Purification procedures extraction

The properties of a botanical gum are determined by its source, the climate, season of harvest, and extraction and purification procedures. Table 6 illustrates one of the important basic properties of all gums, ie, the relationship between concentration and solution viscosity. The considerable viscosity variation observed among gums from different sources determines, in part, their uses. 

The batch and fed-batch procedures are used for most commercial antibiotic fermentations. A typical batch fermentor may hold over 150,000 Hters. When a maximum yield of antibiotic is obtained, the fermentation broth is processed by purification procedures tailored for the specific antibiotic being produced. Nonpolar antibiotics are usually purified by solvent extraction procedures water-soluble compounds are commonly purified by ion-exchange methods. Chromatography procedures can readily provide high quaHty material, but for economic reasons chromatography steps are avoided if possible. 

One can readily appreciate the usefulness of pK value in purification procedures, e.g. as when purifying acetic acid. If acetic acid is placed in aqueous solution and the pH adjusted to 7.76 [AcOH]/[AcO ] with a ratio of 0.1/99.9], and extracted with say diethyl ether, neutral impurities will be extracted into diethyl ether leaving almost all the acetic acid in the form of AcO in the aqueous solution. If then the pH of the solution is adjusted to 1.67 where the acid is almost all in the form AcOH, almost all of it will be extracted into diethyl ether. 

Metal impurities can be determined qualitatively and quantitatively by atomic absorption spectroscopy and the required purification procedures can be formulated. Metal impurities in organic compounds are usually in the form of ionic salts or complexes with organic compounds and very rarely in the form of free metal. If they are present in the latter form then they can be removed by crystallising the organic compound (whereby the insoluble metal can be removed by filtration), or by distillation in which case the metal remains behind with the residue in the distilling flask. If the impurities are in the ionic or complex forms, then extraction of the organic compound in a suitable organic solvent with aqueous acidic or alkaline solutions will reduce their concentration to acceptable levels. 

Other purification procedures include the formation of the picrate, prepared in benzene soln and crystd to constant melting point, then decomposed with warm 10% NaOH and extracted into ether the extract was washed with water, and distd under reduced pressure. The oxalate has also been used. The base has been fractionally crystd by partial freezing and also from aq 80% EtOH then from absolute EtOH. It has been distd from zinc dust, under nitrogen. 

Most purification procedures for a particular protein are developed in an empirical manner, the overriding principle being purification of the protein to a homogeneous state with acceptable yield. Table 5.5 presents a summary of a purification scheme for a selected protein. Note that the specific activity of the protein (the enzyme xanthine dehydrogenase) in the immuno-affinity purified fraction (fraction 5) has been increased 152/0.108, or 1407 times the specific activity in the crude extract (fraction 1). Thus, xanthine dehydrogenase in fraction 5 versus fraction 1 is enriched more than 1400-fold by the purification procedure. 

The most common source of commercial heparin has become pig intestinal-mucosa, from which somewhat more than 100 mg of heparin per kg of tissue is usually obtained.32 Data on the content of heparin in organs of different animal species (summarized in Refs. 7 and 14) are probably not strictly comparable with each other, because of different extraction and purification procedures used in different laboratories, and supposedly different recoveries from different tissues. 

In the UV filter analyses, clear extracts were obtained which did not need any further time- and labor-consuming cleanup when using PLE with in-cell purification. In contrast, a thorough purification procedure was required for the cleanup of the extracts obtained by solid-liquid extraction. Columns packed with silica gel (previously activated) in hexane having Na2S04 at the top of the column were employed for the purification of the extracts. The two first fractions obtained with 20 mL of hexane and 20 mL of hexane/diethyl ether (9/1, v/v) were discarded and 4-MBC, EHMC, and OC were collected with 50 mL of hexane/diethyl ether (9/1, v/v). OT was recovered with 70 mL of hexane/diethyl ether (3/2, v/v). 

A minimalist extraction buffer is often recommended for protein purification [25], since most additives provide only a marginal improvement in yields, but will increase costs significantly. The removal of phenolics, for example by tangential-flow ultrafil-tration/diafiltration [25], is an important step that should be carried out as early as possible in the purification procedure, since these molecules can become covalently linked to amino acid side chains and can oxidize certain residual groups [123]. 

The checkers found that the purification of the pseudopelletierine could be simplified, at least in those preparations in which commercial acetonedicarboxylic acid was used. Thus, the crude product obtained by evaporation to dryness of the methylene chloride extracts can be sublimed directly. Two sublimations give pseudopelletierine of m.p. 62-64°, in 58-62% yield, comparable to the product obtained after the more extended purification procedure described in the text. 

The assembly of tetrapeptide 19 that contains all possible 0-dipeptide bonds, (03-03)-, (03-02)-, and (02-03), and also a turn inducing 03-(R)-Ala-02-(R)-Val element was achieved employing a Boc-strategy (Scheme 5). A fluorous benzyl group was incorporated in the first amino acid to streamline the purification procedure by fluorous solid phase extraction (LSPE) (Lilippov et al. 2002 de Visser et al. 2003). Thus, the assembly of the fully protected tetrapeptide commenced with the construction of the first 03-03-peptide bond by applying the previously established conditions. A residence time of 3 min at 90°C provided the Boc-protected dipeptide 15 in 91% isolated yield after LSPE. Notably, the product precipitated in the collection flask, which was kept at ambient temperature, indicating the poor solubility of this class of compounds (Hessel et al. 2005). 

As an alternative purification procedure, the checkers have esterified the crude acid by refluxing it for 2 hours with three times its weight of methanol and 2 ml. of 98% sulfuric acid. The solution is poured into 10 volumes of water and extracted with the minimum amount of chloroform required to give a clean separation of layers. The chloroform solution is washed with water, dried over calcium chloride, and distilled from a Claisen flask with an indented neck. Methyl 1-adamantanecarboxylate is collected at 77-79° (1 mm.) m.p. 38-39°. Hydrolysis of the ester with the calculated amount of 1A potassium hydroxide followed by acidification yields 1-adamantanecarboxylic acid m.p. 175-176.5° 90% overall recovery. 

These findings lead to (he conclusion that the reduction of MHb by its reductase requires a natural cofactor, which is abolished during the purification procedure and can be replaced by methylene blue (G5, H22, H23, K8, K14). Since methylene blue and the other effective dyes are redox intermediates, it is obvious that the postulated cofactor interacts in the electron transport sequence of the MHbR reaction (H23). This is confirmed by the finding that oxygen and cytochrome c serve as well as terminal electron acceptor as does MHb (H22, H23, K14). Nevertheless, it had been possible to separate a cytochrome c reductase from MHbR in yeast extracts (A6). 

Standard purification procedures including chromatography, liquid-liquid extraction and crystallization can be time consuming and difficult to scale up. In many cases supported reagents have distinct advantages over their solution-phase counterparts, including increased selectivity,... 

Miller DN, Bryant JE, Madsen EL, Ghiorse WC. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl. Environ. Microbiol. 1999 65 4715 1724. 

Pure M-hexane is widely used in laboratories as an extractant for nonpolar compounds and in calibrating instruments for analyses of volatile organic compounds (VOC) or total petroleum hydrocarbons (TPH) (Kanatharana et al. 1993). Since such analyses may require very high levels of purity, laboratories sometimes carry out their own fractional distillation or other pretreatment-purification procedures to remove petroleum hydrocarbon impurities found in commercially available grades of M-hexane (Kanatharana et al. 1993). See Chapter 6 for more information about testing for -hexane. 

This chapter and Chapters 12 and 13 describe modem analytical separation science. First, purification procedures known as recrystallization and distillation will be described. Then the separation techniques of extraction and chromatography are discussed. This is followed by, in Chapters 12 and 13, instrumental chromatography techniques that can resolve very complicated samples and quantitate usually in one easy step. 

An internal standard must always be introduced into the sample before any extraction or purification procedures are undertaken as this will compensate for losses in the analysis as a result of these processes. 

Quantitation of lipids may require an initial extraction step. This should neither degrade the lipids nor extract any non-lipid components, such as carbohydrates, amino acids, etc. Individual requirements will dictate how rigorous any extraction and purification procedure must be but several fundamental precautions must always be taken in order to minimize the possibility of errors. 

When using purified triolein, most samples are amenable to bioassay after di-alytic enrichment. For example, Microtox bioassay of dialysates of SPMDs shows that the SPMDs made with the purified triolein have lower acute toxicities than dialysates from SPMDs made from unpurified triolein (Johnson, 2001). Finally, examination of the dialysates using the yeast estrogen screen (YES) assay (Routledge and Sumpter, 1996) demonstrated that the purification procedure removes all background estrogenic activity (Lebo et ah, 2004). Use of triolein purified by this process expands the potential applicability of SPMD sample extracts to include numerous bioassay procedures (see Chapter 6) and GC-MS as a standard analysis technique. 

A. bronchisepticus was cultivated aerobically at 30 °C for 72 h in an inorganic medium (vide supra) in 1 liter of water (pH 7.2) containing 1 % of polypeptone and 0.5 % of phenylmalonic acid. The enzyme was formed intracellularly and induced only in the presence of phenylmalonic acid. All the procedures for the purification of the enzyme were performed below 5 °C. Potassium phosphate buffer of pH 7.0 with 0.1 mM EDTA and 5 mM of 2-mercaptoethanol was used thoughout the experiments. The enzyme activity was assayed by formation of pheylacetic acid from phenylmalonic acid. The summary of the purification procedure is shown in Table 2. The specific activity of the enzyme increased by 300-fold to 377 U/mg protein with a 15% yield from cell-free extract [9]. One unit was defined as the amount of enzyme which catalyzes the formation of 1 mmol of phenylacetic acid from phenylmalonic acid per min. 

The HbHnl is obtained from the leaves of the rubber tree plant and a crude extract is easily prepared by homogenisation of the frozen leaves, followed by centrifugation [38-40]. A 5-step purification procedure of this crude extract (with over a 100-fold purification factor) to yield a homogenous HbHnl has... 

Methods for determining the parent compound, 2-hexanone, in water, air, and waste samples are available (Badings et al. 1985 EPA 1986 Flawthorne et al. 1985 NIOSFI 1984). Sampling methodologies for compounds such as 2-hexanone continue to pose problems such as nonrepresentative samples, insufficient sample volumes, contamination, and labor-intensive, tedious extraction and purification procedures (Green and Le Pape 1987). It would be helpful to have means to measure organic compounds such as 2-hexanone in situ in water and other environmental media without the need for sampling and extraction procedures to isolate the analyte prior to analysis. 

The capacity of each step of the extraction and purification procedure to remove and/or inactivate contaminating substances derived from the host cell or culture medium, including, in particular, virus particles, proteins, nucleic acids and added substances, must be validated. 

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