is analytically challenging. Here we report a high-throughput salting-out assisted liquid/liquid extraction (SALLE) method with acetonitrile and mass spectrometry compatible salts for simultaneous LC-MS/MS analysis of SS and SSA. The sample preparation of a 96-well plate using SALLE was completed within 20 min, and the SALLE extract was diluted and injected into an LC-MS/MS system with a cycle time of 2.0 min/sample. The seamless interface of SALLE and LC-MS eliminated drying down step and thus potential sample exposure to room or higher temperature. The stability of SS and SSA in various concentration ratios in plasma was evaluated at room and low (4 °C) temperature and the low temperature (4 °C) was found necessary to maintain sample integrity. The short sample preparation time along with controlled temperature (2-4 °C) and acidity (pH 4.5) throughout sample preparation minimized the conversion of SS → SSA to ≤0.10% and the conversion of SSA → SS to 0.00% The method was validated with a lower limit of quantitation (LLOQ) of 0.094 ng mL⁻¹ for both SS and SSA and a sample volume of 100 μL. The method was used for a bioequivalence study with 4048 samples. Incurred sample reproducibility (ISR) analysis of 362 samples from the study exceeded ISR requirement with 99% re-analysis results within 100 ± 20% of the original analysis results.     

Rapid determination of adenine nucleotides in brain tissue by ion-paired reversed-phase column liquid chromatography under isocratic conditions

A rapid method for analysis of adenine nucleotides (AMP, ADP and ATP) in nervous tissue based on ion-paired reversed-phase column liquid chromatography under isocratic conditions is described. An optimal composition of elution buffer was 25 mM potassium phosphate and 4% triethylamine adjusted to pH 6.5 with phosphoric acid. Typical separation time did not exceed 10 min with a 10-cm long compact glass cartridge packed with 5-microns silica C18. The method was employed to determine ATP, ADP and AMP concentrations in rat brain extracts and values thus obtained were compared with those published elsewhere.     

Stability of diazocarbonyl compounds under the conditions of gas chromatography and chromatography-mass spectrometry analysis

The gas chromatographic analysis of alkyldiazoacetates N₂CHCO₂R (R = CH₃ ? C₄H₉), ??-aliphatic diazoketones RCOCHN₂ (R = C₃H₇, C₅H₁₁, and C₉H₁₉), and aryl-substituted diazoketones Ph (CH₂) _n COCHN₂ (n = 0?C2) is shown to be possible when their retention temperatures are below the boiling points of compounds of this series at atmospheric pressure without decomposition (about 140°C). At higher temperatures occurs partial or complete decomposition of ??-diazoketones in chromatographic columns to form ketenes. Among the impurities in the reaction mixtures at the diazotization of corresponding alkyl glycinates were identified for the first time the nitrate esters of glycolic acid O₂NOCH₂CO₂R, as well as the dimeric products. All diazocarbonyl compounds and the impurities were characterized by mass spectra. For the first time their gas chromatographic retention indices were determined.     

Comparative study on camphor enantiomers behavior under the conditions of gas-liquid chromatography and reversed-phase high-performance liquid chromatography systems modified with alpha- and beta-cyclodextrins

The dependence of retention and selectivity parameters of camphor enantiomers on the concentration of alpha- and beta-cyclodextrins were studied under conditions of GLC (matrix solvent: Glycerol, 95 degrees C) and RP-HPLC (matrix solvent: Aqueous methanolic, 20 degrees C). It has been found that beta-cyclodextrin forms complex of 1:1 stoichiometry and does not recognize enantiomers of camphor. In contrast alpha-CD forming complexes of 1:2 stoichiometry appeared to be very efficient chiral selector of (+) and (-)-camphor. Relatively considerable differences have been observed between stability constants determined by GLC and RP-HPLC, what may be explained by the various natures of the matrix solvents and the various temperatures of the measurements. On the contrary, the enantioseparation factor alpha observed at higher concentrations of alpha-cyclodextrin stabilizes on the very similar value alpha+/-(GLC) approximately = alpha-/+(HPLC) approximately = 1.6. Simple theoretical considerations focusing on the differences in the mechanisms of the studied processes have been performed. According to them the enantiomer forming the more stable complex with the cyclodextrin should be eluted from the RP-HPLC column first and GLC column last. This fact has been confirmed experimentally.     

High-performance liquid chromatography separation of enantiomers of flavanone and 2'-hydroxychalcone under reversed-phase conditions

A reversed-phase high-performance liquid chromatography (HPLC) method was developed for evaluating the chiral discrimination ability of Chiralpak IA chiral stationary phase (CSP) towards flavanone. The effect of the nature and pH buffer as well as nature of alcohol modifier on enantioselectivity was investigated. Comparative study of enantioseparation in reversed-phase and polar organic conditions indicated a significative improvement in resolution when aqueous-based eluents were used. The developed reversed-phase chromatographic method was able to separate the enantiomers of flavanone from its isomeric form, the 2'-hydroxychalcone. The stereochemical stability of flavanone was studied by classical off-column HPLC kinetic procedures in aqueous and non-aqueous media. It was clearly demonstrated that the 2'-hydroxychalcone was involved as intermediate in the on-column and off-column enantiomerization process of flavanone.     

Liquid chromatography of polyoxyethylenes under critical conditions: a thermodynamic study

Polyethylene glycols (PEG), fatty alcohol ethoxylates (FAE) and fatty acid polyglycol esters were analyzed by liquid chromatography under critical conditions (LCCC) on typical reversed phase columns in mobile phases consisting of methanol-water and acetone-water in the region of the critical adsorption point (CAP) for the oxyethylene unit. Critical conditions can be adjusted by varying the mobile phase composition or temperature. In methanol-water the temperature dependence is almost negligible, while it is much more pronounced in acetone-water. Critical conditions for the oxyethylene unit were realized on octadecyl phases in methanol-water containing 85.8 wt.% methanol and in acetone-water at 89.6 wt.% acetone. In the van't Hoff plots the logarithm of the distribution coefficient was plotted versus the reciprocal temperature. In all plots straight lines were found, from slope and intercept of which the entropy and enthalpy changes were calculated. The relation of the thermodynamic parameters -deltaG/RT, -deltaH/RT, and deltaS/R to the numbers of A and B chain units in block copolymers AB and BAB at the CAP for A is studied both theoretically and experimentally. Various approaches for obtaining the critical temperature are discussed.     

Predicting the behaviour of polydisperse polymers in liquid chromatography under isocratic and gradient conditions

In this paper we describe how the existing theories to describe retention and peak width in isocratic and gradient-elution liquid chromatography can be expanded to describe the retention behaviour of natural and synthetic repetitive polymers, which feature distributions of molecules with different masses (and often different structures) rather than unambiguous molecular formulas. For polydisperse samples, it is vital that the model accommodates (isocratic) elution of sample components before the onset of a gradient, elution during the gradient, and elution after the completion of the gradient. The expanded models can readily be implemented in standard spreadsheet software, such as Excel. We have created such spreadsheets based on the conventional model for retention in reversed-phase liquid chromatography (RPLC) and on two different models for retention in normal-phase liquid chromatography. The implementation allows an easy visualization of the theoretical concept. Up to three different polymeric series can be entered, with a total of up to 100 peaks being computed and displayed in isocratic or gradient-elution chromatograms. Also visualized are "retention models" (diagrams of isocratic retention vs. composition) and "calibration curves" (retention or elution composition vs. molecular mass or degree of polymerization). The coefficients in the isocratic retention model may be correlated, as has often been observed in RPLC. It is shown that under certain conditions such a correlation corresponds to the existence of so-called critical (isocratic) conditions, at which all the members of a given polymeric series (same composition and end groups, different number of repeat units) show co-elution.     

Stability study and quantitative determination of mebeverine hydrochloride in tablets by means of reversed-phase high-performance liquid chromatography

Summary A rapid and specific reversed-phase high-performance liquid chromatographic method (RPHPLC) is described for the determination of mebeverine hydrochloride in tablets. Elution was performed on an octyl silane column with a methanol-water mixture (75-25), containing 0.05% hexylamine as silanol-blocking agent, adjusted to pH 5.0 with phosphoric acid. The method gave accurate, precise and reproducible results. The mean recovery of the drug from six synthetic tablet mixtures was 100.0% with a relative standard deviation (RSD) of 0.94%. In order to test the specificity of the method, the interference of the degradation compounds of mebeverine hydrochloride and of the intermediates from the synthesis was investigated. None of them did interfere. By means of mass spectrometry and UV-spectrophotometry, the degradation compounds of mebeverine were identified as veratric acid and as 4-|ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino| 1-butanol. The stability study proved that mebeverine hydrochloride is very stable in tablets; the tablets still contain more than 95% of the declared drug potency after storage for more than one year at 50°C.Colofac; Duspatal; Duspatalin     

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.