Feasibility of Phospholipids Separation by Packed Column SFC with Mass Spectrometric and Light Scattering Detection

Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) have been separated by supercritical fluid chromatography coupled with evaporative light scattering and mass spectrometric detection. Four different silica based stationary phases were investigated: diol, cyanopropyl, 2-ethylpyridine, and 4-ethylpyridine. A gradient mobile phase of pressurized carbon dioxide modified with methanol was employed. The individual effect of basic (isopropylamine), acidic (trifluoroacetic acid), and ionic (ammonium acetate) additives incorporated into the primary modifier on the separation of these four lipids from the four stationary phases was investigated. No additive was required to effect the elution of PC and PE (which differed only in polar head group) from any of the four phases. When the two ethylpyridine phases were used, near baseline resolution of two species in each lipid case that differed in fatty acid content were seen. PS and PI could be successfully eluted from each phase when isopropylamine was added to the mobile phase, although the PS peak was always weak and very broad. Ammonium acetate was a more effective additive for phospholipid separation than trifluoroacetic acid. All chromatographic separations were reproducible and required less than 15 min.     

Study of the elution mechanism of sodium aryl sulfonates on bare silica and a cyano bonded phase with methanol-modified carbon dioxide containing an ionic additive

The elution mechanism of sodium sulfonates on both Deltabond cyanopropyl and bare silica stationary phases with an isocratic mobile phase composed of methanol-modified CO2 wherein an ammonium salt additive was dissolved in the methanol has been studied. The presence of the additive was crucial concerning elution of the sulfonate salts. Solid state 29silicon nuclear magnetic resonance spectroscopy provided some insight concerning the interaction of the mobile phase additive with the silica-based stationary phase. Computational calculations concerning the charge distribution on various ammonium salts were performed in an effort to explain the elution behavior. Ammonium ions are believed to deactivate available silanol sites on both phases. In addition, ammonium ion is speculated to interact with the cyano groups on the bonded phase. For concentrations of additive greater than 2 mM, stationary phase coverage of ammonium ion is anticipated to exceed one monolayer for both bare and bonded silica. The acetate counter-ion is thought to facilitate elution of the anionic sulfonates from the positively charged stationary phase in a pseudo ion exchange mechanism.     

Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.     

Effect of increasing concentration of ammonium acetate as an additive in supercritical fluid chromatography using CO2–methanol mobile phase

The effects of increasing concentrations of ammonium acetate additive in supercritical fluid chromatography were studied on silica, 2-ethyl-pyridine and endcapped 2-ethyl-pyridine stationary phases. The study involved the addition of increasing concentrations of the ammonium acetate either in the mobile phase modifier (methanol) or in the sample solvent. The effects of ammonium acetate on retention and peak shape of the analytes were evaluated. Compounds that exhibited satisfactory chromatographic behaviour in the absence of the additive were virtually unaffected by its presence in the mobile phase or sample solvent. Nevertheless, compounds that exhibited late elution and strongly tailing peak shapes when pure methanol was used showed dramatically improved chromatographic behaviour in the presence of the additive. Shorter retention was observed not only when the modifier was introduced in the mobile phase but also when it was in the sample solvent.     

Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol‐modified CO2 and water as the polar additive

The separation of four water soluble nucleobases (thymine, uracil, adenine, and cytosine) via supercritical fluid chromatography with a CO₂‐based mobile phase containing an alcohol modifier and additive is described. Methanol, ethanol, 1‐propanol, and 1‐butanol were examined in conjunction with water as a neutral additive. Packed column stationary phases included silica bonded diol, cyanopropyl, and 2‐ethyl pyridine. Thymine and uracil eluted with good peak shapes without additive, while adenine and cytosine yielded late eluting, severely tailing peaks. The addition of up to 5% water to each of the five alcohols gave rise to much sharper peaks that eluted under gradient conditions in less than 10 min with no baseline noise. Results with water under identical chromatographic conditions were compared with formic acid and ammonium acetate as additives. Water proved to be much superior to formic acid, and it was comparable to ammonium acetate. The role of water was speculated to not only enhance the solvating power of the binary mobile phase for water soluble analytes, but the common elution pattern exhibited by each of the three stationary phases suggested that water had altered the surface chemistry of the packed phase.     

Packed column supercritical fluid chromatography of isomeric polypeptide pairs

The characterization and determination of peptides is of great importance in the pharmaceutical industry as is the ability to rapidly perform targeted determinations of bioactive peptides in complex matrices. The purpose of the presented work is to assess the feasibility of packed column supercritical fluid chromatography (SFC) for the separation of two-pairs of water soluble peptides of identical mass, composition and charge that differ only in amino acid sequence. Upon evaluating a variety of conditions, trifluoroacetic acid (HTFA) in conjunction with methanol as the modifier proved to be, in general, the most successful mobile phase additive for elution of the two isomeric peptide pairs from all nitrogenous stationary phases. In contrast, water and ammonium acetate gave distorted peak shapes and therefore proved to be less satisfactory as neutral additives. The basic additive, iso-propylamine (IPAm), coupled with HA-Pyridine yielded the highest resolution factor for the complete study. Aminopropyl and HA-Pyridine columns with 5 μm particle size and 60 Å pore size were found to be best for resolution of each peptide pair. Bare silica and phenyl-hexyl stationary phases did not afford any separation. The primary roles of the carbon dioxide and methanol modifier are believed to provide (a) stationary phase solvation and (b) peptide solubility and transport; while, HTFA is postulated to fully protonate each peptide and form ion pairs between its conjugate base and cationic peptide analyte. The separation process, therefore, is best viewed as ion pair supercritical fluid chromatography (IP-SFC). For the case where IPAm gave good resolution on the HA-Pyridine column, the peptides are probably in the neutral state.     

Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry

The chromatographic behaviour of 44 polar compounds (23 beta-adrenergic agents, 11 stimulants, 4 narcotics and 6 phenolalkylamines) included in the list of prohibited substances and methods of the World Anti-Doping Agency, has been investigated under hydrophilic interaction liquid chromatography conditions by application of different mobile phase compositions (percentage of the organic solvent, type and amount of mobile phase additive and ionic strength) and column temperatures. Detection of analytes was performed by a triple quadrupole mass spectrometer in positive ionization mode and selected reaction monitoring acquisition mode after liquid/liquid extraction. Data collected using as stationary phase type-B silica materials from different producers, showed that the best chromatographic conditions in terms of peak shape, selectivity and chromatographic retention were obtained using an initial percentage of acetonitrile of 90%, a column temperature of 35 °C, a mobile phase pH of 4.5 and ammonium acetate (5 mM) and acetic acid (0.1%) as mobile phase additives. The selected chromatographic conditions were used to develop screening and confirmation analytical procedures to detect polar compounds in human urine for antidoping purpose. The developed methods were validated in terms of specificity, matrix effect, linearity, precision, accuracy, sensitivity, robustness and repeatability of retention times and relative ion abundances. Such methods offer attractive alternatives and considerable advantages over traditional approaches especially for the analysis of the phenolalkylamines.     

The use of ammonium hydroxide as an additive in supercritical fluid chromatography for achiral and chiral separations and purifications of small, basic medicinal molecules

This study describes using 0.1% of a 28-30% ammonium hydroxide solution as an additive to alcohol modifiers in SFC to improve chromatographic peak shapes for basic molecules. Ammonium hydroxide's high volatility leaves no residual additive in the purified sample unlike classical additives in preparative chromatography such as diethylamine and triethylamine. We demonstrate that the silica support is stable despite having ammonium hydroxide in the modifier by running a durability study for over 350 h (105 L of solvent, 105,000 column volumes) on an analytical Chiralcel OJ column and a second study for 30 h (7.2 L, 14,400 column volumes) on an analytical Lux Cellulose-1 column. The peak shape of small, basic molecules is greatly improved with the use of ammonium hydroxide and this improvement is very similar to those having 0.1% diethylamine as a mobile phase additive. Electrospray ionization is also enhanced with the presence of ammonium hydroxide compared with that of diethylamine. We have found that the age of the 28-30% bottle of ammonium hydroxide solution can have significant effects on the chromatography and we describe how this can be overcome. Finally, we analyzed 23 racemic and basic compounds on six different chiral stationary phases and found there to be very little chiral selectivity difference between ammonium hydroxide and diethylamine, triethylamine, ethanolamine and isopropylamine.     

Effect of pH and mobile phase additives on the chromatographic behaviour of an amide‐embedded stationary phase: Cyanocobalamin and its diaminemonochloro‐platinum(II) conjugate as a case study

Several mobile phase additives (i.e., organic acids and their ammonium salts) were used to modulate the chromatographic retention of cyanocobalamin and its cis‐diaminemonochloroplatinum(II) conjugate, depending on the specific nature of the stationary phase. Regardless of the mobile phase additive, the positively charged cyanocobalamin‐cis‐diaminemonochloroplatinum(II) conjugate was systematically less retained than cyanocobalamin on a conventional octadecyl‐silica column. In contrast, the amide‐embedded C18 column exhibited a progressive increase in the conjugate retention time upon changing the mobile phase additive from organic (acetic, formic and trifluoroacetic) acids to ammonium salts, ultimately leading to an inversion of the elution order. This change of retention was interpreted by invoking the interplay between hydrophobic interactions, hydrogen bonding between the conjugate and the polar amide groups and the ion‐pairing ability of the lyophilic counterions, whereby the acetate anion was found to be the most suitable to control the solute retention.     

超临界流体色谱对吴茱萸中吲哚类生物碱的快速分析

建立了超临界流体色谱快速分析吴茱萸中吲哚类生物碱的方法。以标准品混合物和复杂样品为对象比较4种色谱柱的分离效果,进行色谱柱的筛选;考察了进样体积、改性剂、添加剂、温度和背压对保留行为的影响。结果表明,进样体积对峰形影响显著;添加剂对保留时间和色谱峰形影响有限;改变改性剂能使保留时间显著改变;降低温度,升高背压,保留时间减小。经过优化,确定采用Waters ACQUITY UPC² BEH色谱柱,以甲醇为改性剂,在35 ℃柱温和2.07×10⁷ Pa背压条件下,15 min内完成复杂样品的分析。同时采用超高效液相色谱完成复杂样品的快速分析。结果表明,超临界流体色谱可用于天然产物的高效快速分析,同时该方法与超高效液相色谱在分离选择上的差异有助于天然产物分析方法的拓展。     

Comparison of Reversed Stationary Phases for the Chromatographic Separation of Inorganic Analytes Using Hydrophobic Ion Mobile Phase Additives

Abstract

Alkyl-modified silica (RSi) and polystyrenedivinylbenzene (PRP-1) stationary phases are compared for the chromatographic separation of inorganic analyte anions and cations using hydro-phobic ions of opposite charge as mobile phase additives. Tetra-alkylammonium salts were used for anion separations and alkyl sulfonate salts for cation separations. Two major equilibria influence the retention of analyte ions on PRP-1. These are: retention of the hydrophobic ion on PRP-1 and an ion exchange selectivity between the hydrophobic counterion and the analyte ion. When using RSi retention is also influenced by ion exchange at residual silanol groups, which act as weak cation exchange sites. Mobile and stationary phase variables that influence analyte retention are identified. Optimization of these provides favorable eluting conditions for the separation of inorganic ionic analytes. Of particular interest is the potential use of PRP-1 and RSi columns for the separation of inorganic cations; conditions for the separation of alkali metals and alkaline earths are discussed.     

Development of a supercritical fluid chromatography high-resolution separation method suitable for pharmaceuticals using cyanopropyl silica

A method has been developed for the analysis of a broad spectrum of pharmaceuticals using packed column supercritical fluid chromatography (pSFC) on a cyanopropyl silicagel stationary phase. Five 25 cm x 4.6 mm I.D., 5.0 microm columns were coupled to generate ca. 100000 plates. The selectivity was tuned by varying the nature and concentration of various modifiers and additives in the carbon dioxide mobile phase. It was noted that pressure influences both efficiency and selectivity of the chromatographic process. Final method conditions are: outlet pressure 100 bar, flow 2.0 mL/min, temperature 40 degrees C, organic modifier program from 5% (1 min) to 40% at 2.0%/min, organic modifier composition methanol:acetonitrile in a ratio of 3:1 (variable according to sample composition) with peak symmetry additives trifluoroacetic acid and diisopropylamine both at levels of 0.5%.     

Effect of ionic liquid additives to mobile phase on separation and system efficiency for HPLC of selected alkaloids on different stationary phases

The silica-based stationary phases with favorable physical characteristics are the most popular in liquid chromatography. However, there are several problems with silica-based materials: severe peak tailing in the chromatography of basic compounds, non-reproducibility for the same chemistry columns, and limited pH stability. Ionic liquids (ILs) as mobile phase components can reduce peak tailing by masking residual free silanol groups. The chromatographic behavior of some alkaloids from different classes was studied on C18, phenyl, and pentafluorophenyl columns with different kinds and concentrations of ionic liquids as additives to aqueous mobile phases. Ionic liquids with different alkyl substituents on different cations or with different counterions as eluent additives were investigated. The addition of ionic liquids has great effects on the separation of alkaloids: decrease in band tailing, increase in system efficiency, and improved resolution. The retention, separation selectivity, and sequence of alkaloid elution were different when using eluents containing various ILs. The increase of IL concentration caused an increase in silanol blocking, thus conducted to decrease the interaction between alkaloid cations and free silanol groups, and caused a decrease of alkaloids retention, improvement of peak symmetry, and increase of theoretical plate number in most cases. The effect of ILs on stationary phases with different properties was also examined. The different properties of stationary phases resulted in differences in analyte retention, separation selectivity, peak shape, and system efficiency. The best shape of peaks and the highest theoretical plate number for most investigated alkaloids in mobile phases containing IL was obtained on pentafluorophenyl (PFP) phase.     

Ionic liquids as mobile phase additives in high-performance liquid chromatography with electrochemical detection: Application to the determination of heterocyclic aromatic amines in meat-based infant foods

The beneficial effects of several ionic liquids (ILs) as mobile phase additives in high-performance liquid chromatography with electrochemical detection for the determination of six heterocyclic aromatic amines (HAs) have been evaluated for first-time. The studied ionic liquids were 1-butyl-3-methylimidazolium tetrafluoroborate (BMIm-BF₄), 1-hexyl-3-methylimidazolium tetrafluoroborate (HMIm-BF₄) and 1-methyl-3-octylimidazolium tetrafluoroborate (MOIm-BF₄). Several chromatographic parameters have been evaluated in the presence or absence of ILs, or using ammonium acetate as the most common mobile phase additive, with three different C18 stationary phases. The effect of the acetonitrile content was also addressed. In general, best resolution, lower peak-widths (up to 72.1% lower) and lower retention factors are obtained when using ILs rather than ammonium acetate as mobile phase additives. The main improvement was obtained in the baseline noise, being 360% less noisy for BMIm-BF₄, 310% for HMIm-BF₄, and 227% for MOIm-BF₄, when compared to ammonium acetate at +1000 mV. Different chromatographic methods using the best conditions for each IL were also evaluated and compared. Finally, the best chromatographic conditions using 1 mM of BMIm-BF₄ as mobile phase additive, the Nova-Pak^® C18 column, 19% (v/v) of acetonitrile content in the mobile phase, and +1000 mV in the ECD, have been applied for the chromatographic analysis of six HAs contained in meat-based infant foods. The whole extraction method of meat-based infant foods using focused microwave-assisted extraction and solid-phase extraction has also been optimized. Extraction efficiencies up to 89% and detection limits ranged between 9.30 and 0.165 ng g⁻¹ have been obtained under optimized conditions.     

Hydrophilic interaction chromatography in nonaqueous elution mode for separation of hydrophilic analytes on silica-based packings with noncharged polar bondings

Chromatographic effects of dedicated stationary and mobile phase variations in hydrophilic interaction chromatography (HILIC) were investigated using a set of nucleobases, nucleosides and deoxynucleosides as polar test solutes. Retention and selectivity profiles were comparatively mapped on four in-house developed silica materials modified with short alkyl chains (C4, C5) which carry hydroxyl functionalities (including diol motifs) as well as embedded sulphide or sulphoxide groups. These data were complemented by results obtained with two commercially available diol-type phases and a bare silica column. Besides elucidation of packing-related aspects this work concentrated specifically on extending aqueous HILIC (AQ-HILIC) to nonaqueous polar-organic elution conditions herein termed NA-HILIC. The exchange of the polar modifier water by various alcohols in ACN-rich mobile phases containing 5 mM ammonium acetate decreased the eluotropic strength of the resulting eluents. The gain in retention largely followed the order ethanol (EtOH)>methanol (MeOH)>1,2-ethanediol (Et(OH)2) and was accompanied by distinct effects on chromatographic selectivity. For example, on the most polar home-made packing the purine nucleoside selectivity guanosine/adenosine increased from 2.25 in the AQ-HILIC (kguanosine=8.3) to 7.33 (kguanosine=59) in the NA-HILIC mode when EtOH was employed as NA modifier while this value was 5.84 and 2.93 with MeOH and Et(OH)2, respectively (eluent: 5 mM ammonium acetate in ACN/modifier 90:10 v/v). Besides the type of protic modifier its percentage as well the retention and selectivity effects upon varying the ammonium acetate concentration and column temperature, respectively, were also investigated. Notable inter-column differences were found for all of these elution parameters. A mixed-mode retention model composed of partitioning and adsorption is proposed for both AQ- and NA-HILIC retention processes. The potential of (i) the implementation of novel polar bondings (such as ones containing sulphoxide functionalities) and (ii) the comprehensive exploitation of elution variables (type of protic modifiers, salt, etc.) for providing new selectivity increments to the separation of polar analytes in HILIC is emphasised.     

Comparison of separation selectivity in capillary electrokinetic chromatography using a cationic linear polymeric pseudo-stationary phase or monomeric additives of similar structure

The retention properties in electrically driven systems with monomeric additives were compared to an electrokinetic chromatographic system with a linear, charged polymer of similar chemical structure (all additives are quaternary tetraalkyl ammonium ions). The monomeric additives were tetramethylammonium (TMA), tetraethylammonium (TEA) and dimethylpyrrolidinium (DMP), respectively, the polymeric additive was poly(diallyldimethyl)ammonium (PDADMA). The additive concentration in the background electrolyte was 2 and 4% (w/w). The retention characteristics were based on the apparent mobilities of 10 non-charged analytes with different chemical functionality, which were transported by the anodic electroosmotic flow in the dynamically coated capillary, and retained by the counter-flowing cationic additives. From these data capacity factors were derived, which ranged up to 0.8. Association constants were calculated, and were found between 10 and 170. Roughly, the association constants increased for a given analyte in the sequence TMA相似文献   

Effect of amine mobile phase additives on chiral subcritical fluid chromatography using polysaccharide stationary phases

Increased retention and selectivity in the subcritical fluid chromatography (SFC) of various amine compounds on polysaccharide chiral stationary phases (CSP) was observed upon incorporation of cyclic amines into the modifier. The retention increases are most pronounced with 2-propanol and are almost absent when methanol is used as modifier. This suggests that the effect may arise from a restriction to the modifier access to the binding site required to effect elution. The effect of the amine additives in SFC does not remain after their removal from the mobile phase. Findings were applied to the development of a 5 min separation of amphetamine and methamphetamine enantiomers.     

Elution of Cationic Species with/without Ion Pair Reagents from Polar Stationary Phases via SFC

Traditionally, the application of supercritical fluid chromatography has been limited to compounds of relatively low polarity. Thus, we report here that one secondary amine and two quaternary amine salts were successfully eluted from a Deltabond Cyano-bonded silica column with the addition of sodium alkylsulfonate to the methanol-modified, carbon dioxide-based mobile phase. A possible ion-pairing interaction between the positively charged analytes and the anionic part of the sulfonate additive has been proposed. In another set of experiments, the three amine salts readily eluted from both Ethyl-pyridine-bonded silica and Amino-bonded silica phases without the need of additive although the peak shapes were less than desirable. The addition of sulfonate salt to the mobile phase again sharpened the peaks. In the presence of a mixture of methanol and CO₂, we suggest that these stationary phases are positively charged. We describe here also for the first time the employment of a strong silica-based anion exchange (SAX) column for supercritical fluid separation of cationic species. Two elution mechanisms were proposed for the three amine salts depending upon whether the ionic additive was incorporated into the mobile phase. Separation as the ion-pair was proposed with an ionic additive in the mobile phase; whereas separation of the intact amine salt from the positively charged basic stationary phases was suggested to be operational without the ionic additive.