Silver (I)-dimercaptotriazine functionalized silica: A highly selective liquid chromatography stationary phase targeting unsaturated molecules.

Silver(I)-mercaptopropyl (Ag-MP) functionalized silica gel has demonstrated its effectiveness in separating various unsaturated organic compounds including unsaturated fatty acid ethyl esters (FAEEs), triglycerols (TAGs) and long-chain alkyl ketones (alkenones). While Ag-MP stationary phase displays many advantages over the conventional silver ion-impregnated silica gel (e.g., stability, high recovery, etc.), potential drawbacks of Ag-MP include relatively low retentions for unsaturated molecules, which could limit chromatographic resolutions under certain circumstances. In this study, we evaluate a new silver-thiolate stationary phase: silver(I)-dimercaptotriazine (Ag-DMT) functionalized silica gel targeting the separation of unsaturated compounds. We show Ag-DMT affords substantially higher retention factors, peak resolutions and capacities for TAGs and FAEEs than Ag-MP does. Ag-DMT also yields higher purity eicosapentaenoic acid (EPA) from fish oil FAEE mixtures than Ag-MP. In addition, Ag-DMT resolves double bond positional and cis/trans-isomers of C18:1 fatty acid methyl esters (FAMEs) as well as unsaturated methyl/ethyl alkenones with different number of double bonds. Based on van't Hoff plots, enthalpy changes during the adsorption of unsaturated FAEEs onto Ag-DMT are ~2 times higher than those on Ag-MP. Such difference may be attributed to the stronger electron-withdrawing effect of the thiol group on DMT, which results in more positively charged silver ions hence greater interactions with unsaturated molecules. The stronger interaction between double bonds and Ag-DMT is further corroborated by density-functional theory (DFT) calculations. Ag-DMT shows its high stability for repeated uses in the separation of TAGs over 319 runs, with peak resolutions decreasing by < 3%. Collectively, our data demonstrate the exceptionally high efficiency of Ag-DMT column for separating unsaturated molecules.

An efficient approach to eliminate steryl ethers and miscellaneous esters/ketones for gas chromatographic analysis of alkenones and alkenoates.

Long-chain alkenones (LCAs) and alkenoates (LCEs) are highly valuable biomarkers for paleotemperature reconstructions. A major problem, however, for accurate quantification of these compounds using gas chromatography (GC) is co-elution with steryl ethers, wax esters, saturated ketones and other numerous mid-polarity compounds frequently encountered in marginal marine and lake sediments. Co-elution during GC separation is prevalent, particularly if the full homologous series of alkenones and alkenoates are to be analyzed. Taking advantage of the presence of two or more double bonds in LCAs and LCEs, the conventional silica gel impregnated with silver nitrate has previously been used to remove co-eluting compounds for LCAs. However, this conventional argentation chromatography is hampered by the extreme instability of silver nitrate, poor reproducibility, low recovery and short lifetime. Here we demonstrate a highly efficient flash chromatographic approach based on silver thiolate chromatographic material (AgTCM) that overcomes the shortcomings of the traditional argentation chromatography and allows repeated sample preparation (up to 62 samples in one test) with little loss in separation efficiency. AgTCM selectively extracts LCAs and LCEs and effectively eliminates co-eluting compounds including steryl ethers and wax esters for the subsequent gas chromatography (GC) analysis. This new method, therefore, allows low-cost and high-throughput sample preparation for comprehensive quantification of the full homologous series of LCAs and LCEs in marine and lake sediments.

Discovery of alkenones with variable methylene-interrupted double bonds: implications for the biosynthetic pathway.

Alkenones (C37 -C40 ) are highly specific biomarkers produced by certain haptophyte algae in ocean and lacustrine environments and have been widely used for paleoclimate studies. Unusual shorter-chain alkenones (SCA; e.g., C35 and C36 ) have been found in environmental and culture samples, but the origin and structure of these compounds are much less understood. The marine alkenone producer, Emiliania huxleyi CCMP2758 strain, was reported with abundant C35:2 Me (∆12, 19 ) alkenones when cultured at 15°C (Prahl et al. 2006). Here we show, when this strain is cultured at 4°C-10°C, that CCMP2758 produces abundant C35:3 Me, C36:3 Me, and small amounts of C36:3 Et alkenones with unusual double-bond positions of ∆7, 12, 19 . We determine the double-bond positions of the C35:3 Me and C36:3 Me alkenones by GC-MS analysis of the dimethyl disulfide and cyclobutylamine derivatives, and we provide the first temperature calibrations based on the unsaturation ratios of the C35 and C36 alkenones. Previous studies have found C35:2 Me (∆14, 19 ) and C36:2 Et (∆14, 19 ) alkenones with three-methylene interruption in the Black Sea sediments, but this is the first reported instance of alkenones with a mixed three- and five-methylene interruption configuration in the double-bond positions. The discovery of these alkenones allows us to propose a novel biosynthetic scheme, termed the SCA biosynthesis pathway, that simultaneously rationalizes the formation of both the C35:3 Me (∆7, 12, 19 ) alkenone in our culture and the ∆14, 19 Black Sea type alkenones without invoking new desaturases for the unusual double-bond positions.

Identification of double-bond positions in isomeric alkenones from a lacustrine haptophyte.

RATIONALE: Measurements of alkenone unsaturation ratios are widely used for paleotemperature reconstructions in ocean and lake environments. Previously, we reported the discovery of a series of tri-unsaturated alkenone positional isomers (Δ(14, 21, 28) ) from oligosaline and freshwater lakes in Greenland and Alaska. In this work we provide a detailed analysis of the structures and isotopic compositions (δ(13) C and δ(2) H) of the alkenones produced by the "Greenland haptophyte". METHODS: Alkenones were extracted from sediments of Lake BrayaSø, Greenland. Alkenone double-bond positions were determined by GC/EI-MS analysis of alkenone dimethyl disulfide and cyclobutylimine derivatives. Alkenones were purified by semi-preparative HPLC using a silver(I) thiolate stationary phase. Carbon and hydrogen isotope analysis was performed by gas chromatography/isotope ratio mass spectrometry (GC/IRMS). RESULTS: A series of novel tri-unsaturated alkenone positional isomers were identified among four alkenone homologues (i.e. C37 Me , C38 Me , C38 Et , and C39 Et ) with double-bond positions at Δ(14, 21, 28) . The hydrogen isotope compositions (δ(2) H, VSMOW) of the tri-unsaturated positional isomers from C37 Me and C38 Et were slightly depleted (~ -11 ‰) relative to the common tri-unsaturated alkenone. The carbon isotope composition (δ(13) C, VPDB) of the tri-unsaturated positional isomers from the C37 Me , C38 Me , C38 Et , and C39 Et alkenones were significantly enriched (~ +4 ‰) relative to the common alkenones (di-, tri-, and tetra-unsaturated). CONCLUSIONS: The novel tri-unsaturated alkenone positional isomers produced by the Greenland haptophyte possess Δ(14, 21, 28) double-bond positions, instead of the common Δ(7, 14, 21) double-bond positions. The hydrogen isotope values suggest the novel tri-unsaturated positional isomers could be biosynthetic precursors to the tetra-unsaturated alkenones (Δ(7, 14, 21, 28) ). However, the significantly higher carbon isotope values of the tri-unsaturated positional isomers relative to the common di-, tri- and tetra-unsaturated alkenones suggest these positional isomers may have different/additional biosynthetic precursors.

Purification of omega-3 polyunsaturated fatty acids from fish oil using silver-thiolate chromatographic material and high performance liquid chromatography.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have become increasingly popular in dietary supplements worldwide and global demand for higher purity ω-3 PUFAs in clinical applications has been rising rapidly in the recent years. Traditional methods for isolating ω-3 PUFAs however, generally require multiple, cumbersome steps to obtain high purity (>95%) products. In this study, we report an efficient liquid chromatography method for purifying individual omega-3 fatty acid ethyl esters (FAEEs), eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6), at >95% purity using a silver(I)-mercaptopropyl stationary phase, otherwise known as silver-thiolate chromatographic material (AgTCM). We demonstrate the key variables controlling separation of FAEEs with a silver(I)-thiolate stationary phase and examine important similarities and differences between silver-thiolate and silver-ion chromatography in regards to the separation mechanism taking place. We demonstrate the separation behavior of FAEEs under various conditions such as mobile phase composition, flow rate and sample injection amount. Purification of EPA and DHA was carried out on an analytical and semi-preparative scale and the resulting purity was determined by GC-FID. We report high attainable purity for EPA (>95%) and DHA (>99%) from a single isocratic separation of fish oil FAEEs within 5-10min using a mobile phase of heptane/acetone (95:5, % v/v).

The unique liquid chromatographic properties of Group 11 transition metals for the separation of unsaturated organic compounds.

Silver(I) and copper(I) are known to form reversible complexes with π bonds, which have been exploited in LC for separating unsaturated organic compounds. Prominent examples include the use of AgNO3-impregnated silica gel in LC, and the use of copper(I) salts for selective extraction of alkenes from hydrocarbon mixtures. The Dewar-Chatt-Duncanson model is often invoked to explain the interaction between Ag(I) and Cu(I) and π bonds. However, it is unclear if such a reversible interaction is directly related to their d(10) outer electronic configurations. Particularly, Au(I) has not been reported to separate olefins with different numbers of double bonds in LC. Also, there has not been a systematic comparison of the liquid chromatographic properties of other d(10) transition metal salts (e.g., Zn(II), Cd(II)), making it difficult to fully understand the observed reversible interactions of Ag(I) and Cu(I) with π bonds. We demonstrate for the first time that silica gel impregnated with all three Group 11 transition metals with 1+ oxidation state strongly and similarly retain olefin compounds in LC, while transition metals from Groups 10 and 12 do not. We also tested a range of functionalized silica gels to improve the stability of Cu(I) and Au(I) ions on the surface of the silica.

Thin layer chromatography in the separation of unsaturated organic compounds using silver-thiolate chromatographic material.

We report the use of silver-thiolate chromatographic material (AgTCM) as a stable material for use in TLC. The AgTCM stationary phase operates under the same principles as silver-ion chromatography, separating compounds by degree of unsaturation; however, the AgTCM stationary phase shows considerable advantages over Ag-TLC in terms of light stability and shelf lifetime. We demonstrate the light stability of the AgTCM-TLC and its application for separations based on the degrees of unsaturation using fatty acid methyl esters (FAMEs) and polycyclic aromatic hydrocarbons (PAHs).

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.

Efficient liquid chromatographic analysis of mono-, di-, and triglycerols using silver thiolate stationary phase.

We show that the characterization of mono-, di- and triglycerols can be readily accomplished by high performance liquid chromatography (HPLC) with silver(I)-mercaptopropyl modified silica gel, or silver thiolate chromatographic material (AgTCM), which can be used with evaporative light scattering detection (ELSD) or atmospheric pressure chemical ionization mass spectroscopy (APCI-MS). Separation of triglycerols varying by degrees of unsaturation and cis/trans configuration in common oil samples can be achieved using a simple linear gradient of hexane and acetone. In addition to double bonds, AgTCM also displays major selectivity for compounds with different levels of polarity, allowing for efficient separation between mono-, di- and triglycerols. In comparison to conventional reversed phase columns, AgTCM produces simple chromatograms for rapid assessment of degrees of unsaturation and the amount of trans fats in triglycerides, which are central issues to food quality determination. In comparison to previous silver-ion based HPLC separations, AgTCM-HPLC based column offers greatly enhanced stability, inertness, durability, and reproducibility allowing routine coupling of the HPLC with a mass spectrometer for detection.