Reversed-phase chromatography also called RPC , reverse-phase chromatography , or hydrophobic chromatography includes any chromatographic method that uses a hydrophobic stationary phase. In the s, most liquid chromatography was performed using a solid support stationary phase also called a column containing unmodified silica or alumina resins. This type of technique is now referred to as normal-phase chromatography. Since the stationary phase is hydrophilic in this technique, molecules with hydrophilic properties contained within the mobile phase will have a high affinity for the stationary phase, and therefore will adsorb to the column packing. Hydrophobic molecules experience less of an affinity for the column packing, and will pass through to be eluted and detected first.

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An assessment of the retention behaviour of polycyclic aromatic hydrocarbons on reversed phase stationary phases : selectivity and retention on C 18 and phenyl-type surfaces. In this manuscript the retention and selectivity of a set of linear and non-linear PAHs were evaluated on five different reversed-phase columns. Overall, the results revealed that the phenyl-type columns offered better separation performance for the linear PAHs, while the separation of the structural isomer PAHs was enhanced on the C 18 columns.

The Propyl-phenyl column was found to have the highest molecular-stationary phase interactions, as evidenced by the greatest rate of change in 'S' 0. Interestingly, the Synergi polar-RP column, which also is a phenyl stationary phase behaved more ' C 18 -like' than 'phenyl-like' in many of the tests undertaken. This is probably not unexpected since all five phases were reversed phase.

A mixed-mode chromatographic stationary phase , C 18 -DTT dithiothreitol silica SiO2 was prepared through "thiol-ene" click chemistry.

The obtained material was characterized by fourier transform infrared spectroscope, nitrogen adsorption analysis and contact angle analysis. Chromatographic performance of the C 18 -DTT was systemically evaluated by studying the effect of acetonitrile content, pH, buffer concentration of the mobile phase and column temperature. Additionally, the stability and column-to-column reproducibility of the C 18 -DTT material were satisfactory, with relative standard deviations of retention factor of the tested analytes verapamil, fenbufen, guanine, tetrandrine and nicotinic acid in the range of 1.

Finally, the application of C 18 -DTT column was demonstrated in the separation of non-steroidal anti-inflammatory drugs, aromatic carboxylic acids, alkaloids, nucleo-analytes and polycyclic aromatic hydrocarbons. All rights reserved. Reversed-phase HPLC analysis of levetiracetam in tablets using monolithic and conventional C 18 silica columns. Development and validation of an RP-HPLC method for determination of levetiracetam in pharmaceutical tablets is described.

The separation and quantification of levetiracetam and caffeine internal standard were performed using a single analytical procedure with two different types of stationary phases , conventional Phenomenex Gemini C 18 x 4.

The analyte peaks were detected at nm using a diode array detector with adequate resolution. Validation studies were performed using the method recommended by the International Conference on Harmonization, the U.

Levetiracetam and caffeine were detected in about 7 min using the conventional column, whereas less than 5 min was required when the monolithic column was used. Calibration plots had r values close to unity in the range of 0. Assay of levetiracetam in a tablet formulation was demonstrated as an application to real samples.

Effect of pressure on the selectivity of polymeric C 18 and C30 stationary phases in reversed-phase liquid chromatography. Increased separation of isomeric fatty acid methyl esters, triacylglycerols, and tocopherols at high pressure. With respect to isomeric C 18 FAMEs with one cis-double bond, ODS-P phase was effective for recognizing the position of a double bond among petroselinic methyl 6Z-octadecenoate , oleic methyl 9Z-octadecenoate , and cis-vaccenic methyl 11Z-octadecenoate , especially at high pressure, but the differentiation between oleic and cis-vaccenic was not achieved by C30 phase regardless of the pressure.

A monomeric C 18 phase InertSustain C 18 was not effective for recognizing the position of the double bond in monounsaturated FAME, while the separation of cis- and trans-isomers was achieved by any of the stationary phases. For some isomer pairs, the ODS-P and C30 provided the opposite elution order, and in each case higher pressure improved the separation. The two stationary phases showed selectivity for the isomers having rigid structures, but only the ODS-P was effective for differentiating the position of a double bond in monounsaturated FAMEs.

The results indicate that the improved isomer separation was provided by the increased dispersion interactions between the solute and the binding site of the stationary phase at high pressure.

C 18 columns for the simultaneous determination of oxytetracycline and its related substances by reversed-phase high performance liquid chromatography and UV detection. Simultaneous determination of oxytetracycline, 4-epioxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline on C 18 columns has been accomplished using a high performance liquid chromatographic method with UV detection. These columns were equilibrated with mobile phases consisted of methanol-acetonitrile The flow rate was 1.

Both methods were applied to oxytetracycline raw material, human and veterinary formulations, where the excipients did not interfere. External standard calibration curves were linear for 4-epioxytetracycline, oxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline in the concentration range of 0.

Concerning the first column, limits of detection and quantification of the above compounds were in the concentration ranges of nM and nM, respectively, whereas on the second column these ranges became nM and nM, respectively. A chromatographic estimate of the degree of surface heterogeneity of reversed-phase liquid chromatography packing materials II-Endcapped monomeric C 18 -bonded stationary phase.

The effect of the length of the bonded alkyl chain was investigated. PubMed Central. The procyanidins the most common type of proanthocyanidin or condensed tannin from cell suspension cultures derived from cotyledons of Douglas Fir have been compared with those isolated from leaves of strawberry and avocado.

Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at Higher temperature Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase Inertsil C30 at A monomeric C 18 stationary phase did not show any separation at Published by Elsevier B.

Extrathermodynamic interpretation of retention equilibria in reversed-phase liquid chromatography using octadecylsilyl-silica gels bonded to C1 and C 18 ligands of different densities.

First, the four tests proposed by Krug et al. These tests showed that a real EEC of the retention equilibrium originates from substantial physico-chemical effects. The new model indicates how the slope and intercept of the LFER are correlated to the compensation temperatures derived from the EEC analyses and to several parameters characterizing the molecular contributions to the changes in enthalpy and entropy.

The estimated K values are in agreement with the corresponding experimental data, demonstrating that our model is useful to explain the variations of K due to changes in the RPLC conditions. Extension of the carotenoid test to superficially porous C 18 bonded phases , aromatic ligand types and new classical C 18 bonded phases.

The recent introduction of new stationary phases for liquid chromatography based on superficially porous particles, called core-shell or fused-core, dramatically improved the separation performances through very high efficiency, due mainly to reduced eddy diffusion. However, few studies have evaluated the retention and selectivity of C 18 phases based on such particles, despite some retention order change reported in literature between some of these phases.

The carotenoid test has been developed a few years ago in the goal to compare the chromatographic properties of C 18 bonded phases. Based on the analysis of carotenoid pigments by using Supercritical Fluid Chromatography SFC , it allows, with a single analysis, to measure three main properties of reversed phase chromatography stationary phases : hydrophobicity, polar surface activity and shape selectivity.

Previous studies showed the effect of the endcapping treatment, the bonding density, the pore size, and the type of bonding monomeric vs. It was applied to ten ODS superficially porous stationary phases , showing varied chromatographic behaviors amongst these phases. As expected, due to the lower specific surface area, these superficially porous phases are less hydrophobic than the fully porous one.

In regards of the polar surface activity residual silanols and to the shape selectivity, some of these superficially porous phases display close chromatographic properties Poroshell , Halo C 18 , Ascentis Express, Accucore C 18 , Nucleoshell C 18 on one side and Aeris Wide pore, Aeris peptide and Kinetex XDB on the other side , whereas others, Kinetex C 18 and Halo peptide ES C 18 display more specific ones.

Besides, they can be compared to classical fully porous phases , in the goal to improve method transfer from fully to superficially porous particles. By the way, the paper also report the extension of. For the first time, we synthesized multiple embedded polar groups EPGs containing linear C 18 organic phases. To correlate the NMR results with temperature-dependent chromatographic studies, standard reference materials SRM b and SRM e , a column selectivity test mixture for liquid chromatography was employed.

A single EPG containing the C 18 phase was also prepared in a similar manner to be used as a reference column especially for the separation of basic and polar compounds in reversed-phase liquid chromatography RPLC and hydrophilic interaction liquid chromatography HILIC , respectively. Detailed chromatographic characterization of the new phases was performed in terms of their surface coverage, hydrophobic selectivity, shape selectivity, hydrogen bonding capacity, and ion-exchange capacity at pH 2.

Furthermore, peak shapes for the basic analytes propranolol and amitriptyline were studied as a function of the number of EPGs on the C 18 phases in the RPLC. Evaluation of the phase ratio for three C 18 high performance liquid chromatographic columns. In addition, the boundary depends not only on the nature of the stationary phase , but also on the composition of the mobile phase.

The results obtained in the study provide further support for the new procedure for the evaluation of phase ratio. Effects of elevated temperature and mobile phase composition on a novel C 18 silica column.

A novel polydentate C 18 silica column was evaluated at an elevated temperature under acidic, basic, and neutral mobile phase conditions using ACN and methanol as the mobile phase organic modifier. The temperature range was degrees C. The maximum operating temperature of the column was affected by the amount and type of organic modifier used in the mobile phase. Under neutral conditions, the column showed good column thermal stability at temperatures ranging between and degrees C in methanol-water and ACN-water solvent systems.

At pH 2 and 3, the column performed well up to about degrees C at two fixed ACN-buffer compositions. Under basic conditions at elevated temperatures, the column material deteriorated more quickly, but still remained stable up to degrees C at pH 9 and 60 degrees C at pH The results of this study indicate that this novel C 18 silica-based column represents a significant advancement in RPLC column technology with enhanced thermal and pH stability when compared to traditional bonded phase silica columns.

Labeled carbon dioxide C 18 O2 : an indicator gas for phase II in expirograms. Carbon dioxide labeled with 18O C 18 O2 was used as a tracer gas for single-breath measurements in six anesthetized, mechanically ventilated beagle dogs. C 18 O2 is taken up quasi-instantaneously in the gas-exchanging region of the lungs but much less so in the conducting airways. Its use allows a clear separation of phase II in an expirogram even from diseased individuals and excludes the influence of alveolar concentration differences.

Phase II of a C 18 O2 expirogram mathematically corresponds to the cumulative distribution of bronchial pathways to be traversed completely in the course of exhalation. The derivative of this cumulative distribution with respect to respired volume was submitted to a power moment analysis to characterize volumetric mean position , standard deviation broadness , and skewness asymmetry of phase II.

Position is an estimate of dead space volume, whereas broadness and skewness are measures of the range and asymmetry of functional airway pathway lengths. The effects of changing ventilatory patterns and of changes in airway size via carbachol-induced bronchoconstriction were studied. Increasing inspiratory or expiratory flow rates or tidal volume had only minor influence on position and shape of phase II.

Under all circumstances, position of phase II by power moment analysis and dead space volume by the Fowler technique agreed closely in our healthy dogs. Overall, power moment analysis provides a more comprehensive view on phase II of single. Preparative separation of the polar part from the rhizomes of Anemarrhena asphodeloides using a hydrophilic C 18 stationary phase. The goal of this study was to develop a method that utilized a hydrophilic C 18 stationary phase in the preparative high performance liquid chromatography to isolate the polar part from the rhizomes of Anemarrhena asphodeloides.

The results showed that an initial mobile phase of pure water for the separation could greatly increase the retention and solubility of the polar compounds at the preparative scale. Introducing polar groups on the surface of the hydrophilic C 18 column together with the use of optimized mobile phase compositions improved the column separation selectivity for polar compounds.

Eleven previously undescribed compounds in Anemarrhena asphodeloides were obtained, indicating that the method developed in this study would facilitate the purification and separation of the polar part of traditional Chinese medicines. Pterins are a class of potential cancer biomarkers. New methods involving hydrophilic interaction liquid chromatography HILIC and reversed phase RP high-performance liquid chromatography have been developed for analysis of eight pterin compounds: 6,7-dimethylpterin, pterin, 6-OH-methylpterin, biopterin, isoxanthopterin, neopterin, xanthopterin, and pterincarboxylic acid.

Finally, LiChrospher C8 RP column with fluorescence detection was selected for further validation of the method. The first phase , bridge ethylene hybrid BEH , is neutral at all pHs whereas the second, charged surface hybrid CSH , contains a protonated ligand at W W pH C 18 at 20mV and by the subsequent decrease of the equilibrium constant of weak adsorption sites C 18 environment and removal of the strong adsorption sites accessible silanols.

The experimental data were then successively fitted to an empirical heterogeneous Langmuir-Moreau LM explicit isotherm. A new analytical methodology for the determination of fully methoxylated flavones FMFs in citrus juices is described.

Signals at wavelengths , , and nm bandwidth 4 nm are simultaneously acquired, stored, plotted, and integrated.


Reversed-phase chromatography

Introduction The reversed phase of proteins and peptides in TFA is the standard method of choice for initial method development. Too often no other buffers are ever tried. If the separation of closely related molecules is the goal, the buffer systems listed below should be tried with a single column and solvent gradient before further method development is started. This holds true for analytical and preparative method development. Also, if the final method needs to be validated and subsequently pass FDA inspection and only TFA has been investigated, then the FDA inspector may decide that the validation package will not be acceptable until other buffer systems have been tried.







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