Non-aqueous Reversed-phase Chromatography Research Articles

Determination of heavy polycyclic aromatic hydrocarbons by non-aqueous reversed phase liquid chromatography: Application and limitation in refining streams

The heavy polycyclic aromatic hydrocarbons (HPAHs) cause detrimental effects to hydrocracker operations by deactivating the catalysts and depositing in the downstream of the reactor/ exchangers. Therefore, it is essential to continuously monitor the accumulation of HPAHs in a hydrocracker unit. To accurately measure the concentration of HPAHs, the development of a fast and reliable analytical method is inevitable. In this work, an analytical method based on non-aqueous reversed phase chromatography in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was developed. As a first step, five different types of stationary phases were evaluated for the separation of HPAHs in non-aqueous mode and the best suited phase was further used for the fractionation of HPAHs in a fractionator bottom sample obtained from a refinery hydrocracker unit. The eight major fractions or peaks obtained from the separation were further characterized by UV spectroscopy and FT-ICR MS and the compounds in the fractions were tentatively confirmed as benzoperylene, coronene, methylcoronene, naphthenocoronene, benzocoronene, dibenzoperylene, naphthocoronene and ovalene. The developed liquid chromatography method can be easily adapted in a refinery laboratory for the quantitation of HPAHs in hydrocracking products. The method was further tested to check the interference of sulfur aromatics and/or large alkylated aromatic hydrocarbons on the determination of HPAHs in hydrocracking products.

Selection and Identification of Molecular Gold Clusters at the Nano(gram) Scale: Reversed Phase HPLC-ESI-MS of a Mixture of Au-Peth MPCs.

Recent advances in cluster synthesis make it possible to produce an enormous variety molecule-like MPCs of size, composition, shape, and surface-chemical combinations. In contrast to the significant growth in the synthetic capability to generate these materials, progress in establishing the physicochemical basis for their observed properties has remained limited. The main reason for this has been the lack of the analytical capability to generate and measure samples of suitably high (molecular) purity; such capability is also essential to support therapeutic and diagnostic MPC development. In order for MPC products to get to market, especially those products that are medical-field related, characterization is required to identify and quantify all components present in a material mixture. Here, we show results from analysis of several synthetic mixtures of gold MPCs by nonaqueous reversed-phase chromatography coupled with mass spectrometry detection. The additional or hidden components, revealed to be present in these mixtures, provide novel insights into their comparative stability and interactions.

Capillary Liquid Chromatography Mass Spectrometry Analysis of Intact Monolayer-Protected Gold Clusters in Complex Mixtures

In some respects, large noble-metal clusters protected by thiolate ligands behave as giant molecules of definite composition and structure; however, their rigorous analysis continues to be quite challenging. Analysis of complex mixtures of intact monolayer-protected clusters (MPCs) by liquid chromatography mass spectrometry (LC-MS) could provide quantitative identification of the various components present. This advance is critical for biomedical and toxicological research, as well as in fundamental studies that rely on the identification of selected compositions. This work expands upon the separate LC and MS results previously achieved, by interfacing the capillary liquid chromatograph directly to the electrospray source of the mass spectrometer, in order to provide an extremely sensitive, quantitative, and rapid means to characterize MPCs and their derivatives far beyond that of earlier reports. Here, we show that nonaqueous reversed-phase chromatography can be coupled to mass-spectrometry detection to resolve complex mixtures in minute (∼100 ng) samples of gold MPCs, of molecular masses up to ∼40 kDa, and with single-species sensitivity easily demonstrated for components on the level of sub-10 ng or picomole (1 pmol).

A Simultaneous Analytical Method to Profile Non-Volatile Components with Low Polarity Elucidating Differences Between Tobacco Leaves Using Atmospheric Pressure Chemical Ionization Mass Spectrometry Detection

Summary A comprehensive analytical method using liquid chromatography atmospheric pressure chemical ionization mass spectrometry detector (LC/APCI-MSD) was developed to determine key non-volatile components with low polarity elucidating holistic difference among tobacco leaves. Nonaqueous reversed-phase chromatography (NARPC) using organic solvent ensured simultaneous separation of various components with low polarity in tobacco resin. Application of full-scan mode to APCI-MSD hyphenated with NARPC enabled simultaneous detection of numerous intense product ions given by APCI interface. Parameters for data processing to filter, feature and align peaks were adjusted in order to strike a balance between comprehensiveness and reproducibility in analysis. 63 types of components such as solanesols, chlorophylls, phytosterols, triacylglycerols, solanachromene and others were determined on total ion chromatograms according to authentic components, wavelength spectrum and mass spectrum. The whole area of identified entities among the ones detected on total ion chromatogram reached to over 60% and major entities among those identified showed favorable linearity of determination coefficient of over 0.99. The developed method and data processing procedure were therefore considered feasible for subsequent multivariate analysis. Data matrix consisting of a number of entities was then subjected to principal component analysis (PCA) and hierarchical clustering analysis. Cultivars of tobacco leaves were distributed far from each cultivar on PCA score plot and each cluster seemed to be characterized by identified non-volatile components with low polarity. While fluecured Virginia (FCV) was loaded by solanachromene, phytosterol esters and triacylglycerols, free phytosterols and chlorophylls loaded Burley (BLY) and Oriental (ORI) respectively. Consequently the whole methodology consisting of comprehensive method and data processing procedure proved useful to determine key-components among cultivars of tobacco leaves, and was expected to additionally expand coverage that metabolomics study has ensured. [Beitr. Tabakforsch. Int. 27 (2016) 60-73]

Search for Non-Volatile Components with Low Polarity Characterizing Tobacco Leaves Using Liquid Chromatography / Atmospheric Pressure Chemical Ionization Mass Spectrometry Detector

SUMMARY There has been focus on the components with low polarity in tobacco leaf resin due to their probable relation with taste and aroma of tobacco products, the lack of a feasible analytical method and instrument has long been an obstacle to identifying the components with low polarity. The author thereby paid attention to the analysis employing nonaqueous reversed-phase chromatography hyphenated with a photo diode array detector and an atmospheric pressure chemical ionization mass spectrometry detector which has been considered applicable to the separation of significant but unknown non-volatile. The application succeeded in simultaneously separating, detecting and quantifying more than 100 non-volatile components with different low polarity such as solanesols, triacylglycerols, phytosterols, and chlorophylls. However, their compositional differences among various tobacco leaves still remained partial knowledge based on targeted analysis instead of global knowledge based on comprehensive analysis. No investigation searching for key components elucidating different tastes, aromas, species, cultivars, curing processes, and growing districts among tobacco leaves has been carried out so far. For this reason, all the quantification data were consolidated to form complete multidimensional matrix and were statistically processed to observe the categories and the key components of various tobacco leaves by principal component analysis and hierarchical clustering analysis. Tobacco leaves were first classified into three categories consisting of flue-cured Virginia, air-cured leaf, and Oriental. Solanesyl esters, phytosteryl esters and solanachromene contributed to the category of flue-cured Virginia, while air-cured leaf was characterized by free phytosterols. Oriental was featured by chlorophyll in addition to the contributory components to flue-cured Virginia. Non-volatile components with low polarity seemed to be degraded during curing process and to therefore characterize the different curing processes among various tobacco leaves.

A method for simultaneous analysis of phytosterols and phytosterol esters in tobacco leaves using non aqueous reversed phase chromatography and atmospheric pressure chemical ionization mass spectrometry detector

While numerous analytical methods for phytosterols have been reported, the similar polarity and large molecules of phytosterol esters have made the methods lengthy and complicated. For this reason, an analytical method that could completely separate phytosterol esters including the higher fatty acids such as palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid in addition to phytosterols without preliminary separation was developed. The separation was accomplished by non-aqueous reversed phase chromatography technique using only acetone and acetonitrile. An atmospheric pressure chemical ionization/mass spectrometry detector configured at selected ion monitoring mode was hyphenated with the separation system to detect phytosterols and phytosterol esters. Twenty-four types of these were consequently separated and then identified with their authentic components. The calibration curve was drawn in the range of about 5 to 25,000ng/mL with a regression coefficient over 0.999. The limit of detection and limit of quantification, respectively, ranged from 0.9 to 3.0ng/mL and from 3.0 to 11.0ng/mL. Recovery rates ranged from 80 to 120%. The quantification results were subjected to statistical analysis and hierarchical clustering analysis, and were used to determine the differences in the amounts of phytosterols and phytosterol esters across tobacco leaves. The newly developed method succeeded in clarifying the whole composition of phytosterols and phytosterol esters in tobacco leaves and in explaining compositional differences across the variety of tobacco leaves.

Expanded separation technique for chlorophyll metabolites in Oriental tobacco leaf using non aqueous reversed phase chromatography

An improved separation method for chlorophyll metabolites in Oriental tobacco leaf was developed. While Oriental leaf still gives the green color even after the curing process, little attention has been paid to the detailed composition of the remaining green pigments. This study aimed to identify the green pigments using non aqueous reversed phase chromatography (NARPC). To this end, liquid chromatograph (LC) equipped with a photo diode array detector (DAD) and an atmospheric pressure chemical ionization/mass spectrometer (APCI/MSD) was selected, because it is useful for detecting low polar non-volatile compounds giving green color such as pheophytin a. Identification was based on the wavelength spectrum, mass spectrum and retention time, comparing the analytes in Oriental leaf with the commercially available and synthesized components. Consequently, several chlorophyll metabolites such as hydroxypheophytin a, solanesyl pheophorbide a and solanesyl hydroxypheophorbide a were newly identified, in addition to typical green pigments such as chlorophyll a and pheophytin a. Chlorophyll metabolites bound to solanesol were considered the tobacco specific components. NARPC expanded the number of detectable low polar chlorophyll metabolites in Oriental tobacco leaf.

A novel method for analyzing solanesyl esters in tobacco leaves using atmospheric pressure chemical ionization/mass spectrometer

A direct and simple method for analyzing solanesyl esters found in tobacco leaves was developed. Sample preparation was performed by accelerated solvent extractor 200 (ASE200™) using n-hexane followed by evaporating solution in vacuo and dissolving residue with acetone. The separation of analytes was conducted through high-performance liquid chromatography (HPLC) equipped with an SIL-C18/5C™ column and the non-aqueous reversed phase chromatography (NARP) technique using acetone and acetonitrile as the mobile phase with a linear gradient. Atmospheric pressure chemical ionization/mass spectrometer (APCI/MS) in positive mode was used to detect solanesyl esters in the following conditions: capillary voltage 4000 V, corona current 10 μA, drying gas flow 5 mL/min, fragmentor voltage 200 V, nebulizer pressure 60 psi, and vaporizer temperature 500 °C. Each solanesyl ester was identified by the comparison of analyte with synthesized solanesyl esters. Quantification was conducted by selected ion monitoring (SIM) mode in order to detect the specific product ion (613.6 m/ z) fragmented from solanesyl ester. The calibration curve was made in the range of 0.1–40 μg/mL with a regression coefficient over 0.999 on almost all solanesyl esters. The limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.01 to 0.05 μg/mL and from 0.03 to 0.15 μg/mL, respectively, on the SIM mode of MS for quantification. Recovery (%) ranged from about 80 to 120%. The direct quantification using the developed method succeeded in showing a different amount and composition of solanesyl esters among various tobacco leaves.

Analysis of triacylglycerols in fat body of bumblebees by chromatographic methods

Triacylglycerols (TAGs) from the fat body of several bumblebee species ( Bombus lucorum, B. terrestris, B. lapidarius, B. hypnorum, B. hortorum, and B. confusus) were studied using chromatographic techniques. Semi-preparative thin-layer chromatography was used to isolate the TAGs from the tissue extract. Gas chromatography (GC) enabled us to identify the fatty acids (FAs) that form bumblebee TAGs and to quantify their relative proportions. The TAGs were subsequently analysed by high-performance liquid chromatography–atmospheric pressure chemical ionisation mass spectrometry. Two chromatographic systems, including non-aqueous reversed-phase chromatography and silver ion chromatography on cation exchange resin in silver (I) ionic form, were optimised and their performance compared. The most abundant fatty acids in bumblebees TAGs contained 18 or 16 carbon atoms; oleic acid predominated in most samples. TAGs were found to be a complex mixture of isomers; some of them, e.g. OLnO, PLnO, PoPoO, PoPoP, POO, or OOO (where Po is palmitoleic, P is palmitic, Ln is linolenic, and O is oleic acid) were abundant in particular species. The composition of both FAs and TAGs was found to be species-specific. Only minor differences were found among specimens of the same species.

Use of principal component analysis for investigation of factors affecting retention behaviour of ceramides on porous graphitized carbon column

The potentiality of a porous graphitized carbon stationary phase was assessed for ceramide analysis in nonaqueous reversed phase chromatography with an evaporative light scattering detection. The influence of the nature of the mobile phase was first performed under isoeluotropic isocratic conditions. Eight mobile phase compositions in the presence or absence of triethylamine/formic acid were tested with a set of structurally relevant ceramides. Values obtained under these conditions for the peak heights, retention factors, plate numbers, asymmetry factors, relative retentions and Time-Corrected Calibrated Normalised Resolution Product were analysed by Principal Component Analysis. Principal Component Analysis was effective in attributing relationships between the chromatographic behaviour and analytical conditions. The relationship between the increase of the detector response and the presence of triethylamine/formic acid has been confirmed, and the main change of selectivity has been attributed to interactions provided by the weak solvent of the mobile phase. These conclusions have allowed the development of gradient conditions for the analysis of complex ceramide mixtures on porous graphitized carbon column.

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography.

Influence of the modifiers on the nature of the stationary phase and the separation of carotenes in sub-critical fluid chromatography

Sixteen solvents were tested as modifiers in the separation of α- and β-trans/cis carotenes by sub-critical fluid chromatography. The results obtained indicate that the retention behaviour is related to solubility parameters and the dielectric constant of the solvents. The study of the selectivity of thetrans/cis isomers of β-carotene leads to a better understanding of the role of the solvents in determining the nature of the stationary phase at the mobile phase interface. The results obtained point out the difference in the conformation of the alkyl chains with three classes of solvents: methanol, acetonitrile, and THF or methylene chloride. The analogy between non aqueous reversed-phase chromatography (NARP) and sub-critical fluid chromatography has also been discussed.

Effect of the sample solvent on band profiles in preparative liquid chromatography using non-aqueous reversed-phase high-performance liquid chromatography

If a solvent with a higher elution strength than the mobile phase is used to dissolve poorly soluble samples in preparative chromatography, significant deformation of the band profiles occurs, especially when the column is overloaded. Eventually, band splitting may take place. This behavior was observed in non-aqueous reversed-phase chromatography of cholesterol and other low-polarity sample compounds dissolved in a non-polar solvent, such as dichloromethane, at concentrations exceeding their solubilities in the mobile phase, a higher polarity solvent such as acetonitrile. In this chromatographic system, the dependence of the band deformation and of its splitting on the volume and concentration of the sample, the composition of the mobile phase and the column temperature were investigated. A model taking into account the dependence of the solubility of the sample components in the sample solvent and in the eluent and of the equilibrium isotherm of these components between the stationary and mobile phases on the composition of these two solvents (eluent and sample solvent) was worked out. The possible formation of supersaturated solutions was also considered in the model. This model permits the computer simulation of the band profiles for a single compound or for mixtures dissolved in a solvent different from the mobile phase. The results of the simulations are in qualitative agreement with the behavior observed experimentally.

Separation of trans/ cis- α- and β-carotenes by supercritical fluid chromatography : I. Effects of temperature, pressure and organic modifiers on the retention of carotenes

A partial separation of the isomers of α- and β-carotenes has previously been achieved by non-aqueous reversed-phase chromatography on octadecyl silica columns eluted with mixtures of hexane and polar modifiers. We show that improved results are obtained by the use of mobile phases based on liquid or supercritical carbon dioxide. Polar modifiers have similar effects on chromatographic selectivity in the non-aqueous reversed-phase and supercritical fluid chromatography systems, suggesting that the retention mechanism is the same in both cases (predominantly partition). Withthe carbon dioxide-based mobile phases, it was possible to optimize chromatographic efficiency by varying the temperature and pressure. The use of both binary and ternary mixtures was investigated.

Determination of polymeric hindered amine light stabilizers in polyolefins by high-performance liquid chromatography

Thin-layer and high-performance liquid chromatography procedures were used for the identification and quantitative determination of the polymeric hindered amine light stabilizers Chimassorb 944 and Cyasorb UV 3346 in polyolefins. After extraction from the polymer by the appropriate solvents, the hindered amine light stabilizers solution was qualitatively tested by thin-layer chromatography and successively analysed with a separation technique described as size-exclusion non-aqueous reversed-phase chromatography using traditional high-performance liquid chromatography equipment. The stationary phase was a non-polar styrene—divinylbenzene copolymer. The mobile phase was tetrahydrofuran containing diethanolamine. Both hindered amine light stabilizers were eluted at times ranging from 10 to 19 min and detected by UV at 239 nm, also showing a specific molecular mass distribution of their telomers. Interference due to other stabilizers was determined by a second elution of the same extraction solution after selective treatment on silica-gel cartridge.

Qualitative and quantitative changes in the carotenoids during development of the brine shrimp Artemia.

In order to study the biological fate of all-trans- and cis-canthaxanthin in the brine shrimp Artemia, a quantitative method was developed for the determination of both carotenoids and their metabolic precursors in encysted embryos (cysts), nauplii, whole animals, organs, and subcellular fractions. This method is based on nonaqueous reversed-phase chromatography, two new exhaustive extraction procedures, and the determination of proteins in the extracted residue. Hydration of Artemia cysts caused a reversible conversion of part of the all-trans- to cis-canthaxanthin. During further pre-emergence embryonic development, there was little change in the levels of both isomers. After hatching of cysts, cis-canthaxanthin was progressively isomerized to the all-trans form, while the total (all-trans + cis) canthaxanthin to protein ratio tended to remain constant. Cis-canthaxanthin rapidly became undetectable in animals fed on algae and reappeared in females at an advanced stage of the reproductive cycle. All-trans-canthaxanthin remained present during the whole Artemia life cycle in addition to its metabolic precursors echinenone and beta-carotene. The carotenoid distribution in organs and subcellular fractions indicated high affinity of cis-canthaxanthin for the female reproductive system, oocytes in general, and yolk in particular. A role for cis-canthaxanthin is suggested at an early developmental stage, i.e., in cysts, before hatching.

Triglyceride analysis by combined argentation/nonaqueous reversed phase high performance liquid chromatography

AbstractFull analysis of triglycerides of natural fats and oils has been investigated by the combination of argentation high performance liquid chromatography (HPLC) with nonaqueous reversed phase (NARP) HPLC. An infrared detector was used in argentation HPLC, because it indicated molar responsibility to all triglycerides. After peak trapping with argentation HPLC, each triglyceride fraction was analyzed with NARP chromatography using the glyceride‐selective post‐column reactor detector. The results of the analyses of triglycerides of palm oil and cocoa butter by the proposed method agreed well with those reported earlier.

Nonaqueous Reverse Phase Liquid Chromatographic Determination of Vitamin D2 in Multivitamin Tablets, Using Vitamin D3 as Internal Standard

The technique of nonaqueous reverse phase chromatography has been successfully applied to the separation of lipophilic vitamins D2 and D3. Separation is achieved with a Zorbax-ODS column and a mobile phase of 10% methanol in acetonitrile. A predetermined amount of vitamin D3 internal standard is added to a portion of a powered tablet sample, which is then extracted and partitioned with a mixture of dimethyl sulfoxide, water, methanol, and hexane. A portion of the hexane extract is concentrated, and purified by passing it through a disposable silica cartridge to remove interfering materials. The method should also be applicable to formulations containing vitamin D3 where vitamin D2 can then be used as the internal standard. A system suitability test is included in the method.

Application of Gel Permeation Chromatography and Nonaqueous Reverse Phase Chromatography to High Performance Liquid Chromatographic Determination of Retinyl Palmitate and α-Tocopheryl Acetate in Infant Formulas

A high performance liquid chromatographic (HPLC) method was developed for determining retinyl palmitate and alpha-tocopheryl acetate in infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in a solvent mixture of isopropanol and methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using high pressure gel permeation chromatography (HP-GPC) followed by quantitation using non-aqueous reverse phase (RP)-HPLC. Three muStyragel (100A) columns connected in series were used for HP-GPC fractionation of sample extracts in methylene chloride. A Zorbax ODS (6 micron) column and methylene chloride-acetonitrile-methanol (30 + 70 + 0.2) were used for RP-HPLC quantitation. The 32 commercial infant formulas that were analyzed represent a wide variety of formulations manufactured at various fortification levels. Vitamin A results ranged from 89 to 242% of the declared levels. Vitamin E values, determined as the supplemental form alpha-tocopheryl acetate, ranged from 83 to 272% of the declared levels. Determination of vitamin A in 6 samples and vitamin E in one sample by this method and the official AOAC method gave comparable results. This method, which requires no saponification, was successfully used to determine vitamins A and E in ready-to-use, liquid concentrated, and powdered infant formulas.

Applications of reversed-phase chromatography and nephelometric detection to analysis of non-polar mixture at microgram level

Non-aqueous reversed-phase chromatography with light-scattering detection is suitable for analysis of various non-polar mixtures. Microprecipitations are conveniently used in solute recovery for further investigations by auxillary chromatographic or spectral techniques. Described applications include determination of the triglyceride and fatty acid composition of vegetable oils, hydrocarbons in petroleum wax, and terpenoids in natural mixtures.