Titania Column Research Articles

Simultaneous determination of synthetic edible pigments in beverages by titania-based RP-HPLC

A rapid method for simultaneous determination of five synthetic edible pigments (SEPs) including tartrazine (TA), ponceau 4R (PO), sunset yellow (SY), brilliant blue (BB) and erythrosine (ER) in beverages with titania-based RP-HPLC has been developed. The good linear relationships were obtained in the concentration ranges of 2.5–40 μg mL−1 for TA, PO and SY, 0.75–12 μg mL−1 for BB, and 1.25–20 μg mL−1 for ER, respectively. The detection limits (LODs) of five SEPs were 0.042, 0.021, 0.042, 0.0005, and 0.021 μg mL−1, respectively. The average recoveries were between 92.2% and 106.3%. Relative standard deviations (RSD, n = 7) for five SEPs were less than 1.18%. The precision and accuracy of the method can meet the requirements of HPLC analysis. In addition, the thermodynamic parameters of retention of the pigments in the titania column such as enthalpy (ΔHο), entropy (ΔSο) and Gibbs free energy (ΔGο) were also determined.

Development of Mo recovery and concentration column processes for production of 99Mo from accelerator-driven irradiation of aqueous uranyl-sulfate solution

ABSTRACTRecovery and concentration columns were designed for separation of Mo from highly concentrated uranyl–sulfate solution. Molybdenum uptake on titania sorbent, from concentrated uranium solutions, is superior to alumina and enables use of low-enriched uranium (LEU, <20% U-235) for 99Mo production. Compared to highly enriched uranium (HEU), LEU requires five times more uranium to produce the same amount of Mo. Isotherm parameters were examined for Mo, Te, and Tc. The VERSE (VErsatile Reaction SEparation) model was used to generate profiles for metal ion concentrations in the columns and determine the minimum column length required for 99.9% Mo capture. Column designs were verified with an irradiated uranyl sulfate solution in a pilot-scale process, where 1.4 Ci of 99Mo was produced, recovered, and purified. Partitioning of fission products on titania columns is also presented.

Exploring the phosphoproteome profiles during Xenopus egg activation by calcium stimulation using a fully automated phosphopeptide purification system.

To explore the phosphoproteome profiles duringXenopusegg activation by Ca(2+)-stimulation, an automated phosphopeptide purification system involving a titania column was improved by introducing 4-step elution with phosphate buffers. The number of detected phosphopeptides in the tryptic digest of aXenopusegg cytosol fraction on mass spectrometry (MS) was increased 1.5-fold and the percentage of multiply phosphorylated peptides increased from 17 to 24% with introduction of the 4-step elution method. Phosphopeptides were purified by the improved method from tryptic digests of cytosol fractions ofXenopuseggs without and with a Ca(2+)-stimulus, and then, analysed by MS. One thousand three hundred and seventy-five and 994 phosphopeptides were reproducibly detected on duplicate MS, respectively. They included 818 and 437 phosphopeptides specific to each digest, respectively. A method involving isobaric tags for relative and absolute quantitation (iTRAQ) was also applied to compare the phosphorylation levels inXenopuseggs without and with a Ca(2+)-stimulus, the ratios for 112 phosphopeptides in tryptic digests of these egg cytosol fractions being obtained. It was suggested from all the results that the phosphorylation sites and levels change duringXenopusegg activation for many known and unknown sites on structural proteins, signalling related proteins, cell cycle-related proteins and others.

Separation of xanthines in hydro-organic and polar-organic elution modes on a titania stationary phase

Hydrophilic interaction LC was investigated in hydro-organic and nonaqueous elution modes on a titania column by using a set of N-methyl xanthines as neutral polar probes. To get information regarding the mechanisms that are behind the discrimination of these analytes in hydrophilic interaction, we focused our study on the type and amount of organic modifier as a critical yet rarely explored mobile phase parameter. Several alcohols such as methanol, ethanol, and isopropanol were studied as substitutes to acetonitrile in hydro-organic elution mode. Compared to silica, the investigation of the eluotropic series of these alcohols on titania highlighted a different implication in the retention mechanism of the xanthine derivatives. At low amounts of protic solvents, the adsorption mainly characterized the retention of analytes on bare silica; whereas mixed interactions including adsorption and ligand exchange were identified on native titania. To investigate the peculiar behavior of alcohols on the metal oxide, methanol, ethanol, and ethylene glycol were tested in replacement of water in polar-organic elution mode. Distinctive effects on the chromatographic retention and selectivity of xanthines were noticed for the dihydric alcohol, which was found to be a stronger eluting component than water on titania.

Preparation and Characterization TiO2 Microspheres for the Liquid Chromatography Stationary Phase

TiO2 microspheres with a uniform particle size distribution were synthesized by hydrothermal method. After being sintered, the titania microspheres that are obtained have an average diameter of 5 μm, a surface area of 383.5 m2/g, an average pore volume of 0.25 cm3/g, and an average pore diameter of 35.9 nm. Normal phase chromatography was separated the mixture of benzene, nitrobenzene and nitro-anisole, three substances were separated well on the titania column. The microspheres possess enough rigidity to withstand high packing pressure and are very useful as a new kind of chromatographic packing material for high performance liquid chromatography (HPLC).

Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection

The emergence of antiviral drug-resistant influenza viruses highlights the need for alternative therapeutic strategies. Elucidation of host factors required during virus infection provides information not only on the signaling pathways involved but also on the identification of novel drug targets. RNA interference screening method had been utilized by several studies to determine these host factors; however, proteomics data on influenza host factors are currently limited.In this study, quantitative phosphoproteomic analysis of human lung cell line (A549) infected with 2009 pandemic influenza virus A (H1N1) virus was performed. Phosphopeptides were enriched from tryptic digests of total protein of infected and mock-infected cells using a titania column on an automated purification system followed by iTRAQ labeling. Identification and quantitative analysis of iTRAQ-labeled phosphopeptides were performed using LC–MS/MS.We identified 366 phosphorylation sites on 283 proteins. Of these, we detected 43 upregulated and 35 downregulated proteins during influenza virus infection. Gene ontology enrichment analysis showed that majority of the identified proteins are phosphoproteins involved in RNA processing, immune system process and response to infection. Host–virus interaction network analysis had identified 23 densely connected subnetworks. Of which, 13 subnetworks contained proteins with altered phosphorylation levels during by influenza virus infection. Our results will help to identify potential drug targets that can be pursued for influenza antiviral drug development.

Capture chromatography for Mo-99 recovery from uranyl sulfate solutions: Minimum-column-volume design method

Molybdenum-99 (Mo-99), generated from the fission of Uranium-235 (U-235), is the radioactive parent of the most widely used medical isotope, technetium-99m (Tc-99m). An efficient, robust, low-pressure process is developed for recovering Mo-99 from uranyl sulfate solutions. The minimum column volume and the maximum column length for required yield, pressure limit, and loading time are determined using a new graphical method. The method is based on dimensionless groups and intrinsic adsorption and diffusion parameters, which are estimated using a small number of experiments and simulations. The design is tested with bench-scale experiments with titania columns. The results show a high capture yield and a high stripping yield (95±5%). The design can be adapted to changes in design constraints or the variations in feed concentration, feed volume, or material properties. The graph shows clearly how the column utilization is affected by the required yield, loading time, and pressure limit. The cost effectiveness of various sorbent candidates can be evaluated based on the intrinsic parameters. This method can be used more generally for designing other capture chromatography processes.

Determination of Melamine in Liquid Milk and Milk Powder by Titania-Based Ligand-Exchange Hydrophilic Interaction Liquid Chromatography

A simple and rapid high-performance liquid chromatography (HPLC) procedure for the analysis of melamine in liquid milk and milk powder has been developed. Decrease of acetonitrile percentage and phosphate buffer concentration in mobile phase, and lowering of buffer pH and column temperature would benefit the retention of melamine on titania. Taking advantage of the ligand-exchange and hydrophilic interaction mixed retention mode on bare titania column, neither complex pretreatment nor ion-pair reagent was required. The whole analysis for one sample including sample pretreatment and HPLC analysis could be accomplished within 30 min. The method presented good linearity (R 2 = 0.9998) in a wide range of 0.02–10 μg mL−1. The limit of detection (3σ) and limit of quantification (10σ) of the method were 6 and 20 μg L−1, respectively, which were equivalent to 15 and 50 μg kg−1 melamine in liquid milk, 60 and 200 μg kg−1 melamine in milk powder, respectively. Such sensitivity could be compared with those obtained by HPLC with solid-phase extraction or HPLC coupled with tandem mass spectrometry and was adequate for the screening of melamine in tainted dairy products. The repeatability (RSDs) of the retention times and peak areas of 11 replicate detections of 1.0 μg mL−1 melamine were 0.32% and 2.5%, respectively. The intermediate precision on three consecutive days (RSDs, n = 6) of the retention times and peak areas were 1.1% and 2.3%, respectively. The recovery of spiked melamine in dairy samples ranged from 95.2% to 105%. The simplicity, sensitivity and rapidity of the proposed method make it an effective alternative detecting technique for melamine.

Zirconia, Titania, Silica, and Carbon Columns for the Group-Type Characterization of Heavy Gas Oils Using High Temperature Normal Phase Liquid Chromatography

The hydrocarbon group-type composition of fossil fuels affects their performance and environmental properties. Normal phase high performance liquid chromatography (NPLC) is commonly used to determine the proportion of saturates, mono-, di-, tri-, and polyaromatics in fuel and gas oil samples. It is often difficult to achieve sufficient resolution between group-types using conventional normal phase columns and conditions. This incomplete resolution can negatively impact the accuracy of the group-type analysis. This paper investigates the use of titania, zirconia, carbon, silica, and aminopropyl bonded silica columns to improve group-type resolution using high temperature NPLC. Better group-type selectivity and faster separations were obtained by increasing column temperatures from 35 to 200 °C. A high resolution hydrocarbon group-type analysis method was developed using titania and silica columns with valve-switching and dual gradients to analyze three heavy gas oils (boiling range > 350 °C). A titania column at a high flow rate of 5.0 mL min–1 yielded separations in only 3 min.

Selective separation of flavonoid glycosides in Dalbergia odorifera by matrix solid‐phase dispersion using titania

Dalbergia odorifera contains high concentrations of flavonoid aglycones and trace flavonoid glycosides. In this study, trace flavonoid glycosides were separated from D. odorifera by titania with matrix solid-phase dispersion (MSPD). Before the MSPD experiment, four standards, including two isoflavone glycosides (genistin and formononetin-8-C-apiosyl (1-6)-glucoside) and their aglycones (genistein and formononetin), were used to compare their retention on a titania column. The effect of acetonitrile concentration and pH on their retention was investigated and a conclusion was drawn that high acetonitrile concentration and pH lead to the greatest difference in the retention of flavonoid as glycosides and aglycones. Besides hydrophilic interaction and ligand-exchange interaction may exist between sugar moiety of flavonoid glycoside and titania, so that flavonoid glycosides have stronger retention than that of aglycones. Based on the chromatographic rule of flavonoid as glycosides and aglycones on the titania column, the MSPD method was optimized to elute high concentration flavonoid aglycones first with 90% acetonitrile and 10% water containing 100 mM ammonium acetate buffer, and then to elute trace flavonoid glycosides with 20% acetonitrile and 80% water containing 1% trifluoroacetate (TFA). Isolated flavonoid glycosides were further analyzed by UPLC-MS/MS, and their fragmentation in MS(2) showed they are C-glycosyl flavonoids.

Capillary monolithic titania column for miniaturized liquid chromatography and extraction of organo-phosphorous compounds

A new sol-gel protocol was designed and optimized to produce titanium-dioxide-based columns within confined geometries such as monolithic capillary columns and porous-layer open-tubular columns. A surface pre-treatment of the capillary enabled an efficient anchorage of the monolith to the silica capillary wall during the synthesis. The monolith was further synthesized from a solution containing titanium n-propoxide, hydrochloric acid, N-methylformamide, water, and poly(ethylene oxide) as pore template. The chromatographic application of capillary titania-based columns was demonstrated with the separation of a set of phosphorylated nucleotides as probe molecules using aqueous normal-phase liquid chromatography conditions. Capillary titania monoliths offered a compromise between the high permeability and the important loading capacity needed to potentially achieve miniaturized sample preparations. The specificity of the miniaturized titania monolithic support is illustrated with the specific enrichment of 5'-adenosine mono-phosphate. The monolithic column offered a ten times higher loading capacity of 5'-adenosine mono-phosphate compared with that of the capillary titania porous-layer open-tubular geometry.

A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis

For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including >90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.

Determination of phosphatidylcholine in soybean lecithin samples by high performance liquid chromatography on titania

High performance liquid chromatography (HPLC) on titania column was performed for phosphatidylcholine (PC) separation and analysis. To fully understand the retention mechanism of PC on titania, the effect of mobile phase type, %acetonitrile (%ACN), phosphate buffer concentration, buffer pH and flow rate were all studied. Separation depended on the eluent conditions and PC retention was attributed to the cooperation of hydrophilic interaction and ligand exchange. PC in soybean lecithin products was completely separated from other components within 6 min and quantitative analysis was achieved by an external standard method, with a correlation coefficient (R2) of 0.9947. The method precision and detection limit of PC were 3.40% and 0.2 μg, respectively, with the recovery ranging from 91% to 101%. Moreover, PC and glycerol monostearate standard mixture was successfully isolated by the proposed method, which underlined the selective affinity of titania for phosphorylated lipids. With the superiority of simplicity, rapidity and accuracy, this HPLC method based on titania is a competent method for the determination of PC in lecithin samples with prospects in the field of organo-phosphates analysis.

Separation of carboxylates by hydrophilic interaction liquid chromatography on titania

Hydrophilic interaction liquid chromatography (HILIC) can be performed on titania. To better understand the retention mechanisms on titania, a series of model carboxylates were used. Increasing acetonitrile above 60% dramatically increased the retention and efficiency for carboxylates. The effect of buffer type, buffer concentration, buffer pH and column temperature were also studied. Multiple retention mechanisms are operative on titania, and whether electrostatic repulsion, ligand exchange or HILIC dominates retention and separation depends on the eluent conditions. Guidelines for separations on titania are: (1) higher %ACN most improves retention and efficiency; (2) higher salt concentration increases retention; (3) elution strength is in the order acetate<<malate<methyl phosphonate<<phosphate; (4) electrostatic repulsion (ERLIC) is more operative at low %ACN than high %ACN. A bare titania column (150mmx4.6mm I.D., 5mum) was used for the separation of diphenylacetate, 4-nitrobenzoate, benzoate, 4-aminobenzoate, 4-hydroxybenzoate, phthalate, 3-aminophthalate, 1,3,5-benzenetricarboxylate, 1,2,4-benzenetricarboxylate, 1,2,4,5-benzenetetracarboxylate, benzenepentabenzoate and mellitate under HILIC conditions based on these guidelines, with efficiencies of 2800-55,000 plates/m.

Hydrophilic interaction chromatography of nucleotides and their pathway intermediates on titania

Nucleotides and their pathway intermediates play important roles in all living species. They are essential cellular components in energy transfer, metabolic regulatory processes and biosynthesis. Titania (TiO 2) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of nucleotides. Herein a bare titania column (150 mm × 4.6 mm I.D., 3 μm) with UV detection at 254 nm was used for the separation of a set of nucleotides (AMP, ADP, ATP, UMP, UDP, UTP, GMP, GDP, GTP, CMP and CTP) and their intermediates (NAD, NADH, UDP-Glu and UDP-GluNAc). Addition of phosphate to the eluent suppresses the ligand-exchange interactions with the titania surface such that hydrophilic interaction chromatography (HILIC) separations may be performed. Increasing the %ACN resulted in increasing retention and efficiency (up to 13,000, 9500 and 4500 plates/m for AMP, ADP and ATP, respectively). The effects of pH, buffer concentration and other eluent anions (fluoride and acetate) were also studied. Fifteen nucleotides and their intermediates were separated in 26 min ( R minimum > 1.3) using an one-step gradient.

Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of β-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from β-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.

Comparison of titania, zirconia, and silica stationary phases for separating diesel fuels according to hydrocarbon group-type by supercritical fluid chromatography

Silica, zirconia, and titania columns were compared for their ability to separate diesel samples into saturates, mono-, di-, tri-, and polyaromatics by supercritical fluid chromatography (SFC) using pure CO 2 according to ASTM method D 5186-03. A titania column coupled in series to a silica column was found to provide the highest overall group-type resolutions based on 20 model compounds, with resolutions as high as 14.7 for saturates versus monoaromatics and 11.9 for monoaromatics versus diaromatics. An oilsands-derived Synfuel light diesel, a commercial Ontario diesel, and a heavy Shell Canada Ltd. diesel blending feedstock were studied on a titania–silica coupled column as well as on a conventional silica column. The Synfuel results were similar (within 0.5 mass%) on both the conventional bare silica column and the titania–silica coupled column. The heavier commercial diesel and diesel blending feedstock samples yielded different results on the titania–silica coupled column compared to the silica column alone, demonstrating the importance of achieving the highest possible resolutions when baseline separation of group-types is not obtained.

Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn.

A methodology of phosphopeptide-selective analysis coupled with column-switching HPLC utilizing titania as precolumn media is presented. Phosphopeptides were selectively enriched on titania packing within a protein/peptide mixture without any additional procedure, and analyzed by column-switching high-performance liquid chromatography. First, phospho-compounds were separated from complex mixtures by trapping them under acidic conditions on a titania packing, where non-phosphorylated compounds were effused out of the precolumn. Subsequently, phospho-compounds were desorbed from the titania column under a specific condition and analyzed. The behavior of phospho-compounds on a titania surface, especially adsorption/desorption, was precisely examined and optimized. A phosphoric buffer was successively employed for the elution of phosphopeptides on a titania surface by competition with the free phosphate group. From the successes of a selective concentration/analysis of phosphopeptides with column-switching HPLC with a titania precolumn, a novel phosphopeptide-selective RP-HPLC analysis has been shown to have an application possibility as a tool for phosphoproteomics.

ION CHROMATOGRAPHIC SEPARATION OF ANIONS AND CATIONS ON A TITANIA PACKED COLUMN

A column packed with titania stationary phase was evaluated for the separation of ionic solutes. The retention behaviour of anions, cations and benzoic carboxylic acids were investigated using various carboxylic acids as the eleunts. Factors that strongly influenced solute retention were the type of eluent, eluent pH and concentration. The titania column exhibited significant ion exchange and ligand exchange behaviour, which depended strongly on eluent pH and types of eluents. Separation of anions, cations and simultaneous separation of anions and cations could be obtained by careful selection of the conditions of the eluent.

Determination of Organic Phosphates by Column-Switching High Performance Anion-Exchange Chromatography Using On-Line Preconcentration on Titania

A highly selective organic phosphates analyzer was developed based on a column-switching technique using a titania (TiO2) precolumn and an anion-exchange analytical column. In the preliminary examination, it was proved that the titania column strongly retains the organic phosphates under acidic conditions while it shows no retention in alkaline eluents. Based on these findings, the organic phosphates were selectively trapped on the titania precolumn in an acidic eluent, transferred to an anion-exchange column by a basic eluent and detected by coloration after a postcolumn phosphomolybdic acid reaction. When 25.0μM each of phospho-amino acids solution was determined (injection volume, 100μl), the relative standard deviations were 2.3-2.8% (n=5). The detection limits (S/N=3) of the phospho-amino acids were ca. 2.0μM as the injected concentration. The present analyzer was successfully applied to the analysis of the phosphorylation products of a heptapeptide (kemptide).