Silver-ion Liquid Chromatography Research Articles

Impact of column temperature on triacylglycerol regioisomers separation in silver ion liquid chromatography using heptane-based mobile phases

This work presents the investigation of the use of heptane as an alternative and less toxic mobile phase to the most used hexane for triacylglycerols (TAGs) analysis in silver ion high-performance liquid chromatography (Ag+-HPLC). The impact of column temperature (in the 5 °C-35 °C range) on the retention and resolution of five pairs of regioisomers relevant for the confectionery industry was investigated using a heptane-based mobile phase modified with acetonitrile (ACN). The retention behaviour was compared for a standard TAG mixture and an interesterified cocoa butter. The temperature effect previously observed with hexane-based mobile phases was confirmed for this new system, and it was also observed that the ACN concentration had an important impact on the strength of the temperature effect, with a higher ACN concentration leading to a lesser impact of temperature on the TAGs’ elution behaviour. In general, the study allowed to conclude on the equivalence of hexane and heptane for TAGs regioisomers separation in Ag+-HPLC, independently of the used temperature or the ACN concentration. In addition, the applicability of heptane-based mobile phases for the separation of TAGs regioisomers was demonstrated on three other confectionary fat samples, namely palm olein, interesterified palm olein, and interesterified shea olein.

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.

Identification of Conjugated Linoleic Acid (CLA) Isomers by Silver Ion-Liquid Chromatography/In-line Ozonolysis/Mass Spectrometry (Ag+-LC/O3-MS)

A novel method for the identification of conjugated linoleic acid (CLA) isomers has been developed in which silver ion liquid chromatography is coupled to in-line ozonolysis/mass spectrometry (Ag(+)-LC/O3-MS). The mobile phase containing CLA isomers eluting from the Ag(+)-LC column flows through a length of gas-permeable tubing within an ozone rich environment. Ozone penetrating the tubing wall reacts with the conjugated double bonds forming ozonolysis product aldehydes. These, and their corresponding methanol loss fragment ions formed within the atmospheric pressure photoionization (APPI) source, were detected by in-line MS and used for the direct assignment of double bond positions. Assignment of positional isomers is based entirely on the two pairs of diagnostic ions seen in the in-line O3-MS mass spectra. In this way, de novo identification of CLA positional isomers, i.e. without requiring comparison to CLA standards, was achieved. The Ag(+)-LC/O3-MS method was applied to the analysis of CLA isomers in a commercial CLA supplement, milk fat, and the lipid extract from a Lactobacillus plantarum TMW1460 culture. The results demonstrate how Ag(+)-LC/O3-MS can be used for the direct and fast determination of CLA isomers at low concentrations and in complex lipid mixtures.

The Content of Conjugated Linoleic Acid (CLA) Isomer Groups in Milk of two Polish Sheep Breeds Determined by Silver Ion Liquid Chromatography (Ag+-HPLC)

Sheep milk is rich in CLA isomers which are biologically active components influencing human health. There are four geometric CLA isomer pairs: cis, trans; trans, cis; trans, trans and cis, cis. The aim of the present study was the analysis of CLA isomer groups content by Ag+-HPLC in milk fat of Zelazneńska (ZS) and Wrzosówka (WS) sheep breeds. The ewes of both breeds were kept under the same environmental and nutritional conditions. Milk samples were collected from 60 suckling ewes (30 from each breed), at the age of 3-4 years and in their 4th week of lactation. A higher total amount of all CLA isomer groups was obtained in milk of ZS ewes, however, this result was statistically insignificant. The percentage of the main c9, t11 isomer in total CLA was higher in Wrzosówka milk (68% vs. 74%). The content of the trans, trans isomer group in milk fat of the studied breeds was similar. The percentage of this group in total CLA in milk of WS and ZS constitutes 7.2% and 7.7%, respectively. The amount of cis,cis isomers in milk fat of ZS was higher thanWS (P < or = 0.01).

Extraction, Characterization and In Vivo Neuromodulatory Activity of Phytosterols from Microalga Dunaliella Tertiolecta

In recent years, a great deal of research has been devoted to identify new natural sources of phytosterols and to improve methods for their recovery and purification. In this regard, unexplored natural sources of bioactive ingredients are gaining much attention since they can lead to the isolation of new compounds or bioactivities. The field of available natural sources has been further increased by including algae and, even more interestingly, microalgae. In the present study, a multidisciplinary approach has been used considering, in an integrated view, extraction, chemical composition and bioactivity of phytosterols from the microalga Dunaliella tertiolecta. A novel methodology to extract, separate and characterize microalgal-derived phytosterols has been developed. In addition, recoverable and reusable eluents have been selected in order to reduce the quantities of employed organic solvents. Finally, we addressed the question whether orally administered phytosterols reach the brain and if those interfere with the major neurotransmitter systems, such as the dopaminergic, serotoninergic and noradrenergic ones, in several brain areas of rats. Flash Liquid Chromatography has been used to separate the Total Sterol (TS) fraction, composed of twelve sterols, with a purity of 97.87% and a recovery percentage of 98%, while the "flash version" of Silver Ion Liquid Chromatography has been used to purify the most abundant phytosterols in TS, (22E,24R)- methylcholesta-5,7,22-trien-3β-ol (ergosterol) and (22E,24R)-ethylcholesta-5,7,22-trien-3β-ol (7-dehydroporiferasterol), with a purity of 97.4%. These two combined methods did not need sophisticated technologies but only cheap laboratory supplies. Moreover, the possibility of recovering and recycling the solvents used as eluents made it a cleaner process. Finally, for the first time, a neuromodulatory action of Dunaliella tertiolecta-derived phytosterols has been found in selective brain areas of rats.

Separation of unsaturated organic compounds using silver–thiolate chromatographic material

Separation of organic compounds containing various numbers of double bonds (DB) can be readily achieved by using silver ion impregnated silica gel, often called silver-ion or argentation chromatography. However, the practical application of silver-ion liquid chromatography in analytical and preparative separations has been limited by the concerns about the stability and mobility of silver ions and the widespread use of reversed phase high performance liquid chromatography. Silver covalently anchored onto the thiol moiety of mercaptopropyl modified silica gel has been tested for the separation of polycyclic aromatic hydrocarbons by ring numbers, but has never been shown to separate mixtures of alkenes having different number of double bonds. We report here that silver–thiolate chromatographic material (AgTCM; including, but not limited to, silver(I) mercaptopropyl silica gel) is also highly efficient in liquid chromatographic separation of alkane/alkenes differing by one double bond. AgTCM displays exceptionally high selectivity for unsaturated compounds and high stability under extended heat and light exposure, while silver is virtually immobile during solvent elution. Compared to ionic silver, silver–thiolate interacts with double bonds less strongly, allowing AgTCM to efficiently separate olefins using less polar (and often less viscous and lower cost) solvents. The interaction energy between silver and ethylene is calculated using established computational methods and the results are in full agreement with our experimental results. Importantly, the exceptional stability of AgTCM gives rise to much higher compound recovery than conventional silver-ion silica gel during the chromatographic elution. Our results pave the way for the development of novel covalently bonded, transition metal-containing chromatographic materials.

On-line two dimensional liquid chromatography/mass spectrometry for the analysis of triacylglycerides in peanut oil and mouse tissue

Triacylglycerides (TAGs) are a large class of complex neutral lipids that naturally occur in both plants and animals. In the present work, an on-line comprehensive silver-ion liquid chromatography (silver-ion LC)×reversed-phase liquid chromatography (RPLC) system was constructed to analyze these compounds. A micro bore silver-ion modified column was employed in the first dimension with the commonly used hexane-based mobile phase. After a series of C18 columns were assessed, a wide bore column packed with 1.5μm particles was selected as the second dimension column to reduce the negative effect caused by the large volume and strong solvent injection in the second dimension. The system coupled with mass spectrometry was applied to the analysis of an edible peanut oil and a mouse liver extract. Twenty-eight TAGs from the peanut oil and forty-four from the mouse liver were identified based on the TAGs’ retention behaviors on the comprehensive two-dimensional LC system and their APCI MS fragments.

A new internal standard for HPLC assay of conjugated linoleic acid in animal tissues and milk

A new method for the quantification of underivatized conjugated linoleic acid (CLA) isomers and CLA-metabolites by silver ion liquid chromatography (Ag&lt;sup&gt;+&lt;/sup&gt;-HPLC) with photodiode array detection (DAD) is described. Conjugated fatty acids (CFA) and sorbic acid as the internal standard (IS) were separated on two 5 &amp;mu;m Chrompac ChromSpher Lipids columns (250 &amp;times; 4.6 mm). Biological samples were hydrolyzed with 1M KOH in methanol and 2M KOH in water at room temperature for 12 h. Hydrolyzates were acidified and the free fatty acids were extracted with dichloromethane. The organic solvent was removed and then the residue was re-dissolved in hexane and centrifuged. The supernatant was injected onto the columns. The mobile phase of 1.6% acetic acid and 0.0125% acetonitrile in hexane was chosen as the optimum mobile phase for fractionation of IS, CLA isomers and CLA-metabolites in all assayed biological samples. The use of two silver ion-exchange columns with direct UV detection (Ag&lt;sup&gt;+&lt;/sup&gt;-HPLC-DAD) offers satisfactory precision of the IS quantification and low limits of detection of IS and CLA isomers (0.60 and 0.21&amp;ndash;0.35 ng, respectively). The presented simple Ag&lt;sup&gt;+&lt;/sup&gt;-HPLC-DAD method with sorbic acid as the IS can be used for direct determination of underivatized CLA isomers in specimens of animal origin. &amp;nbsp;

Structure and absolute configuration of an unsaturated anteiso fatty acid from Bacillus megaterium

Gas chromatography in combination with electron ionization mass spectrometry (GC/EI-MS) was used to determine the fatty acids of a membrane lipid from Bacillus megaterium. Special attention was put on the structure and absolute configuration of a monoenoic fatty acid previously described in this sample. GC/EI-MS operated in the selected ion monitoring mode was used to determine twelve fatty acids in the bacterium. Methyl esters were prepared to verify the presence of a 14-methylhexadecenoic acid (a17:1) isomer. The position of the double bond of the a17:1 isomer and four further monoenoic fatty acids was elucidated by means of their picolinyl esters produced by the transesterification of the phospholipid. For the a17:1 isomer, the double bond was located between C-5 and C-6. Silver ion liquid chromatography was used to verify that the double bond was in cis-configuration. The bacterial 14-methylhexadec-5-enoic acid (a17:1Δ5) is chiral due to the stereogenic C-14 carbon. Initial enantioselective measurements were carried out with isomers of a17:1Δ5 which were available in form of racemic and ( S)-enantiopure cis- and trans-isomers of a17:1Δ12 previously synthesized. The cis-a17:1Δ12 enantiomers were partly resolved on a chiral stationary phase coated with 50% heptakis(6-O- tert.-butyldimethylsilyl-2,3-di- O-methyl)-β-cyclodextrin in OV-1701 (β-TBDM). However, resolution of the enantiomers of the trans-isomer of a17:1Δ12 failed. Only one peak was also observed for the a17:1Δ5 isomer from B. megaterium. Thus, it remained unclear whether the compound a17:1Δ5 was racemic or enantiopure in the sample. To clarify this point, we separated the cis-monoenoic fraction from the saturated fatty acids. Then, the monoenoic fraction was hydrogenated in order to transform a17:1Δ5 into 14-methylhexadecanoic acid (a17:0). This chiral fatty acid was known to be sufficiently enantioseparated on the β-TBDM column and was found to be ( S)-enantiopure in the sample. Hence, these measurements verified that the B. megaterium sample contained enantiopure ( S)-a17:1Δ5.

The influence of dietary conjugated linoleic acid isomers and selenized yeast on the fatty acid profile of the spleen, pancreas and kidneys of rats

The influence of 1.2 ppm dietary selenium added in the form of selenized yeast (Se-yeast) and of a mixture of conjugated linoleic acid (CLA) isomers added to a level of 1 or 2%, of cis9trans11CLA (c9t11) added to 1%, and of trans10cis12CLA (t10c12) added to 1% on the body and organ gains of rats and on the concentration of CLA isomers and other fatty acids in the spleen, pancreas and kidneys was investigated. Total fatty acids in the studied organs were separated as methyl esters using long-capillary gas-liquid chromatography with flame-ionization detection, while nonmethylated fatty acids containing conjugated double bonds were examined using silver-ion liquid chromatography with fotodiode detection. Free fatty acids were obtained by mild saponification, whereas their methyl esters, by base- and acid-catalyzed methylation. Feeding t10c12 and Se-yeast resulted in the highest increase in rat growth and organ gain. All diets enriched in CLA isomer(s) significantly elevated the concentration of CLA isomers in the organs; the highest concentration of CLA isomers was found in the pancreas, the smallest, in the spleen. The addition of Se to diets containing CLA isomer(s) usually showed a statistical tendency or increase in the concentration of CLA isomers in the organs. Our results demonstrate that t10c12 was metabolized faster than c9t11 in the pancreas and kidneys, whereas the addition of Se-yeast to the diets with CLA isomer(s) reduced the rate of t10c12 metabolism. A 2% dietary content of a mixture of CLA isomers or of t10c12 usually tended to increase or significantly increased the concentration of linoleic, linolenic, and arachidonic acids and of n-3 long-chain polyunsaturated fatty acids (n-3 PUFA) in the organs. Dietary c9t11 and a 1% content of the mixture of CLA isomers weakly influenced the concentrations of these fatty acids in the organs, whereas the diet enriched simultaneously in c9t11 and Se-yeast resulted in a statistical tendency or increase in the concentration of long-chain n-3 PUFA and the

Properties of trans isomers of eicosapentaenoic acid and docosahexaenoic acid methyl esters on cyanopropyl stationary phases

The trans isomers of 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA) methyl esters were prepared by isomerisation with paratoluenesulfinic acid (PTSA) in dioxane. The isomers were fractionated by silver ion liquid chromatography with baseline resolution between the isomers with different number of trans double bonds. The fractions were analysed by GC–MS and the gas chromatographic properties of the EPA and DHA isomers with one and two trans double bonds were investigated on BPX-70 and SP-2560 cyanopropyl stationary phases. Different temperature and pressure programs were applied to introduce variations in retention indices of the isomers. The retention indices of all the trans isomers showed a strong linear correlation to the retention indices of the equivalent all- cis isomer, but the slopes for corresponding linear regression lines varied with the number of trans double bonds in the molecule. The regression lines were used to predict optimal conditions for the separation of trans isomers from the corresponding all- cis isomers. For DHA on BPX-70, and for EPA on both columns, it was possible to find windows where isomers with one trans double bond can be resolved from the corresponding all- cis isomers with R s > 1.0. In general, BPX-70 seems to have a more suitable selectivity for the analysis of these isomers than SP-2560. Two-dimensional fatty acid retention indices (2D-FARI) were found to be suitable for identification of trans geometry in polyunsaturated fatty acids (PUFA). Although there were substantial overlaps in the range of retention times between the all- cis isomers and isomers with one and two trans double bonds, 2D-FARI separated the isomers into distinct groups according to the number of trans double bonds.

Methods for Analysis of Conjugated Linoleic Acids and trans-18:1 Isomers in Dairy Fats by Using a Combination of Gas Chromatography, Silver-Ion Thin-Layer Chromatography/Gas Chromatography, and Silver-Ion Liquid Chromatography

Conjugated linoleic acids (CLA) are octadecadienoic acids (18:2) that have a conjugated double-bond system. Interest in these compounds has expanded since CLA were found to be associated with a number of physiological and pathological responses such as cancer, metastases, atherosclerosis, diabetes, immunity, and body fat/protein composition. The main sources of these conjugated fatty acids are dairy fats. Rumen bacteria convert polyunsaturated fatty acids, especially linoleic and linolenic acids, to CLA and numerous trans- containing mono- and diunsaturated fatty acids. It has been established that an additional route of CLA synthesis in ruminants and monogastric animals, including humans, occurs via delta9 desaturation of the trans-18:1 isomers. To date, a total of 6 positional CLA isomers have been found in dairy fats, each occurring in 4 geometric forms (cis,trans; trans,cis; cis,cis; and trans,trans) for a total of 24. All of these CLA isomers can be resolved only by a combination of gas chromatography (GC), using 100 m highly polar capillary columns, and silver-ion liquid chromatography, using 3 of these 25 cm columns in series. Complete analysis of all the trans-18:1 isomers requires prior isolation of trans monoenes by silver-ion thin-layer chromatography (TLC), followed by GC analysis using the same 100 m capillary columns operated at low temperatures starting from 120 degrees C. These analytical techniques are required to assess the purity of commercial CLA preparations, because their purity will affect the interpretation of any physiological and/or biochemical response obtained. Prior assessment of CLA preparations by TLC is also recommended to determine the presence of any other impurities. The availability of pure CLA isomers will permit the evaluation and analysis of individual CLA isomers for their nutritional and biological activity in model systems, animals, and humans. These techniques are also essential to evaluate dairy fats for their content of specific CLA isomers and to help design experimental diets to increase the level of the desired CLA isomers in dairy fats. These improved techniques are further required to evaluate the CLA profile in monogastric animals fed commercial CLA preparations for CLA enrichment of animal products. This is particularly important because absorption and metabolism will alter the ingested-CLA profile in the animal fed.