Non-aqueous Reversed-phase Liquid Chromatography Research Articles

Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy

There is a great need for efficient analysis of the composition of vegetable oils and fats, since it affects the physical and technical properties. However, due to the complex nature of these kind of samples, it is often difficult and costly. In the present study, we developed a Non-Aqueous Reversed-Phase HPLC method that can be used to separate and quantify different free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides and triacylglycerides, including regioisomers such as SOS/SSO and 1,2- and 1,3-diolein. Two 25 cm Nucleodur C18 Isis columns in series, sub-ambient column temperature and a mobile phase gradient composed of acetonitrile, acetic acid, isopropanol and heptane were used for the separation. The lipids were detected and quantified using a charged aerosol detector and it was found that the peak shape highly affected the detector response as well as the response uniformity, even when inverse gradient compensation was employed. Thus, calibration and determination of response factors were necessary for reliable quantification. A correlation between response factors and peak width at half peak height was found and used for quantification of non-calibrated components. A quantification approach was suggested including an appropriate selection of calibrated components, depending on sample composition and the accuracy required. It was shown in a complex oil sample that the reduced calibration approach, using only 6 instead of 33 calibrated components, resulted in virtually the same composition, but yielded a more accurate result compared to using relative area that neglects response factors. The method validation showed good reproducibility and accuracy, making it an excellent tool for extensive analysis of complex lipid mixtures.

Ultra high efficiency/low pressure supercritical fluid chromatography (UHE/LP-SFC) for triglyceride analysis: Identification, quantification, and classification of vegetableoils.

Triglycerides of vegetable oils have been extensively studied. Non-aqueous reversed-phase liquid chromatography and silver-ion chromatography are most frequently used to achieve their separation. In previous works, we presented the use of supercritical fluid chromatography with long columns (75cm) packed with fused-core particles to provide ultra-high-performance separations, with a low-toxicity fluid (carbon dioxide) compared to the usual liquid-phase methods. In the present paper, we describe the quantification of triglycerides with supercritical fluid chromatography and evaporative light-scattering detection. Thanks to the isocratic elution mode, this quantification can be simplified, assuming (a) identical response coefficients for compounds having a close structure, (as only triglycerides are quantified), and (b) constancy of the response coefficient along the analysis (no elution gradient). Therefore, the relative concentrations of triglycerides were easily assessed. Only one calibration curve for one reference compound (in this case triolein) was required. The resulting relative concentrations are in good accordance with the numerous publications available. Relative quantification with UV detection at 210nm is also proposed, facilitated by the very low UV absorption of carbon dioxide and with a calibration curve taking account of the variation of UV response according to double bond number. Nineteen vegetable oils are compared. The identification of triglycerides was carried out based on previous knowledge of these oils, but also with the help of a Goiffon retention diagram, based on the relationship between the logarithm of retention factor and the total double bond number. Finally, cluster analyses were computed, based on evaporative light-scattering detection or UV quantification data. They allow a quick comparison of the triglyceride content between the oils, in the goal to exchange one by the other for certain applications, or to compare a new oil to well-known ones.

Ultra-performance chromatographic methods for enantioseparation of liquid crystals based on lactic acid

Fast and efficient ultra-performance chromatographic methods were developed for enantioseparation of new liquid crystals based on lactic acid. Ultra-performance supercritical fluid chromatography and non-aqueous reversed-phase liquid chromatography were applied for the first time for enantioseparation of this type of analytes. Both techniques proved to be suitable and baseline enantioseparation of all compounds was achieved with tris(3,5-dimethylphenylcarbamate) derivative of amylose as chiral selector. The compounds differentiate in number of benzene rings, length of alkyl side chains and presence of chlorine as lateral substituent and the effect of analyte structure on the enantioseparation was assessed. Significant effect of type of mobile phase modifier was observed in both systems including switching the enantiomer elution order based on both the type of the modifier (both techniques) and its portion in mobile phase (non-aqueous reversed-phase mode only).

Lipidomic analysis of Botryococcus (Trebouxiophyceae, Chlorophyta) - Identification of lipid classes containing very long chain fatty acids by offline two-dimensional LC-tandem MS

Very long chain fatty acids (VLCFAs) were identified in four strains of the green alga Botryococcus braunii (Trebouxiophyceae). The algae contained a series of monoenoic fatty acids up to triacontenoic acid and further VLCFAs in amounts around 1% of total fatty acids. The separation of lipid classes using hydrophilic interaction chromatography revealed that the most abundant VLCFAs (28:2, 28:1 and 28:0) were contained in neutral lipids (triacylglycerols and/or diacylglycerols) and in phospholipids (phosphatidic acid and/or phosphatidylcholine). Using non-aqueous reversed-phase liquid chromatography tandem mass spectrometry (NARP-LC/MS2) of the appropriate collected fractions, molecular species of triacylglycerols containing one or two VLCFAs were described and phosphatidylcholines containing VLCFAs were separated for the first time. Because the presence of Botryosphaerella sudetica (Chlorophyceae) as contaminant of Botryococcus braunii strain Droop 1950/807-1 placed some doubts on the results of previous studies, a strain of this green alga of was also analyzed. In contrast to Botryococcus, C16, a substantially lower proportion of C18 polyunsaturated fatty acids and no VLCFAs were detected in Botryosphaerella.

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.

Non-aqueous reversed-phase liquid-chromatography of tocopherols and tocotrienols and their mass spectrometric quantification in pecan nuts

An easy and effective analytical method was developed for the simultaneous quantification of four tocopherols (Ts) and four tocotrienols (T3s) in three pecan nut cultivars (Stuart, Sioux, and Pawnee). The analytes were separated on a C30 column kept at 15°C, under isocratic non-aqueous reversed-phase (NARP) conditions, in only 18min and detected by atmospheric pressure chemical ionization–tandem mass spectrometry (APCI-MS/MS). The HPLC-APCI-MS/MS method was validated, according to the main FDA guidelines, and then applied for the characterization of the real samples. Analytes were extracted by cold saponification with recoveries greater than 87%. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.3–10μg/100mg and 1–30μg/100mg, respectively. Compared to other nuts, vitamin E composition of pecan nuts (Carya illinoinensis) has only partially been elucidated. Results have evidenced the prevalence of γ-forms for both Ts and T3s and clear quantitative differences of the identified vitamers among the studied cultivars. The richest variety in vitamin E was Sioux with a total content of about∼32mg/100g wet weight, followed by Stuart (∼16mg/100g) and Pawnee (∼9mg/100g).

Identification of triacylglycerols in coix seed extract by preparative thin layer chromatography and liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

Here we reported a methodology for identification of triacylglycerols (TAGs) and diacylglycerols (DAGs) in coix seed by preparative thin layer chromatography (prep-TLC) and non-aqueous reversed-phase liquid chromatography (NARP LC)-atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS/MS). Lipid components were extracted from coix seed by reflux extraction using n-hexane for 3 hr. TAGs and DAGs in coix seed extract were effectively purified and isolated from matrix interferences by prep-TLC and then analyzed by LC-APCI-MS and MS/MS for identification. TAGs were effectively identified taking into consideration of their LC retention behavior, APCI-MS spectra patterns, and MS/MS spectra of [DAG] + ions. In MS/MS spectra of TAGs, diacylglycerol-like fragment [DAG] + ions were useful to identify TAGs with isobaric fragment ions. Based on an established method, 27 TAGs and 8 DAGs were identified in coix seed extract. Among them, 15 TAGs and 8 DAGs were for the first time observed in coix seed. Interestingly, some of TAGs isolated by prep-TLC were partly converted into DAGs through probably photolysis process during storing in room temperature. Thus, degradation phenomenon of TAGs should be considered in the quality evaluation and nutritional property of coix seed. LC-APCI-MS/MS combined with prep-TLC will be practical method for precise TAG and DAG analysis of other herbal plants.

Fast non-aqueous reversed-phase liquid chromatography separation of triacylglycerol regioisomers with isocratic mobile phase. Application to different oils and fats.

The distribution of fatty acid species at the sn-1/3 position or the sn-2 position of triacylglycerols (TAGs) in natural fats and oils affects their physical and nutritional properties. In fats and oils, determining the presence of one or two regioisomers and the identification of structure, where they do have one, as well as their separation, became a problem of fundamental importance to solve. A variety of instrumental technics has been proposed, such as MS, chromatography-MS or pure chromatography. A number of studies deal with the optimization of the separation, but very often, they are expensive in time. In the present study, in order to decrease the analysis time while maintaining good chromatographic separation, we tested different monomeric and polymeric stationary phases and different chromatographic conditions (mobile phase composition and analysis temperature) using Non-Aqueous Reversed Phase Liquid Chromatography (NARP-LC). It was demonstrated that mixed polymeric stationary bonded silica with accessible terminal hydroxyl groups leads to very good separation for the pairs of TAGs regioisomers constituted by two saturated and one unsaturated fatty acid (with double bond number: from 1 to 6). A Nucleodur C18 ISIS percolated by isocratic mobile phase (acetonitrile/2-propanol) at 18°C leads to their separations in less than 15min. The difference of retention times between two regioisomers XYX and XXY are large enough to confirm, as application, the presence of POP, SOP, SOS and PLP and no PPO, SPO, SSO and PPL in Theobroma cacao butter. In the same way, this study respectively shows the presence of SOS, SOP and no SSO, PSO in Butyrospermum parkii butter, POP, SOP, SOS and no PPO, PSO and SSO in Carapa oil and finally POP and no PPO in Pistacia Lentiscus oil.

Regioisomeric and enantiomeric analysis of triacylglycerols

A survey of useful methods for separation and identification of regioisomers and enantiomers of triacylglycerols. Gas chromatography, gas chromatography-mass spectrometry, 13C NMR determination of regioisomers by enzymatic methods, and supercritical fluid chromatography are briefly surveyed, whereas a detailed description is given of the analysis of triacylglycerols by liquid chromatography, especially with silver ion (Ag+; argentation), and nonaqueous reversed phase liquid chromatography. Special attention is paid to chiral chromatography. Details of mass spectrometry of triacylglycerols are also described, especially the identification of important triacylglycerol ions such as [M + H-RCOOH]+ in atmospheric pressure chemical ionization mass spectra.

Sample preparation techniques coupled to advanced chromatographic methods for marine organisms investigation

Objectiveof this work was to develop suitable extraction methodologies for the isolation of lipids from fish, mussels and clams from the Mediterranean sea, and their successive analysis by means of advanced chromatographic instrumentation. More specifically, three different sample preparation methodologies were adopted: Folch’s, Bligh & Dyer’s and maceration. The lipidic extracts, after application of two different methylation procedures, were subjected to monodimensional and comprehensive two-dimensional GC analyses, in order to compare the fingerprints of samples derived from different extraction and transesterification methodologies. Triacylglycerols (TAGs) were analyzed by an off-line combination of silver-ion liquid chromatography with non-aqueous reversed phase liquid chromatography. In both LC and GC analyses, mass spectrometric detectors were used, which greatly supported the identification procedure. In particular, with respect to HPLC, mass spectrometry with atmospheric pressure chemical ionization in positive mode was applied.

Comparison of iso-eluotropic mobile phases at different temperatures for the separation of triacylglycerols in Non-Aqueous Reversed Phase Liquid Chromatography.

Triacylglycerols (TAGs) are a large class of neutral lipids that naturally occur in both plant and animal oils and fats. Their analyses in Non-Aqueous Reversed Phase Liquid Chromatography (NARP) require a mixture of weak solvent (mostly acetonitrile) and strong solvent. In the present work, we have established eluotropic solvent strength scale of several binary mobile phases on C18 bonded silica at different temperatures (acetonitrile/methylene chloride, acetonitrile/acetone, acetonitrile/ethyl acetate, acetonitrile/propan-2-ol, and acetonitrile/butan-1-ol at 25°C, 43°C, 63°C and 85°C); it is based on the methylene selectivity and the use of homologous series. We show that this scale is well suited to the TAGs analysis. The analysis of nine seed oils (Aleurites fordii, Calophyllum inophyllum, Glycina max, Olea europea, Orbignya olifeira, Pinus koraiensis, Pistacia lentiscus, Punica granatum and Ribes nigrum) in iso-eluotropic conditions leads to propose unambiguously the couple MeCN/BuOH at 25°C as the best system to separate TAGs. The use of butanol, as strong solvent, provides very good TAGs congeners separations and avoids the use of chlorinated solvents which gave to this day the best separations.

Rapid separation and characterisation of triacylglycerols in ostrich oil by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

The triacylglycerol (TAG) composition of ostrich oil (Struthio camelus) was determined by non-aqueous reversed-phase ultra-performance liquid chromatography–mass spectrometry. Using a column with 150mm in length, the TAGs were separated efficiently with acetonitrile/2-propanol gradient at flow rate of 0.2mL/min for 20min. Detection is performed by positive APCI mass spectrometry, accurate mass measurements (<3ppm) and definite elemental composition for molecular ions and fragment ions were obtained to enable characterisation of 24 TAGs in both ostrich oil and emu oil. In terms of TAGs composition and contents, the ostrich oils are similar to emu oils. This result indicates that the ostrich oil could be a good choice of substitution for emu oil, which is expensive and comparatively rare, in food, dietetics and cosmetics industry.

A study of the ozonolysis of model lipids by electrospray ionization mass spectrometry

The ozonolysis of vegetable oils has been proposed as step to functionalise unsaturated lipids for the production of bio-based chemicals and materials. The observation of ozonolysis products by mass spectrometry will facilitate mechanistic studies that are essential for the further development of industrial processes based on lipid ozonolysis. Ozonolysis of the model lipids methyl oleate and triolein was performed and samples were taken over a range of reaction times. Ozonlysis products were separated by normal and non-aqueous reversed-phase (NARP) liquid chromatography (LC) coupled to electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole-time of flight instrument. Post-column addition of ammonium acetate solutions aided ionization. Volatile reaction products were observed using gas chromatography/electron impact mass spectrometry (GC/MS). Secondary ozonides, reaction intermediates and previously unreported high molecular weight product dimers were observed as intact molecular ammonium ion adducts under positive ESI. The main fragment ions obtained by MS/MS were from cleavage of the trioxolane group, loss of fatty acyl chain and fragmentation between n-8 and n-9 on the fatty acyl chain. The MS/MS spectra and exact mass measurements explain most of the ozonolysis reaction products. LC/MS, LC/MS/MS and GC/MS results demonstrate that the products from the ozonolysis of the model lipids methyl oleate and triolein are consistent with known ozonolysis reaction pathways. LC/MS techniques using non-aqueous chromatography have permitted the direct observation of mono-, di- and tri-1,2,4-triloxanes which are the secondary ozonides formed by the ozonolysis of triolein. It was also shown that intermediates formed during the ozonolysis of triolein can combine to form high molecular weight ozonide dimers.

Analysis of wax ester molecular species by high performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry

High chromatographic resolution of wax esters (WEs) was achieved by non-aqueous reversed-phase liquid chromatography on a Nova-Pak C18 column by optimising the acetonitrile/ethyl acetate mobile phase gradient. The retention behaviour of WEs was studied in this chromatographic system. The WEs eluted according to their equivalent carbon number (ECN) values; within the group of WEs with the identical ECN, the most unsaturated species tended to elute first. The isobaric WEs with different positions of the ester moiety were separated from each other whenever the lengths of the chains were sufficiently different. The methyl-branched esters eluted at shorter retention times than the straight-chained analogues, and the resolution among methyl-branched WEs depended on the position of the branching. The analytes were detected by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) using data-dependent scanning. WEs provided simple full-scan spectra with abundant protonated molecules and low-intensity fragments. Collision-induced dissociation (CID) promoted identification of the WE molecular species. The responses of WEs were found to be dependent on the number of double bonds and on the alkyl-chain length; the limits of the detection ranged from 20 μmol/L to 200 nmol/L. The HPLC/APCI-MS was applied for the analysis of the WEs isolated from honeycomb beeswax, jojoba oil and human hair. Good agreement between reported results and the literature data was achieved, with several novel polyunsaturated WEs also being found.

Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag(+) after their separation by non-aqueous reversed-phase liquid chromatography (NARP-LC) before MS to improve MS sensitivity. Electrospray ionization-MS (ESI-MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO(3)](+) ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS(5) spectra. The observation of ions produced by LC-MS(5) of on-line Ag(+)-cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS(5) experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils.

Development and optimization of a method for analyzing biodiesel mixtures with non-aqueous reversed phase liquid chromatography

Biodiesel (a mixture of fatty acid esters) is normally analyzed using gas chromatography/flame ionization detection, as specified by the ASTM D6584 and EN14105 standards. This paper proposes a binary gradient method for analyzing biodiesel mixtures using non-aqueous reverse phase HPLC with a UV detector capable of overcoming the drawbacks of the gas chromatographic technique normally used. The new analytical method was developed by means of a statistical sensitivity analysis applied to the main parameters influencing the recording, using the full factorial design method combined with the Yates algorithm and the steepest ascent optimization procedure. The present study shows the influence of the main biodiesel mixture separation analysis parameters. The resulting tool proved valid for analyzing not only biodiesel but also any traces of unreacted oil.

Analysis of triacylglycerols on porous graphitic carbon by high temperature liquid chromatography

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb ®, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30–70 °C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC–ELSD and LC-APCI–MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.

Analysis of triacylglycerols with non-aqueous reversed-phase liquid chromatography and positive ion electrospray tandem mass spectrometry.

A non-aqueous reversed-phase liquid chromatographic method coupled to electrospray ionisation (ESI) tandem mass spectrometry was developed for the analysis of triacylglycerols (TGs). The synthetic TGs studied were separated according to their equivalent carbon number with a gradient of methanol (containing 0.01% (v/v) formate adjusted to pH 5.3 with ammonia) and chloroform. ESI mass spectra of TGs yielded positive ion current signals for [M + NH(4)](+) and [M + NH(4)-RCOONH(4)](+). The mass spectra also showed signals believed to arise from [M + K](+). Collision-induced dissociation (CID) of the [M + NH(4)](+) precursor ion yielded [M + NH(4) - RCOONH(4)](+), [RCO + 74](+) and [RCO](+) product ions as aids for the structural elucidation of the TGs. In addition, [RCO - 18](+) and small amounts of [RCO - 2](+) product ions were also found. The latter ions were observed only for TGs containing unsaturated fatty acids. CID of ammoniated 1-stearoyl-2-oleoyl-3-linoleoyl-glycerol (18:0/18:1/18:2) indicated that neutral loss of the sn-2 fatty acid was energetically less favourable than loss of the fatty acid from the sn-1 or sn-3 position.

Retention behaviour of ceramides in sub-critical fluid chromatography in comparison with non-aqueous reversed-phase liquid chromatography

This study was devoted to the development of an analytical method for ceramide analysis in packed subcritical fluid chromatography (pSubFC). Monofunctional grafted silica support was found to be more suitable for ceramide analysis. Five Kromasil columns were coupled and the parameters, temperature, pressure and percentage of organic modifier in CO 2 were optimised, considering selectivity and analysis time. The final conditions were 31°C, 6% of methanol (MeOH) and 13 MPa. In these conditions the selectivity for structural differences (methylene group, unsaturation or two different bases) were studied. As classically observed, the methylene selectivity decreased with the increase of the eluotropic strength. Moreover, unlike in non-aqueous reversed-phase liquid chromatography (NARP–LC), adding a further unsaturation and two further methylene groups on ceramide results to an increase of retention in pSubFC. Moreover, this last technique allowed to separate ceramides with the same total number of carbons containing unsaturated fatty acids, when the distribution of carbon number of the two chain is very different. These results had enabled to plot retention chart in order to predict ceramide structure in view to identify additional ceramide. This retention chart was finally compared with the one already obtained in NARP–LC.

Retention Behavior of Triglycerides in Octadecyl Packed Subcritical Fluid Chromatography with CO2/Modifier Mobile Phases

A study of the retention behavior of vegetable oil triglycerides was carried out in subcritical fluid chromatography (SubFC) with CO(2)/modifier mobile phases. Analyses of 15 oils have enabled us to set up a retention diagram which includes ∼30 triglycerides. This diagram establishes a relation between triglyceride series retention and their unsaturation number. Furthermore, another relationship between retention and the triglyceride carbon number was studied. The retention properties were identical with those reported in nonaqueous reversed-phase liquid chromatography or in SubFC with neat CO(2): the linearity of these relationships and retention order that follows the partition number. Moreover, specific effects of modifier on these relationships or on retention variations were observed. Using octadecyl packed columns, these particular behaviors underline that the compound solubility is different between modified subcritical phases and nonaqueous liquid phases. Finally, these behaviors can be useful to increase the triglyceride separation and the previous relationships can be applied to improve the identification of unknown triglycerides.