Ammonium Acetate Concentration Research Articles

Liquid-phase microextraction with in-drop derivatization combined with microvolume fluorospectrometry for free and hydrolyzed formaldehyde determination in textile samples

A new miniaturized methodology based on the combination of headspace single drop microextraction and microvolume fluorospectrometry is proposed in this work for the determination of free and hydrolyzed formaldehyde in textile samples. The proposed method is based on the extraction and in-drop derivatization of free and hydrolyzed formaldehyde using the Hantzsch reaction. The effect of experimental variables affecting the performance of the proposed method, such as fluorescence parameters, nature of the extractant phase composition (including acetylacetone concentration, pH, ammonium acetate concentration and presence of an organic solvent), sample temperature, NaCl concentration and microextraction time was carefully investigated. Under optimized conditions, instrumental detection and quantification limits were 26 and 87 μg L −1, respectively, whereas procedural detection and quantification limits were 1.0 and 3.5 mg kg −1, respectively. Repeatability, expressed as relative standard deviation, was 4.6% ( n = 9). The method was successfully applied to the determination of free and hydrolyzed formaldehyde in several textile samples, the found results being in good agreement with those obtained with the EN ISO 14184-1:1998 method.

Isolation, screening, and selection of an L-glutaminase producer from soil and media optimization using a statistical approach

A culture isolated from garden soil was found to be a promising L-glutaminase producer. Biochemical identification tests and 16S rRNA sequencing identified this isolate to be Klebsiella oxytoca. Subsequently, media optimization using one-factor-at-a-time approach and response surface methodology was undertaken. A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of maltose, yeast extract, beef extract, and ammonium acetate on glutaminase production. Almost all factors had significant interactive effects on glutaminase production. A medium containing (g/L): maltose, 23.31; yeast extract, 20.0; beef extract, 20.01; ammonium acetate, 10.0; mannitol, 10.0; KH2PO4, 0.4; Na2SO4, 0.4; and MgCl2, 0.4 was optimum for glutaminase production. The applied methodology was validated using this optimized media and enzyme activity of 458.91 ± 9.49 U/L and specific activity of 0.441 ± 0.04 U/mg protein after 42 h of incubation at 33°C were obtained.

Assessment of the gas phase stability of quadruplex DNA using travelling wave ion mobility mass spectrometry

The quadruplex DNA (qDNA) (dT2G5T)4, (dG4T4G4)2, and the intramolecular d[G3(T2AG3)3] (Q4, Q2 and Q1, respectively), were analysed using circular dichroism (CD) spectroscopy and electrospray ionisation travelling wave ion mobility mass spectrometry (ESI-TWIMS). The CD spectra of the three qDNAs showed, in agreement with the literature, that in ammonium acetate, pH 7.0, Q4 exhibited a parallel strand orientation while Q2 and Q1 were predominantly anti-parallel qDNA. At high concentrations of Q2, the CD spectrum was consistent with a mixture of anti-parallel and parallel forms. ESI-travelling wave ion mobility mass spectra of the qDNA sequences were compared under solution conditions where they were known to be stable and under conditions where they were known to be destabilised (low ammonium acetate concentration with acetonitrile present). This comparison enabled the distinction of ions of the same mass-to-charge that corresponded to single-stranded (ss)Q2, [(dG4T4G4)], or bimolecular Q2, [(dG4T4G4)2]. The resolution of the ESI-TWIMS instrument was sufficient to determine that the ions carrying four and five negative charges most likely arose from the folded intramolecular qDNA, Q1, while the ion carrying six negative charges most likely arose from unfolded Q1. This work shows the utility of ESI-TWIMS in distinguishing among different conformations and structures of qDNA.

Improved ethanol precipitation of DNA

In this Short Communication, a shorter version of the standard DNA ethanol precipitation and purification protocol is described. It uses a mixture of 70% ethanol, 75 mM ammonium acetate and different concentrations of different carriers to perform DNA precipitation and washing in only one step.

Analysis of the alcohol biomarker phosphatidylethanol by NACE with on‐line ESI‐MS

Phosphatidylethanol (Peth), a group of aberrant phospholipids formed in cell membranes in the presence of ethanol, has been recently proposed as biomarker of chronic alcohol abuse. The aim of this study was to develop a new analytical method, based on NACE online coupled with a mass spectrometer for the analysis of Peth in blood. For this purpose an ion-trap mass spectrometer equipped with an orthogonal ESI source operating in negative ion mode was used. An alkaline solution of ammonium acetate 5 mM (pH 9) in water/methanol (MeOH) (80:20 v/v) was delivered as coaxial sheath liquid. All experiments were performed using an uncoated fused-silica capillary (90 cm x 75 microm id). The effects of variable percentages of ACN, MeOH, 2-propanol, dichloromethane, along with variable concentrations of ammonium acetate were investigated for the separation of Peth. Collectively, a separation medium composed of ACN (45% v/v), 2-propanol (20% v/v), dichloromethane (20% v/v), MeOH (10% v/v), water (5% v/v), and ammonium acetate (25 mM) was chosen. The estimated LOD was 0.1 microM, while LOQ was 0.4 microM. Within-run (intra-day) and between-run (inter-day) precision was always lower than 15%. The method proved to be robust and reliable. The MS detector allowed the simultaneous identification of several Peth homologues, and the use of an internal standard (phosphatidylbutanol) with similar electrophoretic properties of that of Peth increased quantitation effectiveness.

Evaluation of hybrid hydrophilic interaction chromatography stationary phases for ultra‐HPLC in analysis of polar pteridines

Retention characteristics of ultra-HPLC (UHPLC) hybrid stationary phases (bridged ethyl hybrid (BEH) and BEH Amide) were studied in hydrophilic interaction chromatography system in the group of polar basic pteridines (neopterin, biopterin, dihydroneopterin and dihydrobiopterin). The effect of mobile phase composition, buffer type, pH and concentration on retention of pteridines were examined in detail under UHPLC-fluorescence detection and UHPLC-MS conditions. The selectivity, retention properties and column performance were examined. BEH HILIC did not provide sufficient retention and selectivity for the separation of four pteridines under any tested conditions. BEH Amide provided strong retention for all pteridines especially at high pH values such as 9.8. However, at pH 9.8 the selectivity of separation for the pairs neopterin-dihydroneopterin and biopterin-dihydrobiopterin substantially decreased and resulted in very long analysis time. The best separation of four pteridine derivatives was obtained in the pH range 4.8-7.8 within a reasonable analysis time up to 8 min for UHPLC-fluorescence detection using higher concentrations of ammonium acetate buffer and up to 4 min for UHPLC-MS using lower concentrations of ammonium acetate.

Retention of arsenic species on zwitterionic stationary phase in hydrophilic interaction chromatography

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) was used to study the retention of selected organoarsenicals. The retention behavior of nine organic arsenic species on ZIC-HILIC was investigated to elucidate which is the driving force for their separation, hydrophilic partitioning or adsorption driven by hydrogen bonds with surface H-donor/acceptor groups of the stationary phase. For this, the retention factor of the compounds k was correlated with log P(O/W) and with the calculated strength of hydrogen bonding of the analytes. By examining aliphatic and phenylic compounds separately, improved correlation was received. This indicates that both phenomena contribute to the separation of these arsenic species on ZIC-HILIC. The results obtained evidence that considerable electrostatic interactions also occur on ZIC-HILIC. Retention behavior of arsenic species was investigated by varying the separation conditions, which shows that the composition of the eluent has a strong influence on the retention behavior. It is highly dependent on water/acetonitrile ratio, pH value and salt additives. Dissociation degree and polarity of arsenic species, which are varying with pH, regulate the distribution of arsenic species between stationary and mobile phases in HILIC. Increase in the ammonium acetate concentration leads to shortened or to prolonged retention depending on the structure of the arsenic species.

Effects of Buffer Loading for Electrospray Ionization Mass Spectrometry of a Noncovalent Protein Complex that Requires High Concentrations of Essential Salts

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful method for analyzing the active forms of macromolecular complexes of biomolecules. However, these solutions often contain high concentrations of salts and/or detergents that adversely effect ESI performance by making ion formation less reproducible, causing severe adduction or ion suppression. Many methods for separating complexes from nonvolatile additives are routinely used with ESI-MS, but these methods may not be appropriate for complexes that require such stabilizers for activity. Here, the effects of buffer loading using concentrations of ammonium acetate ranging from 0.22 to 1.41 M on the ESI mass spectra of a solution containing a domain truncation mutant of a sigma(54) activator from Aquifex aeolicus were studied. This 44.9 kDa protein requires the presence of millimolar concentrations of Mg(2+), BeF(3)(-), and ADP, (at approximately 60 degrees C) to assemble into an active homo-hexamer. Addition of ammonium acetate can improve signal stability and reproducibility, and can significantly lower adduction and background signals. However, at higher concentrations, the relative ion abundance of the hexamer is diminished, while that of the constituent monomer is enhanced. These results are consistent with loss of enzymatic activity as measured by ATP hydrolysis and indicate that the high concentration of ammonium acetate interferes with assembly of the hexamer. This shows that buffer loading with ammonium acetate is effective for obtaining ESI signal for complexes that require high concentrations of essential salts, but can interfere with formation of, and/or destabilize complexes by disrupting crucial electrostatic interactions at high concentration.

Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Effects of ammonium ion, acetate and aerobic conditions on hydrogen production and expression levels of nitrogenase genes in Rhodobacter sphaeroides O.U.001

In the present study, expression levels of nitrogenase encoding nifH and control genes nifA and prrA were examined at different physiological conditions in Rhodobacter sphaeroides O.U.001. In addition to variations in expression levels, changes in hydrogen production and growth were also investigated in response to different concentrations of ammonium source, acetate and aerobic conditions. In the present study, increasing concentration of ammonium chloride was found to be caused decrease in hydrogen production. Glutamate containing medium had the capacity for higher hydrogen production. Hydrogen production was observed even in aerobic conditions. The expression levels of nifH and nifA genes decreased with the increasing concentrations of ammonium chloride. Although the expression of nifA was present in the highest concentrations of NH 4Cl under anaerobic conditions, no expression was observed under aerobic conditions of the same culture conditions. This was likely due to transcriptional level inhibition of nitrogenase in the presence of ammonium ion. Negative correlation was observed between the expression levels of prrA gene and its target, nifA gene.

Effect of increasing concentration of ammonium acetate as an additive in supercritical fluid chromatography using CO 2–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.

Multivariate approach for the enantioselective analysis in MEKC‐MS: II. Optimization of 1,1′‐binaphthyl‐2,2′‐diamine in positive ion mode

Enantiomeric separation and detection of 1,1'-binaphthyl-2,2'-diamine (BNA) has been successfully optimized by MEKC-ESI-MS using a polymeric surfactant polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) as a pseudostationary phase. In the first step, MEKC conditions were optimized by a five-factor three-level central composite design (CCD) of experiment. All five MEKC factors (buffer pH, percentage of ACN in the running buffer, concentration of surfactant, concentration of ammonium acetate (NH4OAc), and voltage) were found significant to the responses (measured as the chiral resolution and analysis time). The interactions between MEKC factors were further evaluated using a quadratic model equation which allowed the generation of 3-D response surface image to reach the optimum conditions. To obtain the best S/N, sheath liquid composition and spray chamber parameters were successfully optimized using the same strategy. Baseline enantiomeric resolution in less than 20 min and optimum MS signal of BNA enantiomers (S/N = 45 at 0.4 mg/mL) were ultimately achieved at the optimized conditions. The adequacy of the model was validated by experimental runs at the optimal predicted conditions. The predicted results were found to be in good agreement with the experimental data.

HPLC of fluoroquinolone antibacterials using chiral stationary phase based on enantiomeric (3,3′‐diphenyl‐1,1′‐binaphthyl)‐20‐crown‐6

A residual silanol group-protecting chiral stationary phase (CSP) based on optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 was successfully applied to the resolution of fluoroquinolone compounds including gemifloxacin mesylate. The chiral recognition ability of the residual silanol group-protecting CSP was generally greater than that of the residual silanol group-containing CSP. From these results, it was concluded that the simple protection of the residual silanol groups of the latter CSP with lipophilic n-octyl groups can improve its chiral recognition ability for the resolution of racemic fluoroquinolone compounds. The chromatographic resolution behaviors were investigated as a function of the content and type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase and the column temperature. Especially, the addition of ammonium acetate to the mobile phase was found to be a quite effective means of reducing the enantiomer retentions without sacrificing the chiral recognition efficiency of the CSP.

The Cu(ii) complex of Aβ40 peptide in ammonium acetate solutions. Evidence for ternary species formation

Fluorimetric Cu(ii) titrations of Alzheimer's disease (AD) Abeta40 peptide at various ammonium acetate concentrations demonstrated that ternary Cu(Abeta40)L complexes are formed with buffer components L, thereby providing a novel perspective on Cu(ii)-Abeta interactions.

Using orthogonal array to obtain gradient liquid chromatography conditions of enhanced peak intensity to determine geniposide and genipin with electrospray tandem mass spectrometry

The conditions to determine geniposide and genipin using gradient liquid chromatography–tandem mass spectrometry (LC–MS/MS) via electrospray ionization were obtained using fractional factorial experimental design approaches, guided with Taguchi orthogonal arrays to enhance peak intensity. Geniposide, the major iridoid glycoside component of Gardenia herbs, which has been recognized to have choleretic effects, is transformed to genipin in animals. In this paper, the gradient establishment times, ionization source temperatures, and the concentrations of volatile additive ammonium acetate were investigated under the guidance of experimental designs to obtain LC–MS/MS signals of the highest peak intensity. Using geniposide and genipin standards, the methods are validated at the concentration ranges of 0.5–1000 ng/mL and 10–5000 ng/mL using ammonium adducts. The correlation coefficients of geniposide and genipin standard curves are greater than 0.999. Compared with the sensitivities of previously published LC–MS/MS methods, the methods developed in this work provide 6-fold sensitivity improvement. The lowest concentrations of geniposide and genipin, 0.19 and 2 ng/mL, respectively, to generate detectable LC–MS/MS signal peaks are one order of magnitude lower than the repoered values in previous publications. The measurement accuracy and precision of geniposide are within 23% and 15%, respectively. The accuracy and precision of genipin are within 16% and 12.5%, respectively. When the validated calibration curves of geniposide and genipin are used to determine spiked control samples in rat blood dialysates, the geniposide determination errors are within 15% accuracy and within 5.8% precision, respectively, and the genipin determination errors are within 23% accuracy and within 3.6% precision, respectively.

Chemical Characterization of Metal-Contaminated Soil in Two Study Areas in Finland

The chemical characterization of contaminated soil was assessed in two study areas in Finland contaminated with metals. The aqua regia extractable and/or concentrated nitric acid leachable median concentrations of selected elements within the study areas were first compared to those from various geochemical baseline studies. Based on this comparison, the studied elements were divided into two groups: elements displaying general enrichment in the study areas and those with median concentrations within the range of baseline levels. In addition to comparison with the baseline levels, the distribution of aqua regia extractable concentrations of selected elements was assessed in relation to the Finnish soil screening values. The potential leachability of the selected metallic elements in contaminated, mainly man-made soil was examined by determining the ratio between the median ammonium acetate and median aqua regia extractable concentrations in the two study areas. Aqua regia extractable concentrations are considered to represent the near-total fraction of the elements and ammonium acetate extractable concentrations the ‘bioavailable’ fraction. These ratios were compared with those from non-polluted sites representing geochemical baselines. In addition, water soluble element concentrations were available from the two study areas and the ratio between synthetic rainwater or distilled, de-ionised water extractable and aqua regia extractable concentrations was calculated. The ratio between ammonium acetate extraction and aqua regia extraction indicated that of the studied elements, Cu, Ni, Pb, Zn and V exist in a more leachable form in contaminated soil than in the natural environment. It can be assumed that these elements are also more ‘bioavailable’ in contaminated land and could therefore pose a risk to the environment. The water soluble fraction of the potential harmful elements was generally low in both study areas, but single samples had easily leachable metal concentrations. High concentrations of trace elements were found in the groundwater on the down stream side of the potential pollution source. The chemical characteristics of the soil material in both study areas were defined with hierarchical cluster analysis, with the results presented as dendrograms produced using Ward’s method. Although some clusters were identified from the dendrograms, no special characterization of the fill material was possible. However, suggestive grouping of certain element groups was observed. Similar grouping of elements was found in factor analysis. Cluster analysis as well as factor analysis was found to be feasible for the chemical characterization of soil provided that a sufficient number of samples with appropriate analysis are available.

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.

Activity of some Mucolytics Against Bacterial Adherence to Mammalian Cells

In this article, some mucolytic agents were tested for their activity to prevent bacterial adherence to mammalian cells. Preliminary screening for antiadherent activity showed that ambroxol, bromhexine, ammonium chloride, and ammonium acetate but neither guaiphenesin nor carbocysteine significantly reduced the adherence of the tested clinical isolates to cultured mammalian cells. The antiadherent effect of such agents was observed when mammalian cells were treated with these agents either before or after bacterial adherence, and this effect was exhibited in a dose-dependent manner. The minimum concentrations of ambroxol, bromhexine, ammonium chloride, and ammonium acetate required for mammalian cells treatment to prevent bacterial adherence were 2.5, 5, 50, and 20 ng/ml, respectively, whereas significantly higher mucolytic concentrations were required to eradicate bacteria that adhered to mammalian cells. Upon treatment of mammalian cells with mucolytics, the maximum reduction in adherence of the tested isolates attained by ambroxol, bromhexine, ammonium chloride, ammonium acetate were 99%, 98%, 75%, and 54% to that of control, respectively. Insignificant difference existed between the antiadherent activities of ambroxol and bromhexine, while both agents had significantly higher effect than ammonium chloride and ammonium acetate. Pretreatment of the immobilized mucin with ambroxol, bromhexine, ammonium chloride, or ammonium acetate reduced the adherence of Pseudomonas aeruginosa, Escherichia coli, and staphylococcal isolates to this receptor analogue. A strong correlation was observed for the antiadherent activity of the tested mucolytics in case of mammalian cells and immobilized mucin. Moreover, pretreatment of the immobilized receptor analogues chondroitin sulfate-B and heparin with the abovementioned agents significantly reduced the adherence of Staphylococcus aureus, P. aeruginosa, and E. coli isolates to such immobilized glycoproteins.

Liquid chromatographic direct resolution of aryl α-amino ketones on a residual silanol group-protecting chiral stationary phase based on optically active (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6

A residual silanol group-protecting chiral stationary phase (CSP) based on optically active (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 was successfully applied to the resolution of racemic cathinone and it analogue aryl α-amino ketones. The separation factors ( α) and the resolutions ( R S) for 12 analytes were in the ranges of 2.85–16.12 and 6.49–19.64, respectively. The chromatographic resolution behaviors were investigated as a function of the content and type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase. The practical usefulness of the CSP in the determination of the enantiomeric purity of optically active cathinone and in the preparative resolution of racemic cathinone was demonstrated.

Liquid chromatographic direct resolution of tocainide and its analogs on a (3,3′‐diphenyl‐1,1′‐binaphthyl)‐20‐crown‐6‐based chiral stationary phase containing residual silanol protecting n‐octyl groups

Optically active (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based chiral stationary phase (CSP) containing residual silanol protecting n-octyl groups on silica surface was applied to the liquid chromatographic direct resolution of tocainide and its analogs. The chiral recognition ability of the CSP was excellent, the separation (alpha) and the resolution factors (R(S)) for 15 analytes including tocainide being in the range of 3.02-22.92 and 3.94-20.41, respectively. In addition, the chiral recognition ability of the CSP was much greater than that of (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6-based CSP containing residual silanol groups on the silica surface. The chromatographic behaviors for the resolution of tocainide and its analogs were found to be dependent on the content and the type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase.