Criteria Of Purity Research Articles

Elaboration of high purity iron (RRR>4000) by horizontal zone melting—attempts for chemical characterization

High purity iron bars were prepared by horizontal zone melting in a water cooled copper boat. The bar is zone melted five times under helium and five times under dry hydrogen. In order to characterize the metal, RRR measurements were performed on cold rolled and annealed samples in dry and wet hydrogen. The RRR measurements vary with the water content of hydrogen: 2734 after dry annealing, 4287 after wet annealing. The RRR variation cannot be related to titanium, chromium, copper or manganese internal oxidation, but to that of aluminium and silicon. After chemical polishing, RRR measurements increase by about 2000 points. If we want to adopt the RRR measurement as a purity criterion of the samples, the surface treatments and the annealing conditions have to be stated. We tried to connect the RRR to the chemical purity of the material. Considering the present analysis capabilities and the high purity metal degree involved, it seems difficult to directly connect the RRR measurements to the variation of the chemical impurities contents.

Evidence for the safety of gum arabic (Acacia senegal (L.) Willd.) as a food additive--a brief review.

Scrutiny of the experimental evidence of safety demanded by the international food safety authorities has led them to conclude that no limitation to the use of gum arabic as a food additive need be specified when it conforms to the established criteria of identity and purity. This brief review collates the dietary, toxicological, immunological, chemical and other studies available.

Isolation and Separation of Epidermal and Mesophyll Protoplasts from Rye Primary Leaves — Tissue-Specific Characteristics of Secondary Phenolic Product Accumulation

Abstract We have develop ed a technique for the large-scale isolation of epidermal and mesophyll proto­plasts, as well as the vascular strands, of rye primary leaf blades. Separation of the two types of protoplasts has been successful only from leaves harvested at the end of a 13-h light period, when chloroplasts were enriched in starch. The occurrence of different flavonoid compounds, and amounts, in epidermal and mesophyll protoplasts can be used as criteria for protoplast purity and viability since C-glucosylflavone O-glycosides are characteristic of epidermal protoplasts whereas flavone O-glucuronides and anthocyanins are typical of mesophyll protoplasts. Several non-flavonoid phenolic compounds are found only in the epidermal protoplast. These patterns of secondary product accumulation reflect the high tissue specificity of the rye leaf.

Isolation and characterization of methionine synthetase from human placenta.

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.

1] l-Glutamate decarboxylase from brain

Publisher Summary γ-Aminobutyric acid (GABA) is one of the major neurotransmitters in the vertebrate central nervous system. L-Glutamate decarboxylase (GAD), which catalyzes α-decarboxylation of L-glutamate to form GABA and CO 2 , is the rate-limiting enzyme that normally determines the steady-state levels of GABA in vertebrate and invertebrate nervous systems. There is good correlation between GABA levels and GAD activity in vertebrate nervous system. Hence, GAD is a better marker for GABAergic neurons than GABA per se, which may redistribute or may be metabolized during the preparation of the tissues. This chapter discusses the various purification procedures, assay methods, and criteria of purity of GAD preparations. The immunochemical characterization of anti-GAD serum and immunocytochemical studies of GAD are also discussed. GAD is assayed by a radiometric method based on the formation of 14 CO 2 from either L-[1- 14 C] - or L-[U- 14 C] glutamate.

An evaluation of purity criteria for bovine rod outer segment membranes

The purity criteria of bovine rod outer segments (ROS) purified by different procedures were evaluated. Bovine ROS were purified by flotation and/or sedimentation in a continuous concentration gradient of sucrose. The purity of the different fractions was then evaluated according to four purity criteria: (i) the A 280 ΔA 500 ratio, (ii) the moles of phospholipid per mole of rhodopsin, (iii) the fatty acid composition, and (iv) the interfacial properties of ROS membranes. All the purity criteria, except the A 280 ΔA 500 ratio, were found to be adequate. From our results, the A 280 ΔA 500 ratio cannot be used alone to characterize ROS purity. Furthermore, the phospholipid-to-rhodopsin ratio appears as the best purity criterion because of its reliability, its higher sensitivity, and its ease of achievement. It is noteworthy that mechanical treatment of the retinas dramatically affects the purification of ROS.

Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli

Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy- d-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-ϵ- amino-n- caproyl- d-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 ± 300 Da; the isoelectric point was 6.0–6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E 275 1% of 4.55 was calculated. The molecular activity was 1800 s −1 for the deamination reaction and 455 s −1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on K m for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the K m values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

Application of reversed-phase HPLC in some critical peptide separations.

The application of reversed-phase high performance liquid chromatography in peptide chemistry is illustrated. Its use is described for separation of closely related endorphins, as an optical purity criterion, for rapid determination of the species of origin of insulin preparations, and finally for analysis of small, basic (hydrophilic) peptides.

Spectral analysis of the conformation of polyadenosine diphosphoribose. Evidence indicating secondary structure.

Based on boronic acid chromatography, a rapid method was developed for the purification of polyadenosine diphosphoribose oligomers of various chain lengths. Polyadenosine diphosphoribose oligomers obtained by the new method were distinguished on the basis of spectral criteria. The purity of polyadenosine diphosphoribose was determined by high performance liquid chromatography, which is a highly sensitive method for the detection of contamination by nucleotides derived from other nucleic acids. The A280/A260 ratio, which has been used in the past as one of the criteria of purity of oligomers, was found to be an unsuitable index of purity because it significantly varied as a function of temperature or ionic strength, exhibiting characteristics of temperature- and ionic strength-dependent hypochromicity. Hypochromicity was highly significant at 260 nm but not at 280 nm. The A280/A260 ratio showed a correlation with the oligomer chain length, significantly diminishing below a chain length of 9 adenosine diphosphoribose units, with concomitant loss of hypochromicity. The increase in A280/A260 ratio above a chain length of nine was small and gradual. Circular dichroism of long, medium, and short oligomers was determined at varying temperatures (5-72 degrees C). For long chains, a temperature-dependent change of the major negative theta value and a red shift occurred from 249.5 to 267.5 nm. With medium chain length oligomers, there was no decrease in the theta value at 249.5 nm, only a red shift. In the case of short chain oligomers the major negative theta value was at 267.5 nm and its position was temperature-independent with only a small temperature-related decrease in size. At 72 degrees C, the different patterns of circular dichroism of the three groups of oligomers of differing chain lengths became indistinguishable. These results were interpreted to indicate a significant secondary structure of polyadenosine diphosphoribose of long chain length.

Complete computer system for processing chromatographic data

A complete data processing system for complex chromatogram analysis is described. Although this system is essentially designed to treat high-performance liquid chromatographic results, it can be applied to several other separation techniques. Original methods are indicated for chromatogram peak integration and evaluation of peak areas, even when separation of compounds is incomplete. For gradient analyses, techniques for filtering and correction of baseline are described. The ability to treat radioactive counting data enables the system to be applied to the processing of metabolic arrays. The use of even derivatives to obtain a criterion of peak purity and the problem of information losses during sampling are also discussed.

Analytical Methods for Drugs in the Next Decade

Today's research for new drug substances is moving toward molecules of much larger molecular weight and complexity than the small, modern drug molecules, e.g., polypeptides, interferon, slow-releasing substances, and blood and plasma components. The complexity of natural products derived from human or other animal bodies requires a new analytical chemistry. Simple tests for drug stability, dissolution, and bioavailability will no longer suffice to provide and ensure purity and structural identity of such drugs. During the next decade, the demands on analytical chemists will increase; more biological organic and physical chemistry will be required by analysts to perform their jobs. Criteria for purity will require even greater chromatographic involvement but this will have to be complemented by nuclear magnetic resonance and mass spectrometry. The demands made on AOAC to validate and approve methods to assay drugs and determine their purity will increase enormously in this next decade. The understanding of drug purity will take on a new meaning because of the complex nature of natural products. Stability indication will be significant only when chemical and biological tests can complement and define the active components and the impurities. AOAC will be required to play a major role in assuring the manufacturer and the public that their drugs are adequately tested, safe, and efficacious.

Study of the effect of chloride ion on the ferric ion yield of the Fricke dosimeter in the absence of impurities

Significant chloride ion effects on G(Fe3+), not related to the suppression ofimpurity effects, have been measured for our dosemeter solutions. This observationnecessitates a modification of an earlier recommendation to use the absence of a Cl- effectas a purity criterion. The data reveal a cubic root relationship with the sodium chlorideconcentration. This relationship is shown to hold up to 1 moll-1 Cl-, after suitablecorrection for the increased density of the dosemeter solution and for the effect ofchloro-Fe3+ complexes at high Cl- concentration. The measured G-value decrease cor-responds exactly to the decrease of the initial Hz02 yield in the presence of Cl-, i.e. it equals2ΔGH2O2 : however, a suitable reaction mechanism could not be put forward.

The H-2k k antigen: Isolation using monoclonal immunoadsorbent chromatography and sequence analysis without radioisotopes

Monoclonal immunoadsorbent chromatography has been used to isolate milligram quantities of detergent-solubilized H-2K k antigen. Using the procedure described in this paper 10 12 cells may be processed yielding 10 mg of homogenous H-2K k which represents 70% of the allotypic serological activity present in the original homogenate. NH 2-Terminal sequence data of the first 30 residues of the H-2K k heavy chain are presented. The cell line selected as the source of antigen and the criteria of purity of the antigen have been found to be critical as proteins of molecular weight 42,000 and 12,000 were copurified with H-2K k from the BW5147 cell line. The additional components were observed in gradient gel electrophoresis or twodimensional electrophoresis, but not in conventional Laemmli gel electrophoresis.

Phospholipid composition of chlorophyll-free mitochondria isolated via protoplasts from oat mesophyll cells.

Mitochondria were isolated from oat primary leaves via mesophyll protoplasts and subjected to phospholipid analysis. In mesophyll cells mitochondria account for only small proportions of cellular phospholipids (in the order of 5%) and proteins (in the order of 2%). Contamination by lipids from other membranes was insignificant as indicated by the absence or very low levels of chlorophyll, galactolipids and steryl glycosides. The absence of 3-trans-hexadecenoic acid in phosphatidylglycerol from mitochondria of green cells serves an an additional criterion of purity. The phospholipid mixture extracted from these mitochondria resembles phospholipids in mitochondria from non-green tissues regarding composition as well as fatty acid profiles. Therefore, mitochondria maintain a rather constant lipid profile and in contrast to plastids do not respond at this level to differences in the physiological status of their housing cell. Palmitic acid in mitochondrial phosphatidylcholine and phosphatidylethanolamine is primarily localized at the C-1 position of the glycerol moiety. Two enzymatic activities so far not described in mitochondria, formation of acylgalactosyl diacylglycerol and hydrolysis of acyl-CoA, were found in the purified mitochondrial fraction.

Resolution of conflicting migratory reports in ring expansion of 3-keto steroids to oxygen and nitrogen

The migration of C-2 and/or C-4 to O or N in the Beckmann, Schmidt, and Baeyer–Villiger reactions of 3-keto steroids has been studied with the aid of 13Cmr spectroscopy. Authentic specimens of the eight possible lactone and lactam products from both 5α- and 5β-cholestan-3-one have been prepared; their physical properties and 13Cmr assignments are given. Seven ring expansion reactions reported to give only one product have been found to give both possible migration products. The much studied reactions of 5α-cholestane-3,6-dioneand its derivatives have been reexamined. The results emphasize again the necessity of using 13C spectra both as an analytical tool and as the best criterion of purity in work with these molecules.

Isolation and characterization of immunoreactive somatostatin from fish pancreatic islets.

Using a radioimmunoassay with labeled synthetic tetradecapeptide somatostatin, a large amount of immunoreactive somatostatin was found in the principal pancreatic islet of the channel catfish (Ictalurus punctata). The purpose of these experiments was to isolate and characterize the somatostatin-like material. Extracts of islets were chromatographed on a Bio-Gel P-30 column, and over 90% of the immunoreactive somatostatin migrated with proteins at least twice the size of synthetic tetradecapeptide somatostatin. This fraction was further purified by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose columns. Two peptides were obtained with identical immunoreactivity, which was approximately 25% that of the synthetic somatostatin. Each peptide was judged to be >95% pure by thin-layer electrophoresis, polyacrylamide gel electrophoresis at pH 8.9, and highpressure liquid chromatography. Further criteria of purity included amino-terminal analysis of fraction IV yielding only aspartic acid. A total of 1.3 mg of fraction II, and 3.8 mg of fraction IV somatostatin-like peptides were obtained from 10 g of fresh frozen islets. Characterization of the two peptides revealed both peptides slightly more acidic than synthetic tetradecapeptide somatostatin. Fraction II had an isoelectric point of 8.0-8.3, and fraction IV 8.3-9.0. Molecular weight estimation by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis revealed similar mobility of both peptides, between pancreatic polypeptide (mol wt 4,500) and glucagon (mol wt 3,500). The mobility was not altered by reduction, and was approximately twice the size of synthetic tetradecapeptide somatostatin (mol wt 1,800). This confirmed that the peptides were single polypeptide chains and not aggregates, or somatostatin bound to larger proteins. Molecular weight determination by gel filtration chromatography on Bio-Gel P-6 in 8 M urea gave an estimated mol wt of 3,700. Amino acid analysis of the two immunoreactive somatostatins indicated that they were very similar in composition. Both pancreatic somatostatins (1 muM) had full biological activity relative to synthetic somatostatin measured as inhibition of growth hormone release from rat anterior pituitary cells.

Potential limitations of recrystallization for the definitive identification of radioactive steroids

The usefulness of recrystallization in establishing the radiochemical purity of steroids is widely recognized, but the potential limitations of the technique have received little attention. The current study reports the failure of standard recrystallization procedures using methanol/water as the solvent pair to separate contaminating 14C-17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) from 3H- and 14C-labeled 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) despite ten serial crystallizations. The standard criteria of radiochemical purity were met despite gross impurity of the crystals as evidenced by thin layer chromatography. Thus, recrystallization may, under certain conditions, yield misleading results when employed as the only method for identifying radioactive steroids. These observations illustrate the importance of an optimal choice of solvent and crystallization conditions, and emphasize the need for confirmation by derivative formation and chromatography.

Human renal renin. Complete purification and characterization.

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.

Isolation and characterization of a serine-threonine-rich galactoglycoprotein from normal human plasma.

A serine-threonine-rich galactoglycoprotein was isolated in homogeneous form from pooled normal human plasma. After precipitation of the major portion of the plasma proteins by Cohn's low temperature-low salt-ethanol procedure, the proteins and glycoproteins remaining in the supernatant solution of Cohn Fraction V were concentrated with the aid of DEAE-cellulose. Fractional elution from this ion exchange resin yielded a crude preparation of the galactoglycoprotein which was purified subsequently by gel filtration through Sephadex G-100. Homogeneity of the protein was established by several criteria of purity including disc polyacrylamide gel electrophoresis and COOH-terminal amino acid analysis. The major physicochemical constants of the macromolecule including its molecular weight of 81,000 were measured. The chemical composition of this protein was found to be unusual: the total carbohydrate moiety accounts for 76% of its weight and consists of 23% sialic acid, 23% total neutral sugar, including 20% galactose, 1% fucose, and almost equal amounts of GalNAc and GlcNAc totalling 27%. The polypeptide moiety accounting for 26% of the weight of the glycoprotein distinguishes itself by a high content of serine and threonine.

Criteria of Purity for Zearalenone Standards

Abstract cis-Zearalenone was identified as one of the impurities in commercial zearalenone used as a reference standard. Zearalenone purified chromatographically gave molar absorptivities of 29,512±376, 13,225±15, and 6,162±61 at the absorption maxima of 236, 274, and 316 nm, respectively, in methanol. The precision in the ratio of absorbances for the major spectral peaks for purified zearalenone preparations provided a useful criterion of purity for zearalenone. A comparison of the GLC measurements of various purified zearalenone preparations with the amount of zearalenone in each preparation as determined by absorbance measurements demonstrated the validity of using absorbance measurements for zearalenone quantitation. Results from a collaborative study demonstrated that the concentration of solutions of zearalenone standards can be determined by UV absorbance measurements.