An evaluation of acid digestion methods for amniotic fluid phospholipids

Five methods of acid digestion of amniotic fluid phospholipids for inorganic phosphate release were evaluated at a temperature of 225 °C and a heating time of 30 min. The methods used the following digestants: (A (perchloric acid), B (perchloric acid containing molybdate), C (sulfuric acid, 30% hydrogen peroxide, 5% urea), D (sulfuric-perchloric acids), and E (sulfuric-perchloric acids with vanadium pentoxide). After digestion and dilution with water, a hydrogen ion concentration of about 2.5 mol/ liter was obtained that permitted the use of stannous chloride-hydrazine sulfate as the reducing agent for phosphate color development. Recoveries of the different amniotic fluid phospholipids and aqueous phosphate standards were quantitative for all methods. A comparative study with amniotic fluids showed similar results for all methods. Since all methods were found to be equivalent under the specified conditions described, the choice was dependent upon the user's preference.     

A semi-automated and manual method for serum phospholipids

Five methods of acid digestion of amniotic fluid phospholipids for inorganic phosphate release were evaluated at a temperature of 225 °C and a heating time of 30 min. The methods used the following digestants: (A (perchloric acid), B (perchloric acid containing molybdate), C (sulfuric acid, 30% hydrogen peroxide, 5% urea), D (sulfuric-perchloric acids), and E (sulfuric-perchloric acids with vanadium pentoxide). After digestion and dilution with water, a hydrogen ion concentration of about 2.5 mol/ liter was obtained that permitted the use of stannous chloride-hydrazine sulfate as the reducing agent for phosphate color development. Recoveries of the different amniotic fluid phospholipids and aqueous phosphate standards were quantitative for all methods. A comparative study with amniotic fluids showed similar results for all methods. Since all methods were found to be equivalent under the specified conditions described, the choice was dependent upon the user's preference.     

The effect of barbituric acid concentration in the spectrophotometric determination of cyanide and thiocyanate by the pyridine-barbituric acid method

Summary The König reaction forms the basis of many analytical methods including those for the determination of chlorine, nicotinic acid, thiocyanate and cyanide. The colorimetric methods have, however, been plagued with various problems such as the use of hazardous, often carcinogenic compounds, and the instability of the final color formed as well as of the color reagent itself.Using a mixture of pyridine, hydrochloric acid and barbituric acid as the color reagent, the present study shows the effect of barbituric acid concentration on the intensity and stability of the color complex. The problems associated with the use of barbituric acid can be attributed mainly to the high concentration used by previous workers. A color reagent containing about 0.1% (w/v) barbituric acid shows marked improvements in color intensity and stability as well as reagent stability.     

Blister-colorimetric determination of phosphate ions in water,agricultural samples,and biological samples

A procedure was proposed for the determination of phosphate ions in a blister cell (pellet cartridge) with a dry reagent mixture. The procedure is suitable for the quantitative determination of phosphate in different samples using a dry reagent mixture in an ampule or a blister without dissolving the reagents. After an ampule or a blister cell was opened and several drops of a test liquid were added, a color developed, whose intensity was proportional to the concentration of phosphate ions in the solution. The solution was then diluted to 2 mL with water and analyzed by photometry. The composition of the mixture was determined, and the procedure for the quantitative determination of phosphate ions was proposed; the procedure involves the formation and reduction of phosphomolybdic acid and the use of auxiliary reagents. The error of the colorimetric determination of phosphate ions in aqueous solutions, soil extracts, and urine was estimated with the participation of inexperienced operators.     

Semiquantitative and Quantitative Determination of Trace Amount of Phosphate Ion in Water Using Polyurethane Foam Thin -Layer Colorimetry

Abstract

A simple, sensitive and fast method for the colorimetric determination of phosphate ion in water is described.

The method is based on spectrophotometric measurement of the blue molybdoantimony phosphoric acid species sorbed in a polyurethane foam thin-layer for quantitative determination of phosphate ion, or the visual color comparison technique for rapid semiquantitative determination.

The detection limit for the quantitative procedure is 5 μg/1 and for semiquantitative procedure 20 μg/1 for sample volumes of 100 ml and 25 ml, respectively.     

Determination of phosphatidylcholine in amniotic fluid

A relatively rapid method for measuring phosphatidylcholine (lecithin) quantitatively in amniotic fluid has been described that requires 1 ml or less of sample for fluids having a total phospholipid concentration greater than 25 mg/liter. Following extraction with chloroform-methanol, the solvent is passed through a calcium hydroxyphosphate column which removes the acidic phospholipids and allows passage of the phosphatidylcholine and sphingomyelin. Hydrolysis with periodate-sulfuric acid selectively releases inorganic phosphate from the phosphatidylcholine that is measured by reduction of the formed phosphomolybdate complex to the usual blue color. Various mixtures of phospholipids were carried through the entire procedure with excellent recoveries. Phosphatidylcholine added to an amniotic fluid pool was also quantitatively recovered, so that the method appeared completely suitable for routine clinical laboratory use.     

Determination of trace amounts of phosphate by flow-injection photometry

This paper describes a simple and rapid procedure for determination of traces of phosphate by means of molybdenum blue chemistry. The use of a cost-effective home-made flow cell with a long path length in combination with a light emitting diode (LED) and a photodiode (PD) is demonstrated as a simple absorbance detector for flow-injection analysis. In this method, a sample is injected into the carrier stream through an injection valve and mixed online with mixed reagent (a mixture of molybdate, bismuth, and ascorbic acid in sulfuric acid). The color intensity of the resulting association complex, molybdenum blue, is measured photometrically (_max 875 nm). The proposed method can be used to detect phosphate in the range 0.02–4.0 mg L^–1 and the precision of the proposed procedure is less than 5% at 0.1 mg L^–1 phosphorus as phosphate. The method has been successfully applied to a variety of natural water samples.     

Analysis of acrylamido-buffers for isoelectric focusing by capillary zone electrophoresis

Immobilized pH gradients use a series of weak acrylamido acids and bases (Immobiline) to create a pH gradient along the separation axis. These buffers can be degraded in water by two mechanisms: (i) hydrolysis of the amido bond, with generation of free acrylic acid and either an amino acid or a diamine; (ii) autopolymerization to oligomers and/or n-mers. In order to check for these degradation products, different capillary zone electrophoresis systems for analysis of all Immobilines have been devised. The acidic compounds are resolved in 100 mM acetate, pH 4.0, whereas the alkaline Immobilines are separated in 50 mM phosphate buffer, pH 7.7 (or pH 7.2 for the weaker species). Polymers of alkaline Immobilines are resolved in 50 mM phosphate buffer, pH 2.5, in 1% Ficoll-400. All Immobilines are detected underivatized, by their adsorption at 214 or 254 nm. A calibration curve has been constructed for quantification of acrylic acid contamination. As little as 1 mol% of acrylic acid contamination in Immobiline solutions can be detected, with a sensitivity limit below 0.2 mM (at the injection port).     

Spectrophotometric determination of nitrite

A new reagent for the absorptiometric determination of nitrite ion is used. Nitrite is reacted with 1-amino-4-naphthalenesulfonic acid to form a diazonium ion, which gives an orange color at pH 1.5 with maximum absorption at 310 nm, or an orange-red color at pH 4 with maximum absorption at 485 nm. Diazotation is fast at pH 1.5 but is stable only for 30 min after which the color fades continuously. However, at pH 4, the color develops after 1 hr and remains stable for >40 hr. The time of color development at pH 4 can be reduced to 30 min when heated to 60–70 °C for 5 min, but is followed by reduction of its intensity to half. The described method is found suitable for the determination of 0.1–5.0 ppm of the titled ion.     

Direct serum inorganic phosphate determination

A simple serum microinorganic phosphate determination is described which obviates protein precipitation. Chylous or turbid specimens can also be processed, but a sample blank must be included. The latter treatment is simple and is required only infrequently.Two sets of specimens were investigated for their inorganic phosphate content. One set included clear or slightly opalescent serums while the other set consisted of chylous and turbid specimens. Both sets were analyzed for phosphate content by the new direct technique with the sample correction applied to only the second set. In addition, phosphate concentration was determined in all specimens from both sets using a protein precipitation technique considered here as a reference procedure (3). The results obtained for both sets by the direct technique correlated well with those obtained by the reference method.Jaundiced specimens do not present a problem because the yellow color due to bilirubin does not absorb at the wavelength used. The red hemoglobin color does not interfere for the same reason. However, grossly hemolyzed specimens are not suitable because of the high phosphate content in erythrocytes.     

Removal of sample background buffering ions and myoglobin enrichment via a pH junction created by discontinuous buffers in capillary electrophoresis

Traditional CE sample stacking is ineffective for samples containing a high concentration of salt and/or buffer. We recently reported the use of a discontinuous buffer system for protein enrichment that was applicable to samples containing millimolar concentrations of salt. In this paper, the technique was investigated for samples containing unwanted buffering ions, including TRIS, MES, and phosphate, which are commonly used in biological sample preparation. Using myoglobin as a model protein, the results demonstrated that background buffering ions can be effectively removed or separated from the enriched protein. The key is to use either the acid or the base of the discontinuous buffers to adjust the pH of the sample, such that the net charge of the unwanted buffering ions is near-zero. The successful isolation and enrichment of myoglobin from up to 100 mM TRIS and 50 mM MES was demonstrated. The enrichment factors remained at approximately 200. Removal of phosphate was more challenging because its net charge was anionic in both the acid and the base of the discontinuous buffers. The enrichment was only achievable up to 30 mM of sodium phosphate, the enrichment factors observed were significantly lower, below 50, and the process was delayed due to the higher ionic strength resulted from phosphate. The migration of phosphate during enrichment was studied using a UV-absorbing analogue, phenyl phosphate. In addition, Simul 5.0 was used to simulate the discontinuous buffers in the absence and presence of TRIS and phosphate. The stimulated TRIS and phosphate concentration profiles were generally in agreement with the experimental results. The simulation also provided a better understanding on the effect of phosphate on the formation of the pH junction.     

A severe peak tailing of phosphate compounds caused by interaction with stainless steel used for liquid chromatography and electrospray mass spectrometry

A severe peak tailing was observed for adenosine 5'-monophosphate in flow injection analysis with stainless steel tubing and water/methanol mixture (1:1, v/v) as carrier. The cause of the peak tailing was investigated by focusing on the chemical structure of the analytes, the material used for the analytical systems and the composition of the carrier. We clarified that the peak tailing was caused by the interaction between phosphate residues in the analytes and stainless steel. The severe peak tailing did not occur with stainless steel tubing when the phosphate compounds were analyzed with carrier containing phosphoric acid or phosphate buffer. The findings indicate that such ill peak profiles are usually not considerable in conventional HPLC separation because phosphoric acid or phosphate buffer is quite commonly used in eluents. In LC-MS, however, the use of phosphoric acid and phosphate buffer is usually avoided because of their non-volatility; therefore this interaction between stainless steel and phosphate compound becomes predominant and results in severe peak tailings. We also found an effective method for avoiding the interaction. When stainless parts, such as LC tubing and ESI spray capillary, were treated with phosphoric acid prior to analysis, the peak profiles of the phosphate compounds were dramatically improved, even when non-phosphate buffer is used as carrier.     

Analysis of anthranilic acid by liquid chromatography

Analytical methods for the assay of anthranilic acid and for determination of the impurities methyl anthranilate, anthranoylanthranilic acid and 3- and 4-aminobenzoic acid are described. A Microbondapak C18 column is used for both the assay and the impurity determination. The assay is based on isocratic development with a mobile phase of 35:65 v v methanol/pH-3 phosphate buffer, with benzoic acid as internal standard. The impurities are separated by gradient elution. The standard deviation of the assay method is about 1% and the limit of detection for the impurities is about 0.01%.     

Influence of high‐conductivity buffer composition on field‐enhanced sample injection coupled to sweeping in CE

The aim of this work was to clarify the mechanism taking place in field‐enhanced sample injection coupled to sweeping and micellar EKC (FESI‐Sweep‐MEKC), with the utilization of two acidic high‐conductivity buffers (HCBs), phosphoric acid or sodium phosphate buffer, in view of maximizing sensitivity enhancements. Using cationic model compounds in acidic media, a chemometric approach and simulations with SIMUL5 were implemented. Experimental design first enabled to identify the significant factors and their potential interactions. Simulation demonstrates the formation of moving boundaries during sample injection, which originate at the initial sample/HCB and HCB/buffer discontinuities and gradually change the compositions of HCB and BGE. With sodium phosphate buffer, the HCB conductivity increased during the injection, leading to a more efficient preconcentration by staking (about 1.6 times) than with phosphoric acid alone, for which conductivity decreased during injection. For the same injection time at constant voltage, however, a lower amount of analytes was injected with sodium phosphate buffer than with phosphoric acid. Consequently sensitivity enhancements were lower for the whole FESI‐Sweep‐MEKC process. This is why, in order to maximize sensitivity enhancements, it is proposed to work with sodium phosphate buffer as HCB and to use constant current during sample injection.     

Effects of phosphate and fulvic acid on the sorption and transport of uranium(VI) on silica column

Column experiments were performed, breakthrough curves (BTCs) and displacement curves (DPCs) were obtained of uranium(VI) in the absence and presence of phosphate or fulvic acid individually and simultaneously which demonstrated the effects of phosphate and fulvic acid on the sorption and transport of U(VI) in a silica column at pH 3.7 and U(VI) concentration of 5·10⁻⁶ mol/L. It was found that in the presence of phosphate or fulvic acid sorbed preliminarily on the silica column, the amount of U(VI) sorbed increased significantly and the transport of U(VI) delayed significantly relative to that in the absence of phosphate or fulvic acid. Moreover, in the presence of phosphate and fulvic acids sorbed preliminarily and simultaneously on the silica column, the amount of U(VI) sorbed on the silica column is significantly increased again relative to that in the presence of phosphate or fulvic acid individually. Transport studies of U(VI) are important, since all uranium isotopes are radioactive, and uranium contamination of soils and groundwaters occurs at mining and mill sites. A fundamental understanding of the transport behavior of U(VI) in the water-mineral systems is necessary for accurate risk assessments.     

Polar Red‐Emitting Rhodamine Dyes with Reactive Groups: Synthesis,Photophysical Properties,and Two‐Color STED Nanoscopy Applications

The synthesis, reactivity, and photophysical properties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the amino‐reactive sites (NHS esters or mixed N‐succinimidyl carbonates) are reported. All fluorophores contain an N‐alkyl‐1,2‐dihydro‐2,2,4‐trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary carboxamide group. The absorption and emission maxima in water are in the range of 635–639 and 655–659 nm, respectively. A vastly simplified approach to red‐emitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed. As an example, a phosphorylated dye with an azide residue was prepared and was used in a click reaction with a strained alkyne bearing an N‐hydroxysuccinimid (NHS) ester group. This method bypasses the undesired activation of phosphate groups, and gives an amphiphilic amino‐reactive dye, the solubility and distribution of which between aqueous and organic phases can be controlled by varying the pH. The presence of two hydroxyl groups and a phenyl ring with two carboxyl residues in the dyes with another substitution pattern is sufficient for providing the hydrophilic properties. Selective formation of a mono‐N‐hydroxysuccinimidyl ester from 5‐carboxy isomer of this rhodamine is reported. The fluorescence quantum yields varied from 58 to 92 % for free fluorophores, and amounted to 18–64 % for antibody conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two‐color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting a pair of fluorophores with absorption/emission bands at 579/609 and 635/655 nm enabled two‐color channels with low cross‐talk and negligible background at approximately 40 nm resolution.     

Determination of trace phosphate ion with 3,3',5,5'-tetramethylbenzidine using a flotation-extraction preconcentration method and spectrophotometric detection

A flotation-extraction method for sensitivity enhancement in spectrophotometry is proposed. The method is based on the reaction among molybdate, phosphate and 3,3',5,5'-tetramethylbenzidine (TMB) in acidic medium to form an color charge transfer complex ion. It forms an ion associate with positive ions and floated at the water/benzene interface simultaneously, and then extracted by the DMSO-formic acid directly and determined by spectrophotometry. The proposed method is simple and convenient. The apparent molar absorptivity is 4.03x10(5) L mol(-1) cm(-1) at 458 nm. The proposed method has been applied to the determination of phosphate in wastewater with satisfactory results.     

Non-aqueous spectrophotometric determination of citric acid

A non-aqueous spectrophotometric method is described for the determination of citric acid in the presence of carboxylic acids and lactic acid. The method was developed as a result of critical evaluation of the Furth-Herrmann color reaction in a non-aqueous pyridine-acetic anhydride solution. The optimum conditions are described. The absorbance is read at 389 ± 2 mμ. The minimum concentration of citric acid that can accurately be determined is about 2 μg per sample.     

Metal-ion-coordinating properties of the dinucleotide 2'-deoxyguanylyl(5'-->3')-2'-deoxy-5'-guanylate (d(pGpG)3-): isomeric equilibria including macrochelated complexes relevant for nucleic acids

The interaction between divalent metal ions and nucleic acids is well known, yet knowledge about the strength of binding of labile metal ions at the various sites is very scarce. We have therefore studied the stabilities of complexes formed between the nucleic acid model d(pGpG) and the essential metal ions Mg2+ and Zn2+ as well as with the generally toxic ions Cd2+ and Pb2+ by potentiometric pH titrations; all four ions are of relevance in ribozyme chemistry. A comparison of the present results with earlier data obtained for M(pUpU)- complexes allows the conclusion that phosphate-bound Mg2+ and Cd2+ form macrochelates by interaction with N7, whereas the also phosphate-coordinated Pb2+ forms a 10-membered chelate with the neighboring phosphate diester bridge. Zn2+ forms both types of chelates with formation degrees of about 91% and 2.4% for Zn[d(pGpG)]cl/N7 and Zn[d(pGpG)]-cl/PO, respectively; the open form with Zn2+ bound only to the terminal phosphate group, Zn[d(pGpG)]-op, amounts to about 6.8 %. The various intramolecular equilibria have also been quantified for the other metal ions. Zn2+, Cu2+, and Cd2+ also form macrochelates in the monoprotonated M[H;d(pGpG)] species (the proton being at the terminal phosphate group), that is, the metal ion at N7 interacts to some extent with the P(O)2(OH)- group. Thus, this study demonstrates that the coordinating properties of the various metal ions toward a pGpG unit in a nucleic acid differ: Mg2+, Zn2+, and Cd2+ have a significant tendency to bridge the distance between N7 and the phosphate group of a (d)GMP unit, although to various extents, whereas Pb2+ (and possibly Ca2+) prefer a pure phosphate coordination.     

Rapid isolation method for lipopolysaccharide and lipid A from gram-negative bacteria

A fast, convenient extraction method for lipopolysaccharide (LPS), using a commercial RNA isolating reagent, allows the isolation of LPS or lipid A from low milligram (dry weight) quantities of bacterial cells. The method avoids the use of specialized equipment and has been used for processing relatively large numbers of samples. The major components of the commercial RNA isolating reagent, Tri-Reagent, are phenol and guanidinium thiocyanate in aqueous solution. The bacterial cell membranes are disrupted with guanidinium thiocyanate, which eliminates the need for mechanical cell disruption (e.g. French press) or heating. LPS and its degradation products, with particular attention paid to its bioactive lipid A portion, were measured and compared with those from the most common conventional extraction method, hot phenol-water. Negative ion quadrupole ion trap and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid composition analysis by capillary gas chromatography, total and free phosphate by UV spectrophotometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that LPS and lipid A isolated using the Tri-Reagent approach were cleaner and suffered less degradation through loss of phosphate and (or) fatty acyl side chains from lipid A. The Tri-Reagent extraction method generated low free phosphate contamination, 11% of the total phosphate concentration, whereas the hot phenol-water extraction method gave approximately 58% as free, inorganic phosphate. Similar results were observed for the degradation of fatty acyl side chains. The time required by the new method is considerably shorter (two or three days) than that required by conventional hot phenol-water extraction (about two weeks).