Extractive and Degradative Procedure

With the recognition of the extensive occurrence of amine oxides as alkaloids in the plant there have been developed differential isolation procedures, to separate these from the amines, and reduction procedures, applied to crude extracts, to convert all the amine oxides to amines. For qualitative detection of amine oxides, the red or reddish brown coloration that these compounds give with acetic anhydride serves as a useful test (150). The problem of the separation of the natmnlly occurring amines from the amine oxides has been attacked in different ways. In the first instance of isolation (56-56c), it was found that both trachelanthamine and its, -oxide could be extracted from an aqueous solution by dichloroethane. Sequences of organic extractants such as ether followed by chloroform (116) and chloroform followed by butanol 

If the isolation of the iV-oxides is to be circumvented, the original aqueous solution of the combined alkaloids can be treated with zinc dust and acid, followed by alkalization of the solution and purification in the usual manner (113). This operation may lead to greatly enhanced yields of the known alkaloids (115,117,126). As an extreme illustration. 

Spectral analysis serves as a useful adjunct to paper and column chromatography in the fast determination of the composition of alkaloidal mixtures. A comparative study of the IR-spectra of many pyrrolizidine alkaloids, all very similar to each other, has permitted the designation of a few bands of good intensity in the low-frequency region which are characteristic of certain of the alkaloids and therefore permit the detection of small amounts of impurities in a given sample (83, 84, 120). Thus, seneciphylline has a distinctive band at 902 cm. (s) and another at 992 cm.- (m) senecionine (and its stereoisomers), a band at 757 cm.-i (m) riddelliine, 1120 cm. (m) retrorsine, 1055 cm. (m). 

The degradation of the Senecio alkaloids continues to be effected by saponification or hydrogenolysis. The use of alkali in the hydrolysis does not alter the necine moiety but it may cause a change in the geometrical configuration about an a,j8-oarbon-carbon double bond in the necic acid. Hydrogenolysis provides important information as to the location of the esterified hydroxyl on the necine, and subsequent conversions permit a decision as to which end of a dicarboxylic necic acid (in the cyclic diesters) is attached to the allylic or primary hydroxyl of the necine. 

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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. 

Concentration Level. The level of GA3-like activity has been estimated by the visual comparison of the fluorescence of the appropriate band to that of standard GA3. This technique gives only crude estimates, but does allow a minimum level to be selected based on the intrinsic sensitivity of the test. No correction has been applied for losses in the extraction and purification procedures, for losses due to degradation of GA3 in the chemical steps, or for extract consumed in testing each first-run chromatograph. 

When isolating nucleosides and nucleotides from bacterial cells, it should be borne in mind that extraction and fractionation procedures may degrade or modify the substances under consideration. 

The preparation of the sample prior to its analysis will depend upon the nature of both the sample and the analytical method chosen and may involve the disruption of cells, homogenization and extraction procedures as well as the removal of protein or other interfering substances. It may be necessary to prevent the decomposition and degradation of the carbohydrate content during such treatments or during storage by the addition of antibacterial agents such as thymol or merthiolate, or substances such as fluoride ions, which will inhibit the enzymic transformation of the carbohydrates. 

In our first experiments, we had to find the size gf collagen peptide most amenable to the Quadrol program. The upper limit of the size of a peptide is determined by the amount of peptide that can be introduced into the spinning cup. Thickness of the film resulting from application of more than 10 mg of protein will interfere with the procedures of dissolving, reaction, extraction, and drying. However, the degradation should be started with approximately 300 nM of protein in order that the reaction can be extended to 40—60 identifiable residues. 

To sequence an entire polypeptide, a chemical method devised by Pehr Edman is usually employed. The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact (Fig. 3-25b). The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the amino-terminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluo-roacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The deriva-tized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The use of sequential reactions carried out under first basic and then acidic conditions provides control over... 

A base extraction procedure was developed to minimize the degradation of the performance of fused-silica capillary chromatographic columns used to analyze XAD resin extracts. The degradation of the capillary gas chromatographic column was apparently caused by acidic nonvolatiles called humic materials. The humic materials were absorbed on XAD resins and eluted by nonpolar solvents along with the nonpolar organic compounds of interest in the samples. The base extraction procedure removed approximately 84% of the humic materials present in the ether extract. 

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