Non-aqueous Reversed-phase Liquid Chromatography Research Articles

Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid–sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.

Comparison of the responses of triacylglycerols with an evaporative visible light scattering detector used in conventional, micro and capillary liquid chromatography

During recent years, there has been a growing interest in the development of packed nano- and microchromatographies. It needs coupling the systems used to detectors adapted for microchromatographies. In the reported results, the properties of two different nebulizers of an evaporative visible light scattering detector have been studied under different flow-rate conditions (conventional HPLC, micro-LC and capillary LC). In micro-LC and capillary LC, it is shown that the calibration curves gained for triacylglycerols analyzed in non-aqueous reversed-phase liquid chromatography could be statistically assimilated to a linear relationship even if they rigorously follow a well-known power relationship such as area= a·mass b . This makes the establishment of the calibration curves easier in miniaturized chromatographies than in conventional ones. Depending on the chromatography used, the linear range falls between either 2–50 ng or 14–80 ng. The limit of quantification was 5 ng ( S/ N=10). On the other hand, a study of the response of modified nebulizer versus flow-rate has shown that the modified evaporative visible light scattering detector is more complex than detectors whose response is really proportional to the concentration or to the mass.

Analysis of commercial ceramides by non-aqueous reversed-phase liquid chromatography with evaporative light-scattering detection

This paper describes the development of a chromatographic system for analysis of commercial ceramides structurally similar to those found in the stratum corneum. The ceramides used in this study contain different amine based (phytosphingosine, sphingosine and dihydrosphingosine) and fatty acids of different chain lengths and with different functional groups (hydroxylated and unsaturated). Non-aqueous reversed-phase (NARP) liquid chromatography with evaporative light-scattering detection (ELSD) were the techniques chosen in accordance with the nature of the ceramides. The eluent strength and the potential selectivity of different organic solvents were investigated. On a C18-bonded silica, the most promising chromatographic conditions employed a gradient from ACN-THF, 95∶5, to ACN-THF-PrOH, 35∶5∶60, in 15 min with a constant concentration of TEA (10 mM) and a stoichiometric amount of formic acid.

Computerized optimization of RP-HPLC separation with nonaqueous or partially aqueous mobile phases

“Osiris” optimization software was usedfor optimizing separations with nonaqueous or partially aqueous mobile phases, consisting of 2, 3 or 4 solvents. For nonaqueous eluents used in Nonaqueous Reversed Phase Liquid Chromatography (NARP-LC), classical transfer rules cannot be used to determine isoeluent mobile phase compositions that are generally the starting point of most of the optimization procedures. The ability of this software to determine isoeluent compositions under NARP conditions is demonstrated. This determination is performed by modeling the solute retention from different sets of two gradient runs, each set being carried out for each binary mobile phase studied. The application of retention models to nonaqueous mobile phases is discussed in terms of the accuracy of the predicted retention times. In addition, it is shown that these sets of gradient runs allow selection of the best composition space for the optimum composition search without requiring additional runs. It is demonstrated that such a selection procedure greatly reduces the number of preliminary experiments.

Determination of coenzyme Q by non-aqueous reversed-phase liquid chromatography

A non-aqueous reversed-phase liquid chromatographic method for determination of coenzyme Q 10 in pharmaceutical formulations has been developed. The reversed-phase system provides better reproducibility and better selectivity for the separation of coenzyme Q 10 analogues and degradation products than studied normal-phase systems. Furthermore, the non-aqueous mobile phase showed a very good solubility and provides a greater variety of work-up solvents for the lipophilic formulations than aqueous mobile phases. Validation studies showed detector response linearity over a concentration range of 0.2–100 μg/ml. The lower limit of detection was 2 ng on-column. The intra-assay precision (relative standard deviation) for a soy bean oil formulation was 2.0% ( n = 11).

Profiling and Quantitation of Bacterial Carotenoids by Liquid Chromatography and Photodiode Array Detection

An analytical method for the profiling and quantitative determination of carotenoids in bacteria is described. Exhaustive extraction of the pigments from four selected bacterial strains required treatment of the cells with potassium hydroxide or liquefied phenol or both before the addition of the extracting solvent (methanol or diethyl ether). The carotenoids in the extracts were separated by nonaqueous reversed-phase liquid chromatography in conjunction with photodiode array absorption detection. The identity of a peak was considered definitive only when both its retention time and absorption spectrum, before and after chemical reactions, matched those of a reference component. In the absence of the latter, most peaks could be tentatively identified. Two examples illustrate how in the analysis of pigmented bacteria errors may result from using nonchromatographic procedures or liquid chromatographic methods lacking sufficient criteria for peak identification. Carotenoids of interest were determined quantitatively when the authentic reference substance was available or, alternatively, were determined semiquantitatively.

Planarity recognition of large polycyclic aromatic hydrocarbons by various octadecylsilica stationary phases in non-aqueous reversed-phase liquid chromatography

Planarity recognition of polycyclic aromatic hydrocarbons has been investigated using bonded octadecyl stationary phases synthesized in different ways. Retention results indicate apparent differences among the functionalities of the stationary phases, a fact found useful for identifying the functionality of commercially available octadecylsilica (ODS) phases. Retention behevior can be explained by the slit-like structures of polymeric oDS phases, as evidenced by suspension13C NMR measurements.

Reversed-phase liquid chromatography of astacene

Carotenoids can be efficiently separated by non-aqueous reversed-phase liquid chromatography on Zorbax ODS. However, astancene, an acidic kwetocarotenoid was found to display a peculiar behaviour in that it could not be eluted from this support using mixtures of acetonitrile—methanol—dichloromethane. The importance of astacene as a degradation product of astaxanthin warranted the development of a new liquid chromatographic system. Organic acids promoted the elution of the compound, but failed to suppress the accompanying excessive peak tailing. Efficient chromatography resulted from the incorporation of bis(2-ethylhexyl) phosphate (BEHP), an acid with a high degree of lipophilicity, in the mobile phase. Optimum separation of astacene and astaxanthin was achieved using methanol-free, semi-aqueous eluents, i.e., acetonitrile—water, containing 0.01—0.05 M BEHP. Increasing amounts of BEHP significantly reduced retention while, unexpectedly, water had little effect over the concentration range studied (1–5%). To illustrate the practical usefulness of the new system, astacene was demonstrated in a saponified extract of flower petals of Adonis annua.

Comparison of retention mechanisms of homologous series and triglycerides in non-aqueous reversed-phase liquid chromatography

The retention behaviour of saturated homogeneous triglycerides was compared to that of single chain homologous compounds in reversed-phase liquid chromatography on silica-based octadecyl bonded phases with non-aqueous binary eluents containing chloroform. For both series, in all eluents and at all investigated temperatures, a discontinuity (1–6% slope change) in log k′ vs. the carbon atom number, n c, of each solute hydrocarbonaceous chain was observed for a given critical number of carbon atoms, n c, crit ≈ 12–13. Similar discontinuities were observed in Δ H° and Δ S° vs. n c for the same n c, crit value. These and other phenomena (existence of two convergence points for log k′ vs. n c plotted at different eluent compositions; existence of two convergence temperatures in log k′ vs. 1/T plotted for several members of a given solute series) reflect the mechanism of penetration of the solute aliphatic chains into the bonded layer. The eluotropic strength of acetonitrile—chloroform mobile phases was determined and seen to have a larger rate of variation with the solvent composition than that of some other non-aqueous binary eluents. The ratio of the slope of plots of log k′ vs n c curves for triglycerides and single chain homologous series is not a whole number but has a value lying between 2 and 3. This explains why triglyceride retentions cannot be predicted from retention data for the fatty acid methyl esters. More importantly, this indicates that the triglycerides may interact with the stationary phase using various conformations, one, two or three chains penetrating into the bonded layer. These conformations, in dynamic equilibrium with each other, contribute differently to the retention. This offers, in principle, the possibility to separate unsaturated triglycerides having the same number of carbon atoms and of double bonds but differing in the distribution of the unsaturations along the chains, if the double bonds located in a non-penetrating chain can selectively interact with a mobile phase component.

Elution behaviour of peropyrene-type polycyclic aromatic hydrocarbons in liquid chromatography

The elution behaviour of peropyrene-type polycyclic aromatic hydrocarbons (PAHs) on variously bonded stationary phases in non-aqueous reversed-phase liquid chromatography has been investigated. The results clearly indicate that the retention behaviour is effected by the degree of planarity of the PAHs and by the properties of the stationary phases used. Solute planarity might also be influenced by solvation. Therefore, these factors combine to determine the retention of large PAHs.

Identification of naphtho [8.1.2 abc] coronene in the extract of diesel particulate matter by non-aqueous reversed-phase liquid chromatography coupled with UV multichannel detector

Non-aqueous reversed-phase liquid chromatography coupled with a UV multichannel detector has been used for the identification of large polycyclic aromatic hydrocarbons in the extract from diesel engine particulate matter. The existence of naphtho[8,1,2,abc]coronene is first confirmed by this technique.

Nonaqueous reversed-phase liquid chromatography and fluorimetry compared for determination of retinol in serum.

A new assay for retinol in human serum, based on nonaqueous reversed-phase liquid chromatography, is presented. Sample preparation includes addition of the internal standard, retinyl propionate, deproteinization of 100 microL of serum with acetonitrile, and extraction with hexane. The standard curve is linear up to 2 mg/L. The assay is characterized by excellent sensitivity (detection limit, 15 micrograms/L) and good within-run and between-run precision (CVs of 2.6 and 2.7%, respectively), and results compare favorably with those by fluorimetry. We assayed 135 samples from hospitalized patients by both methods. Although the two sets of values correlated well (r = 0.955) the fluorimetric method occasionally suffers from interferences. In practice, fluorimetry proves valuable as a routine method, while liquid chromatography meets the criteria of a potential reference method.

Isocratic nonaqueous reversed-phase liquid chromatography of carotenoids

Isocratic nonaqueous reversed-phase liquid chromatography of carotenoids

Non-aqueous reversed-phase liquid chromatography : A neglected approach to the analysis of low polarity samples

It has been found that mnay hydrophobic substances such as hydrocarbons and glyceride oils may not completely elute from a reversed-phase liquid chromatographic column when using semi-aqueous mobile phases containing methanol or acetonitrile. Addition of lower-polarity solvents such as methylene chloride or tetrahydrofuran to the carrier causes rapid elution of these non-polar susbtances. By using a highly retentive reserved-phase packing. Zorbax ODS, it has been demostrated that non-aqueous mobile phases provide an effective way of separating water-insoluble, non-polar substances. The enhanced solubility of compounds in these non-aqueous solvents greatly facilitates the detection of compounds which have poor UV absorbance/fluorescence characteristics.

Non-aqueous reversed-phase chromatography of glycerides using infrared detection

Summary Non-aqueous reversed-phase liquid chromatography has been found to provide an effective means of resolving complex mixtures of low-polarity substances present in glyceride-based oils. The most selective elution conditions from a column containing Zorbax ODS chromatographic packing are obtained with mobile phases formed from methylene chloride or tetrahydrofuran and acetonitrile. Separation of the essentially UV-transparent glyceride-type solutes can be monitored by either differential refractive index or infrared detection. The latter method has the advantage that temperature control of the detector is unneccessary and that the wavelength of operation imparts a degree of specificity to the detection system. By using infrared detection, it has been possible to monitor separations carried out under gradient elution conditions.