Skip to Main contentScienceDirectJournals BooksRegisterSign in Sign inRegisterJournals BooksHelpChromatographyHILIC should be considered complementary to SCX rather than an alternative strategy, since the two techniques can lead to the identification of different sets of proteins from those in the sample [62].From: Advances in Biological Science Research, 2019Related terms:Mass SpectrometryTransfer FactorMonospecific AntibodypHBacteriumPurificationMetaboliteView all TopicsDownload as PDFSet alertAbout this pageA Plant Virus with Properties of a Free Ribonucleic Acid: Potato Spindle Tuber VirusT.O. DIENER, in Comparative Virology, 19714 Hydroxyapatite ChromatographyChromatography on hydroxyapatite (Bernardi, 1965) has been shown to be a powerful tool for the fractionation of nucleic acids, and particularly for the separation of single-stranded from double-stranded RNA (Bockstahler, 1967).Hydroxyapatite (Bio-Gel HT, Calbiochem) columns (1 × 10 cm) were equilibrated with 0.01 M phosphate buffer (primary sodium and secondary potassium phosphates), pH 6.7. The columns were loaded with phenol-treated and DNase-digested preparations of PSTV RNA suspended in 0.01 M phosphate buffer, pH 6.7. Elution was carried out with phosphate buffers of increasing molarity.As expected, most of the nucleic acid eluted in 0.10 and 0.15 M phosphate buffers; only traces of nucleic acid eluted at higher molarities [Fig. 8(a)]. Only traces of infectious material eluted in 0.10 M buffer; most of the infectious material eluted in 0.15 M buffer. Elution of infectious material was, however, not complete at 0.15 M; small amounts of it continued to elute upon prolonged washing of the column with 0.15 M buffer and with buffers of higher molarity. Hydroxyapatite chromatography of nucleic acid extracts, prepared in identical fashion from healthy plants, gave identical elution patterns. Obviously, the 0.15 M fraction, although containing the bulk of infectivity, was still heavily contaminated with host nucleic acid. To determine whether this host nucleic acid could be separated from the infectious nucleic acid by a different elution protocol, the fraction that had eluted in 0.15 M buffer was reconcentrated, dialyzed against 0.01 M phosphate buffer, and loaded onto a new column of hydroxyapatite. This time, elution was commenced with 0.11 M buffer, followed by a linear gradient of buffer from 0.11 to 0.15 M. As shown in Fig. 8(b), much of the nucleic acid eluted in 0.11 M buffer; yet only traces of infectivity were associated with this fraction. The bulk of infectious material eluted during gradient elution. The highest level of infectivity, as well as the highest nucleic acid concentration, was contained in a fraction that eluted in 0.125 M buffer; and more infectious material could be recovered by elution with higher molarity phosphate buffer [Fig. 8(b)].FIG. 8. Absorbance profiles and infectivity distributions of eluates from hydroxyapatite columns. (a) Elution profile of a phenol- and DNase-treated PSTV preparation. (b) Elution profile of the pooled, reconcentrated, and dialyzed 0.15 M phosphate buffer eluate of (a). Solid lines, absorbance profiles; broken lines, molarity of phosphate buffer; FR. NO., fraction number; I.I., infectivity indexes of individual fractions.Parallel chromatography with extracts from healthy plants resulted in identical elution profiles. Thus, most of the nucleic acid that eluted in the gradient from 0.11–0.15 M buffer was host nucleic acid. This conclusion was confirmed by analysis of the 0.11–0.15 M gradient fraction by rate-zonal density-gradient centrifugation. Most of the nucleic acid remained at the top of the gradient; whereas the infectious material sedimented to its usual position in the gradient (ca. 10 S). No optical density peak was evident at this position.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780124702608500191FRAGRANCE AND PIGMENTS | Odoriferous Substances and PigmentsH. Schulz, in Encyclopedia of Rose Science, 2003Analytical Characterization of Rose Flowers and Rose OilsFor chromatographic separation and identification of the volatile, odoriferous rose substances it is first necessary to apply optimal clean-up steps. For isolation of the volatile fraction, the following methods have mainly been used:•solvent extraction•steam distillation•various headspace techniques.The major limitation of solvent extraction is that it is useful only on samples that do not contain any lipids. Furthermore, solvent extraction and steam distillation are liable to produce artefacts by isolating nonvolatile decompositions. Therefore, headspace sampling, as a nondestructive technique, which can be used on living plants either in the laboratory or in the field, has been found to be the method of choice. Generally, there exist four distinct methods of different headspace techniques (vacuum headspace technique, closed-loop stripping method, dynamic headspace technique and solid phase microextraction) that are commonly used today for analysing the rose volatile substances. The first one is the ‘vacuum degassing’ or ‘vacuum headspace’ technique in which the volatile constituents are separated from the plant matrix at a high vacuum and frozen out into traps cooled down by liquid nitrogen at −196 °C. The vacuum headspace apparatus consists of a round-bottomed flask filled with the fresh rose flowers, connected to two cold traps via a glass tube with a spherical ground joint. When the air is removed by a vacuum oil pump, the fragrance substances vaporise, together with the water present in the flowers, and are frozen out in the first cold trap. A second cold trap is installed in series with the first one, in connection with an active charcoal tube, to prevent the possibility that impurities from the vacuum pump may adulterate the analyte concentrate. Finally, the collected fragrance substances are passed into an organic solvent and after separation of the condensed water the mixture is separated and identified by coupled gas chromatography–mass spectrometry (GC-MS).Although the vacuum headspace technique may provide a more or less authentic reproduction of the fragrance profile of an individual rose cultivar, it has the crucial disadvantage that flowers can only be analysed after they have been picked. In most cases enzymatic processes, initiated by the damage of plant cells, dramatically change the profile of the volatile fraction and in such cases it is very difficult to study the biochemical pathways occurring in the plant. Therefore, in recent times another technique, the so-called closed-loop stripping method, is usually applied in this context. Here, the fragrance substances released from the rose flower are carried along in a stream of carrier gas and are finally concentrated on a suitable adsorbent, for example, active charcoal or an organic polymer. To prevent impurities from the pump getting into the vapour phase surrounding the flower, a second charcoal tube is placed in series.In order to perform on-site measurements, the rose flowers are placed in a suitable-sized conical flask and the opening of the flask is carefully closed off with a plastic film, taking care not to damage the flower stalks. A small suction pressure pump transports the volatiles in the stripping apparatus. The development of this dynamic headspace technique allows enrichment and reproducible identification of the most relevant volatile rose substances without damaging the flower being analysed. Subsequently, the loaded molecules to be analysed are thermally desorbed from the adsorbent cartridge by slow heating (2 min at 200 °C) and it is possible to collect them in a loop cooled with liquid nitrogen. Finally, this loop is rinsed in helium steam, so that the volatile rose substances can be completely injected on a gas chromatography column. Active charcoal has been found to be an extremely powerful and nonselective adsorbent which can be applied very efficiently to a wide range of organic substances. However, because the adsorption is so strong, it may sometimes be a problem desorbing the molecules to be analysed; furthermore, often numerous artefacts are generated during thermal desorption. Porous synthetic polymers like Tenax™ (poly [2,6-diphenyl-p-phenylene oxide]) also exhibit a high adsorptive capacity towards volatile and semivolatile plant substances but they clearly show fewer problems when the analytes are desorbed.First primitive GC-headspace techniques still implied the treatment of a relatively large quantity of raw material and the use of solvents which could produce artefacts. In comparison to that, dynamic GC headspace collection is much more effective and allows efficient enrichment of volatile, medium-volatile, and even low-volatile minor plant constituents.During recent years, solid-phase microextraction (SPME) using various porous polymers as fibre materials (e.g. polydimethylsiloxane (PDMS)) has been increasingly applied in the analysis of plant volatiles. Compared with conventional liquid-liquid extraction, this method is convenient, easy to use, less time-consuming and requires no solvents. In the general SPME procedure, the coated fused silica fibre is exposed to the sample or its headspace and the volatile analytes present in the sample are adsorbed in the fibre coating. After that, the fibre is directly introduced into the injection port of a gas chromatograph; the extracted volatile substances are thermally desorbed and subsequently separated on an appropriate GC column. For the analysis of living rose flowers, one single blossom still attached to the plant is placed into a suitable glass chamber in such a way that the petals are not injured. This chamber contains sidearms closed with GC septa; the hole at the bottom of the glass flask is sealed with plastic film (Figure 4). Depending on the individual ambient temperature and the rose cultivar, the PDMS fibre is placed inside the glass chamber for 15–30 min. If the fragrant intensity of the rose blossom is high enough, this time is sufficient to extract most of the volatiles released by the plant material to be analysed.Figure 4. Solid-phase microextraction field sampling of a rose blossom for subsequent gas chromatography analysis of the volatile compounds collected.Figure 5 shows the results of SPME-GC analyses performed on five different rose cultivars grown in the Europa-Rosarium in Sangerhausen, Germany. The cultivars ‘Ave Maria’ and ‘Lavender Lassie’ contain as main volatile constituents phenylethyl alcohol and phenylethyl acetate. Other characteristic rose fragrance substances such as hexyl acetate, citronellol, citronellyl acetate, methyleugenol, germacrene-d and heneicosane were also identified in the headspace of the flower. In contrast, the remontant rose reveals a completely different pattern of volatile components; here the main components are nerol, geraniol, citronellol and the corresponding acetates which significantly contribute to the fresh citrus fragrance of this rose cultivar. The individual smell of cultivars ‘Roundelay’ and ‘Kore’ is mainly influenced by the intensely odorous substances β-ionone (fruity character) and 3,5-dimethoxytoluene.Figure 5. Results of nondestructive and rapid solid-phase microextraction gas chromatography on-site measurements on rose flowers.Figure 6 presents the SPME-GC separation of volatile rose substances collected from the flower of the old cultivar ‘Pariser Charme’. The main components in the chromatogram, which have been detected by mass spectrometry, are germacrene-d, hexyl acetate, cis-3-hexenyl acetate, citronellol, citronellyl acetate, nerol and geraniol.Figure 6. Solid-phase microextraction gas chromatography measurement of volatile rose substances collected from the flower of ‘Pariser Charme’. Gas chromatographic separation was performed with a polyethylene glycol column (HP-Innowax, 30 m × 0.25 mm × 0.5 μm) and mass spectrometry detection. 1, hexyl acetate; 2, cis-3-hexenyl acetate; 3, β-caryophyllene; 4, citronellyl acetate; 5, nerol; 6, germacrene-d; 7, neryl acetate; 8, geranial; 9, geranyl acetate; 10, citronellol; 11, nerol; 12, geraniol.Several rose cultivars have been analysed by different GC-headspace techniques presenting clearly differing fragrance types. The volatile fraction of ‘Othello’, a modern English hybrid developed in 1986 by David Austin, contains as its main constituents citronellol, nerol and geraniol; the other fragrances identified by GC-MS occur in amounts of 3% (e.g. p-cymene, cis-hexenyl acetate, β-caryophyllene, neral, germacrene-d, geranial, nonadecene). The fragrance of the dark red blooms of this rose cultivar can be described as strongly rosy and narcotic.The famous old French rose cultivar ‘Duchesse de Montebello’, which has a fresh-floral, typically rosy fragrance, shows a similar pattern of volatile substances. One exception is the relatively high level of β-phenylethyl alcohol (approx. 30%) and β-phenylethyl acetate (approx. 7%). Other constituents in the volatile fraction are only present in lower amounts (e.g. α-pinene, sabinene, myrcene, cis-3-hexenyl acetate, β-caryophyllene, citronellol, nerol). In contrast, the flowers of the bush rose ‘Lichtkönigin Lucia’® give off a floral-green scent (reminiscent to fresh cut grass) with spicy and fruity aspects. The main constituent of the headspace was found to be β-phenylethyl acetate (approx. 37%), but comparatively high concentrations of cis-3-hexenyl acetate, trans-2-hexenyl acetate and trans-2-hexenol were also detected. The last three secondary metabolites in particular are responsible for the characteristic fresh-green fragrance of this rose cultivar. The fruity note of this hybrid is mainly related to the occurrence of the intensely odorous β-ionone. This fragrance material only occurs in amounts of about 0.4% in the volatile fraction but with regard to the very low odour threshold it is important for this individual rose type.Generally, it has to be mentioned here that compounds to be detected in high concentrations are not necessarily the most efficient ones to express the scent; sometimes only a small number of minor compounds have a high impact on the whole fragrance perception.The bright-pink flowers of the cultivar ‘Queen Elizabeth’® (‘Charlotte Armstrong’ × ‘Floradora’) also possesses a very specific fragrance profile. The GC is dominated by 3,5-dimethoxytoluene. Other principal constituents are dihydro-β-ionone, dihydro-β-ionol as well as cis- and trans-theaspirane. Especially, dihydro-β-ionone was found to be responsible for the characteristic fruity and raspberry-like odour of the rose flower. Generally, ionone derivatives and theaspirane substances are known to be produced by enzymatic decomposition of various carotinoids which are predominantly found in hybrid roses in addition to other plant pigments such as flavonoids or anthocyanes.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B0122276205000707RNA AND DNA TUMOR VIRUSES--MECHANISM OF CELL TRANSFORMATION AND ROLE IN HUMAN CANCERMaurice Green, in Membranes and Viruses in Immunopathology, 1972Human (?) RD-114 Virus–Extensive Homology With RNA From Hodgkin s Lymphomas (24)Hydroxylapatite chromatography analysis of complexes formed between RD-114 3H-DNA and Hodgkin s Lymphoma RNA. Reaction mixtures between RNA from 27 human tumors and RD-114 3H-CNA were analyzed by hydroxylapatite chromatography at 60°C (Fig. 6). RD-114 viral 70S RNA formed hybrids with 52% and 60% of the RD-114 3H-DNA. All five RNAs isolated from the lymph nodes and lung of patients with Hodgkin s lymphomas were analyzed in duplicate and formed hybrids with 5 to 10% of the RD-114 3H-DNA product. No significant hybrid formation was detected on hydroxylapatite when RD-114 3H-DNA was annealed with RNA from six other human lymphomas, two breast carcinomas, two lung carcinomas and a variety of other tumors. RNA from one of the three reticulum cell sarcomas that were analyzed formed significant amounts of hybrid with RD-114 3H-DNA (Fig. 6); possibly this tumor is Hodgkin s lymphoma that was misdiagnosed as often occurs. The positive hybridizations with Hodgkin s lymphoma RNA are highly significant for they were consistent, being positive in duplicate, and represented a confidence level of over 99.9% (2.7% duplex equals three standard deviations, i.e., 3 σ in Fig. 6. The extent of hybrid formation are minimal values since when the amount of Hodgkin s RNA annealed was increased from 200 to 400 μg, nearly 20% of 3H-DNA was converted to a hybrid form. RNAs from normal lymph nodes and spleens analyzed simultaneously did not hybridize significantly with RD-114 3H-DNA.Fig. 6. Hydroxyapatite chromatography analysis of RD-114 3H-DNA annealed with RNA from human cancers.Cs2SO4 density gradient analysis of complexes formed between RD-114 3H-DNA and Hodgkin s lymphoma RNA. The amounts of RD-114 3H-DNA complexed with Hodgkin s RNA as detected by equilibrium centrifugation in Cs2SO4 were similar to those obtained by hydroxylapatite analysis. For example, the Hodgkin s RNAs that formed the most and the least hybrid on hydroxylapatite gave peaks in Cs2SO4 representing 12.5% (310 c.p.m., Fig. 7.A) and 3.8% (77 c.p.m., Fig. 7.B) of RD-114 3H-DNA, respectively. RNA from normal lymph nodes and spleens did not yield significant amounts of complex (Fig. 7C and D.Fig. 7. Analysis by equilibrium centrifugation in Cs2SO4 density gradients of hybridization reactions between RD-114 3H-DNA and RNA from Hodgkin s lymphomas, normal spleen, and normal lymph nodes.The complexes formed with Hodgkin s lymphoma RNA have the properties of true DNA-RNA hybrid molecules. The RNAs extracted from Hodgkin s lymphomas formed complexes with RD-114 3H-DNA that behaved as DNA-RNA hybrids on hydroxylapatite and banded at the position of RNA in Cs2SO4 density gradients. Two additional stringent criteria for DNA-RNA hybrids were also examined: (i) Thermal stability consistent with base composition; and (ii) resistance to digestion by nucleases that degrade single-stranded DNA. (i) Complexes between two Hodgkin s RNAs and RD-114 3H-DNA were bound to hydroxylapatite at 60°C in 0.12 M phosphate buffer and dissociated stepwise by raising the temperature in 5°C increments. The mean thermal dissociation temperatures (Tm) were 77.4 and 77.8 (Fig. 8A and B which is consistent with an approximately G+C content of 42-43% for the RD-114 3H-DNA sequences that hybridize with Hodgkin s RNA, values in the range of mammalian mRNA molecules (25). (ii) Neurospora crassa nuclease (26) degraded 97% of single-stranded DNA but DNA-RNA hybrids prepared from RD-114 3H-DNA and viral 70S RNA were resistant. The Hodgkin s DNA-RNA complexes that were isolated on hydroxylapatite and from Cs2SO4 gradients were 80-95% resistant to the action of this nuclease (unpublished data).Fig. 8. Thermal melting profiles of hybrids formed between RD-114 3H-DNA and Hodgkin s RNA.Viral specificity of DNA-RNA hybrids formed with Hodgkin s lymphoma RNA. The Hodgkin s RNA sequences that hybridize with RD-114 3H-DNA were not detected in appreciable quantities in the DNA product prepared from two animal RNA tumor viruses (20,27). As shown in Table 4, five Hodgkin s RNAs hybridized extensively with the RD-114 3H-DNA product but not with the murine sarcoma virus (MSV) or the feline sarcoma virus (FeSV) DNA products. Small amounts of complex with MSV DNA were detected only Cs2SO4 gradients (unpublished data).TABLE 4. Complex Formation Between the 3H-DNA Product of RD-114, MSV(M), and FeSV(G) with RNA from Hodgkin s Lymphomas as Detected by Hydroxyapatite ChromatographySource of 3H-DNARD-114MSV(M)FeSV(G)Source of RNA%hybrid*S.D.**=S.D.=S.D.=± 0.85± 0.93± 1.41Homologous viral70S RNA52, 6060, 6130,31Hodgkin slymphomas: Tumor No.:  C10117.0, 4.10.6, 1.01.4  C692 Z10.1, 9.02.80.6  C191 Y19.4, 9.51.9,-0.4, 1.61.9  C692 08.6, 9.20.42.1  C692 P9.3, 10.82.60.7*Background hybridization, i.e.,3H-DNA bound to hydroxyapatite after annealing with RNA derived from non-Hodgkin s tissues, was subtracted. Background values were 3.2% ± 0.85 for 22 RD-114 hybridizations; 9.4% ± 0.93% for 22 MSV hybridizations; and 3.48% ± 1.41 for 22 FeSV(G) hybridizations. Background values were not significantly different when RNA was omitted and most likely represent the formation of DNA-DNA duplexes.**S.D. = standard deviation.Significance of hybrid formation between RD-114 3H-DNA and Hodgkin s RNA. Recently Cs2SO4 density gradient analysis was used to show that the DNA products of the RNA-directed DNA polymerase of several RNA tumor viruses of animal origin form complexes with RNA from human tumors. These complexes are presumed to be DNA-RNA hybrids and have not been further characterized. Four origins of these complexes may be proposed: (i) The presumed hybrid may not be a true hybrid but may consist of poorly matched, partially homologous sequences formed between human cancer RNA and the DNA product. For example, weak complexes that band in CsCl density gradients are formed at room temperature between viral DNA strands and G-rich polyribonucleotides, (ii) The presumed hybrid may be a complex formed between viral poly d(T) and cellular poly (A); the DNA product may contain poly d(T) sequences copied from the large poly (A) stretches in the genome of RNA tumor viruses (28,29). (iii) The presumed hybrid may be formed between cancer cell RNA sequences and cell DNA sequences in the RD-114 3H-DNA product; RNA tumor virus particles contain cell nucleic acids that may be copied by the viral RNA-directed DNA polymerase. (iv) The presumed hybrid may be a true hybrid formed between virus-specific DNA sequences and complementary RNA sequences that are enriched in certain categories of cancer.Possibility (i) cannot explain the complexes with Hodgkin s RNA which have the properties of true DNA-RNA hybrids, i.e., retention on hydroxylapatite at 60°C, thermal stability, and resistance to nucleases specific for single-stranded DNA. Possibility (ii) is unlikely since complexes between poly 14C-d(T) and poly r(A) melt close at 61°C on hydroxylapatite (unpublished data), but those between RD-114 DNA and Hodgkin s RNA melt at 78°C. Complexes formed with cancer RNAs that are detected on Cs2SO4 gradients but not on hydroxylapatite may have originated from (i) or (ii). To examine possibility iii), i.e., that cell-specific sequences in RD-114 3H-DNA may be hybridizing with Hodgkin s RNA, we copied RD-114 70S RNA directly with the highly purified DNA polymerase prepared from avian myeloblastosis virus (Grandgenett, Gerard, and Green, unpublished data). Hodgkin s RNAs formed hybrids with 2 to 4% of several such \"synthetic” RD-114 3H-DNA products, as detected both by hydroxylapatite chromatography and on Cs2SO4 density gradients (unpublished data). These data indicate that it is virus-specific sequences that hybridize with Hodgkin s RNA, although the possibility that cell specific sequences are integral parts of the viral 70S RNA genome is not excluded at present. The most likely explanation for our results appears to be possibility (iv), i.e., RNA from Hodgkin s lymphomas share sequences with the RD-114 genome.The Hodgkin s RNA sequences that hybridize to RD-114 3H-DNA may have originated from either viral genes or from cellular genes homologous to viral gene sequences. The first possibility implicates infection with RD-114 or a related virus although this data is not sufficient to differentiate between a function as a causative agent or as a passenger virus. Several clinical, pathological, and epidemiological features of Hodgkin s disease are consistent with viral involvement. But an alternative and more attractive explanation for the presence of RD-114 sequences in Hodgkin s lymphomas is the oncogene hypothesis, i.e., specific base sequences common to RD-114 or a related virus are expressed in human cancer. If as suggested by these hybridization studies, RD-114 specific sequences are specifically expressed in some human neoplasms, the further characterization of these RNA sequences, the identification of their cellular functions, and the analysis of the regulation of these functions may lead to advances in understanding and controling cancer.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780122072505500208Infections Due to Non-Clostridial AnaerobesA. TREVOR WILLIS DSc, MD, FRACP, PhD, FRCPath, FRCPA, in Anaerobic Bacteriology (Third Edition), 1977Direct gas liquid chromatographyGas liquid chromatographic analysis of samples of pus provides a rapid (30 min) and reliable means for the presumptive differentiation of anaerobic from aerobic infections (Phillips, Tearle and Willis, 1976; Gorbach et al., 1976). An aliquot of the specimen is processed in the same way as a bacterial culture: 1 ml of the sample is acidified with a few drops of 50% sulphuric acid and the volatile acids extracted in 1 ml of diethyl ether. One μl of the ether layer is withdrawn for injection into the chromatograph. The detection of volatile fatty acids other than acetic acid is strong presumptive evidence of the presence of significant numbers of anaerobes in the specimen. Exudates from sepsis due to facultatively anaerobic bacteria only, contain no volatile acids or acetic acid only.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780407000810500104Examination and Identification of AnaerobesA. TREVOR WILLIS DSc, MD, FRACP, PhD, FRCPath, FRCPA, in Anaerobic Bacteriology (Third Edition), 1977GAS LIQUID CHROMATOGRAPHYIn the past, workers encountered considerable difficulty in establishing the specific identities of a variety of non-sporing anaerobic bacteria. Not unreasonably, emphasis was placed on the habitat, microscopic morphology and conventional biochemical reactions of these organisms for their classification, taxonomic determinants which were (and still are) well suited to the classification of many other groups of bacteria, including the clostridia. Generally speaking, attempts to classify the non-sporing anaerobes by these criteria met with failure, so that once a consistent set of characteristics had been obtained for any particular strain, it was often impossible to assign a specific name to the organism. Thus, Eggerth and Gagnon (1933) isolated 118 strains of fusiform rods from human faeces, and divided them into 18 species, only two of which had been described previously. New species, often represented by only one or two strains were frequently recorded, but were not encountered by later workers. The confusion regarding the Gram-negative anaerobic bacilli was emphasized by Dack (1940) who listed 22 synonyms of F. necrophorum.In their notes on the 36 species (and 6 variants) of anaerobic cocci listed in the seventh edition of Bergey s Manual, Breed, Murray and Smith (1957) admitted that some species, possibly many of them, may be identical with one another. Such uncertainty was due in part to the rarity with which individual strains could be identified with described species, and to the characteristic variation which these organisms showed in their cultural reactions when tested by conventional methods. Referring to this problem, Smith (1955) commented that ‘primarily, the trouble lies in the fact that variability in cultural characteristics is the rule rather than the exception, and, given a sufficient number of isolates, unbroken chains of organisms leading from one species to another may be arranged’.It was suggested by Beerens et al. (1962) that these problems might be resolved by the application of other biochemical tests, in particular the detection of the types of volatile fatty acids produced from fermentation of glucose. This suggestion followed from an earlier observation of Guillaumie et al. (1956) that among the metabolic end products of bacterial growth various combinations of formic, acetic, propionic and butyric acids were characteristic of the different genera of the Bacteroidaceae. In the third edition of his manual, Prévot (1957) made frequent reference to the products of bacterial metabolism although he did not use them as cardinal definitive features.At that time the techniques for the extraction and identification of fatty acids from cultures were laborious and time-consuming (Charles and Barrett, 1963), and this type of analysis was widely regarded in clinical microbiological circles as essentially of academic or of limited taxonomic interest. In 1952, James and Martin developed the technique of gas chromatography; they used a moving gas phase to separate fatty acids on a column containing silicone oil supported on kieselguhr. This experiment initiated a surge of interest in gas chromatography which led to the development of the highly sophisticated gas chromatography systems that are available today.American workers were quick to explore the application of this new technique to the analysis of volatile fatty acids produced by bacterial metabolism, and it soon became evident that the concept of Beerens and his colleagues was of fundamental importance in the characterization of the non-sporing anaerobic bacteria. Largely due to the careful and detailed studies of Professor W. E. C. Moore and his colleagues at the Virginia Polytechnic Institute Anaerobe Laboratory, much valuable information is now available concerning the relationships of metabolic products to the taxonomy of the anaerobic bacteria (see Moore, 1970; Bricknell, Sutter and Finegold 1975; Moore, Cato and Holdeman, 1966, 1969; Holdeman and Moore, 1975; Moore and Holdeman 1972).At present it is fair to say that in the routine clinical laboratory detailed specific identification of most non-sporing anaerobic isolates is not mandatory. For those who wish to ‘speciate’ their isolates, however, chromatographic analysis of volatile metabolic products is essential.Although this may be accomplished by paper chromatographic methods (Slifkin and Hercher, 1974), gas liquid chromatography is much more convenient. In addition to its proven value in the specific identification of pure cultures of anaerobes, gas liquid chromatography also finds a place in the examination of mixed cultures (Mitruka et al., 1973). Of special interest are the studies of Gorbach et al., (1974, 1976) and Phillips, Tearle and Willis, (1976) which show that direct gas liquid chromatographic analysis of clinical specimens (notably pus) enables anaerobic infections to be recognized within a few minutes of their collection.Principle of gas liquid chromatographyGas liquid chromatography (partition gas chromatography) is the process by which a mixture is separated by solution partition between two immiscible phases - a ‘liquid stationary phase’ distributed on a solid inert support material in a column, and a ‘moving gas phase’. The separating principle depends on the difference in the partition coefficients between the liquid and gas phases of the constituents of the mixture. Those constituents which favour the gas phase have low partition coefficients and move quickly through the column, while those that favour the liquid phase have high partition coefficients and are delayed in their transit through the column.In practice, a little of the liquid mixture for analysis is inoculated onto the column which is held at a temperature that ensures vaporization of the volatile components of the mixture; clearly, the stationary phase in the column must be non-volatile at this temperature. A steady flow of carrier gas (commonly nitrogen) carries the gas phase of each volatile component through the column until each is eluted. The volatile components pass through the column at different rates, so that they are eluted from the end of the column at different times. A detector at the end of the column records the emergence of the various volatile constituents of the original mixture, which is thus analysed.The detector most suited to microbiological analyses is the flame ionization detector (FID). In this system the detector is used to measure an ionization current in a hydrogen flame. When an eluted substance is burned in the flame there is an increase in the ionization current; this signal is fed to an amplifier and then to a strip chart recorder. The recorder plots the attenuated detector output against a range of time scales.A recommended specification is the Pye Unicam Series 104 chromatograph, the design concept of which allows for continuing updating of the instrument as requirements expand. A range of add-on modules is available which convert the basic Series 104 chromatograph to varying levels of automation. To the basic instrument in my laboratory has been added the Auto-jector S4, which enables analysis of up to 100 samples to be carried out automatically.In the Series 104 Chromatograph the column packing used for the analysis of volatile acids and methyl derivatives of non-volatile acids in polyethylene glycol adipate (10%) (liquid phase) adsorbed onto Celite (mesh size 100−120) (support). For the analysis of alcohols, the column packing used is polyethylene glycol 400 (10%) (liquid phase) adsorbed onto Celite (mesh size 100–120) (support).Analysis of acid and alcohol products of bacterial metabolismBacterial culturesCultures for gas liquid chromatographic analysis are grown in VL glucose broth in an anaerobic atmosphere containing 10% carbon dioxide. Analysis may be carried out once good growth has developed (24−48 h for many species), although some organisms, notably proteolytic species, require 5 days’ incubation for maximum yields of butyric acid.Analysis of volatile fatty acids and alcoholsAcidify the culture by adding 0.2 ml of 50% H2SO4 to each 12 ml of culture. This ensures that fatty acids are present in the free form, and are therefore soluble in ether.Pipette 2 ml of the acidified culture into a centrifuge tube, add 1 ml of ethyl ether, stopper the tube and mix by inversion. Lightly centrifuge to separate the ether and aqueous phases, and pipette off the ether layer for analysis.Inject 1 µl of the ether extract into the column.Analysis of methyl derivatives of non-volatile fatty acidsNon-volatile fatty acids (pyruvic, lactic, fumaric and succinic acids) are converted to volatile methyl derivatives for chromatographic analysis as follows.Place 1 ml of the acidified culture into a small test tube. Add 0.4 ml of 50% H2SO4 and 2 ml of methanol. Stopper, and heat at 55 ±C in a water bath for 30 min. Add 0.5 ml of chloroform and 1 ml of distilled water, and mix by inversion. If necessary, separate the chloroform and aqueous phases by light centrifugation, and withdraw the chloroform (lower) phase for analysis.Inject 1 µl of the chloroform extract into the column.Standard solutionsEach time chromatographic analyses are carried out it is necessary to prepare ‘control’ chromatograms from mixtures of standard solutions of the different substances being sought.VOLATILE ACID STANDARD STOCK SOLUTIONSNine standard stock solutions are prepared as follows:1.Acetic acid5.7 ml2.Propionic acid7.5 ml3.Isobutyric acid9.2 ml4.Butyric acid9.1 ml5.Isovaleric acid12.7 ml6.Valeric acid12.5 ml7.Isocaproic acid12.6 ml8.Caproic acid12.6 ml9.Heptanoic acid12.6 mlMake up each of these nine standard solutions to 100 ml with distilled water, and keep them well stoppered.Working mixed standard solutionMix together 1 ml of each stock standard, and make up the volume to 100 ml with distilled water. Keep this working standard well stoppered.Analysis of working mixed standardProceed as described under analysis of volatile fatty acids and alcohols in cultures (p. 99).ALCOHOL STANDARD STOCK SOLUTIONSSix standard stock solutions are prepared as follows:1.Ethanol10.0 ml2.Propanol3.5 ml3.Isobutanol0.5 ml4.Butanol1.0 ml5.Isopentanol0.5 ml6.Pentanol0.5 mlMake up each of these six standard stock solutions to 100 ml with distilled water, and keep them well stoppered.Working mixed standard solutionMix together 1 ml of each stock standard, and make up the volume to 100 ml with distilled water. Keep this working standard well stoppered.Analysis of working mixed standardProceed as described under analysis of volatile fatty acids and alcohols in culture (p. 99).NON-VOLATILE ACID STANDARD STOCK SOLUTIONSEight standard stock solutions are prepared as follows:1.Pyruvic acid6.8 ml2.Lactic acid8.4 ml3.Oxalacetic acid6.0 g4.Oxalic acid6.0 g5.Methyl malonic acid6.0 g6.Malonic acid5.0 g7.Fumaric acid6.0 g8.Succinic acid6.0 gMake up each of these standard solutions to 100 ml with distilled water, and keep them well stoppered.Working mixed standard solutionMix together 1 ml of each stock standard, and make up the volume to 100 ml with distilled water. Keep this working standard well stoppered.Analysis of working mixed standardAcidify 12 ml of the working mixed standard solution by adding 0.2 ml of 50 per cent H2SO4. Then proceeds as directed under analysis of methyl derivatives (p. 100).Figure 3.5b. Typical chromatogram of methylated acidsMedium controlSince different batches of uninoculated culture medium may contain varying small amounts of acids, especially acetic, lactic and succinic acids, corrections for these must be made when chromatograms of cultures are interpreted. For this reason a ‘control’ analysis of the uninoculated medium is performed with each new batch of culture medium.Interpretation of chromatogramsThe presence and approximate amounts of acids and alcohols in a culture extract are determined by comparing the culture chromatograms (duly corrected by the chromatograms of the uninoculated medium) with the chromatograms of the working standard solutions (Figures 3.5 and 3.6). Identification of the culture is then accomplished by comparing its chromatograms against those of known species.Figure 3.5a. Typical chromatogram of volatile acidsFigure 3.6. Typical chromatogram of alcoholsChromatographic patterns of many of the clinically important anaerobes are summarized in later chapters. Chromatographic patterns of a much wider range of anaerobes have been published by Holdeman and Moore (1975).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780407000810500074The Isolation and Characterization of Immunoglobulins, Antibodies, and Their Constituent Polypeptide ChainsJaton Jean-Claude, ... Pierre Vassalli, in Immunological Methods, 1979IV FRACTIONATION BY GEL FILTRATION CHROMATOGRAPHYThis technique involves chromatography on Bio-Gel P-300 or Sephadex G-200, which separates proteins or crude Ig mixtures according to the molecular size of the proteins. The method is largely used for quantitative fractionation of the macroglobulin IgM, which can be isolated free of IgG or IgA proteins by one-step chromatography. The IgM of most animal species is excluded from the gel and recovered in the first elution peak in the presence of a high salt concentration (0.5 M NaCl buffered with 0.02 M phosphate buffer, pH 7.3, or 0.05 M Tris–HCl, pH 8.0). Because of the euglobulin properties of many IgM or Waldenström macroglobulins, dialysis of serum containing these macroglobulins against low ionic strength buffers causes the macroglobulins to precipitate. The washed precipitates are redissolved in buffered 0.5 M NaCl and eluted on a Sephadex G-200 column. The void volume fractions from the gel contain the IgM protein. Mouse and rabbit IgM s isolated in this way are often contaminated by α2-macroglobulin, which coelutes with IgM. The contaminant is best removed by subjecting the impure IgM preparations to agarose block electrophoresis: IgM migrates toward the cathode, whereas α2- macroglobulin travels toward the anode (see Section V).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780124427501500073IMMUNOCHEMISTRY OF CATTLE BLOOD GROUP J HAPTENMartin I. Horowitz, Bronislaw L. Slomiany, in Blood and Tissue Antigens, 1970Analytical proceduresMethanolysis, gas liquid chromatography (GLC) and TLC. Methanolysis (12) of the J hapten glycolipid was performed in 1.0 M methanolic HCl at 80 for 18 h. Methyl esters of fatty acids were extracted three times by equal volumes of petroleum ether (bp 30–60°). Fatty acid methyl esters were determined by GLC with the Perkin Elmer 801 gas Chromatograph equipped with glass columns 6 ft. × 14 in. packed with either 3% SE-30 (on Chrom W-AW-DMCS, 80–100 mesh) or with 15% diethylene glycol succinate (DEGS) (on Chrom W-HMDS, mesh 80–100). For analysis of fatty acid methyl esters on SE-30, columntemperature was maintained at 150° for 6 minutes after injection of sample and then increased at 4°/minute to 230°; GC on DEGS was performed isothermally at 148°. Helium gas was used as carrier at a flow of 43 ml/min. Column performance was evaluated with standard mixtures of fatty acid methyl esters (Applied Science, State College, Pa.).The HCl was removed from the methanol phase by passage down a column (1.2 × 25 cm) of Bio Rad AG 3-x4 (20–50 mesh). Methyl glycosides and bases were recovered by washing the column with 50 ml of methanol. The solution containing methyl glycosides and long chain base was evaporated to dryness and 2 ml of 1 N HCl was added. The mixture was hydrolysed at 100 for 3 h in a sealed tube. The HCl was removed by repeated evaporation. TLC for long chain bases was performed on silica gel G plates, 250 μthick, activated at 130 for 1 h and developed with the solvent system chloroform-methanol-2% ammonia (80: 20:2) (13). Spots were visualized by 0.3% ninhydrin in n-butyl alcohol. TLC of sugars was performed on silica gel G plates according to Gal (14). Paper chromatography was performed on Whatman # 1 by descending chromatography with the solvent system n-butyl alcohol-pyridine-H2O (3:1:1) (15). TLC for detection of amino acids was performed on fraction V (16).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780120570508500163Characteristics of the Pathogenic and Related ClostridiaA. TREVOR WILLIS DSc, MD, FRACP, PhD, FRCPath, FRCPA, in Anaerobic Bacteriology (Third Edition), 1977Biochemical characteristicsTypes A, B, E and F ferment glucose, maltose and sucrose; types C and D ferment glucose and maltose, but not sucrose; type G is non-saccharolytic. Cl. botulinum is thus exceptional among the pathogenic clostridia in that it commonly ferments sucrose, but not lactose (Table 4.3). All types produce gelatinase and hydrogen sulphide, but none produces indole.Table 4.3. Some properties of different toxicological types of Cl. botulinumFermentation ofCl. botulinum typeGlucoseMaltoseLactoseSucroseGelatinase (gelatine agar)Proteinase on milk agarLipase on egg yolk agarHaemolysis on horse blood agarPathogenic for menType A++–++++++Type B         Proteolytic+±–++++++Non-proteolytic+±–++–+++Type C++––+–++–Type D++––+–++–Type E+±–+±–+++Type FProteolytic++–++++++Non-proteolytic+±–++–+++Type G––––++––Not known±=Strains varyMetabolic products detected by gas liquid chromatography are as follows:1.All type A strains and proteolytic strains of types B and F produce predominantly acetic and butyric acids, with smaller amounts of propionic, isobutyric and isovaleric acids, and propyl, isobutyl, butyl and isoamyl alcohols (cf. Cl. sporogenes).2.All type E strains and non-proteolytic strains of types B and F produce acetic and butyric acids only.3.All type C and D strains produce predominantly acetic, propionic and butyric acids.4.Type G strains produce predominantly acetic acid with lesser amounts of isobutyric, butyric, isovaleric and lactic acids.Mayhew and Gorbach (1975) suggested that gas liquid chromatographic analysis of foods for short-chain fatty acids may provide presumptive evidence of Cl. botulinum contamination. Unfortunately, Cl. sporogenes produces almost identical metabolic products, so that final identification must still depend on specific toxin assay (see p. 126).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780407000810500086HUMAN TRANSFER FACTOR: EXOGENOUS LABELLING, PURIFICATION, AND ROLE OF RIBONUCLEIC ACID SEGMENT1Gary V. Paddock, ... H. Hugh Fudenberg, in Immunobiology of Transfer Factor, 1983Purification and LabellingWe have previously shown that material contained in HPLC Fraction 7 (TF-H7) can be labelled with 125I. Label and activity co-migrate through both boronate affinity chromatography and cellulose TLC (4). Figure 2 shows an experiment in which HPLC fractionated TF-H7 was iodinated with I-125, and subsequently purified of unattached label by boronate chromatography. The label was distributed primarily in cellulose TLC fractions 1–4 and TF activity was found in fraction 2. All four fractions were then subjected to PEI TLC. A multitude of labeled moieties can be seen. TF activity was found in the region indicated in Figure 2.Fig. 2. I–125 labelling of TF-H7. Track C (figure on left) shows purification of labelled TF-H7 by cellulose TLC developed with methanol: HCl: H2O (70:20:10; v/v/v). Fractions 1–4 indicated by brackets were eluted with water and correspond to tracks 1–4, respectively, in the PEI TLC developed by homochromatography (figure on right). TF activity was found in fraction 2 of the cellulose TLC. TF activity has been narrowed to the bracketed region of the PEI TLC.Figure 3A shows another experiment in which cellulose TLC fractionated TF-H7 was labeled with I–125 and again further purified by boronate affinity chromatography. The labeled material was then repurified by cellulose TLC and fractions assayed for activity. Activity was found to exist in the labeled material migrating in the usual position relative to nucleotide markers (3,4). The material was then further fractionated alongside labeled DNA markers using PEI TLC (Fig. 3B) or acrylamide gel (20%) electrophoresis (Fig. 4). A multitude of labeled moieties were found to separate in each case. Digestion with alkaline phosphatase, pancreatic ribonuclease A, and pronase were employed to help locate TF-H7. Material (undigested) was also eluted from the gel to test for activity (results not yet available). These results show that there are a large number of nucleotides containing moieties in DLE that can be effectively and simply separated. We plan to utilize additional separative techniques in order to determine whether the material present in the active gel fraction is indeed pure or requires further fractionation.Fig. 3. Cellulose and PEI TLC of I–125 labelled TF-H7. Figure 3A shows an autoradiograph cellulose TLC of I–125 labelled TF-H7. Both the water and acetic acid eluates from the prior boronate chromatography step are shown. TF activity was found only in the bracketed region of the water eluate (see arrow). The circles were dram on the X-ray film autoradiograph to match with nucleotide markers located by UV absorbance. The circles were photographed for a longer time than the remainder of the autoradiograph to increase visability. B denotes base, N denotes nucleoside, 3′P denotes nucleoside 3’ monophosphate and 5′P denotes nucleoside 5’ monophosphate. The four circles in ascending order are always G,A,C and U except that a slow moving contaminant is always found in the nucleoside mix. Figure 3B shows PEI TLC of the active material eluted fran the cellulose TLC in Figure 3A. Tract 1 is undigested material and tracks 2–4 correspond to digestion with BAP, pancreatic RNase A, and pronase, respectively. To the right are 32P-labelled DNA size markers obtained from DNA sequencing reactions on duck genome recombinant DNA fragments (16).Fig. 4. (see previous page). Acrylamide gel electrophoresis of TF-H7. Material eluted from the region indicated in Figure 3A was electrcphoresed in 20% acrylamide as follows. Part A shows undigested material in tracts A-E. Regions 1–7 were separately excised from tracks A-D and were eluted with 0.5M sodium acetate for further study. Part B shows a comparison of 1) undigested material, 2) BAP digested material, 3) pancreatic RNase A digested material, or 4) pronase digested material. Part C shows 32P-labelled DNA size markers (16) with base identity and nucleotide number indicated to the right. The bromphenol blue dye marker was run 12 cm for this 40 cm gel.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780124098503500103Technological advancements in industrial enzyme researchVazhakatt Lilly Anne Devasia, ... Usha D. Muraleedharan, in Advances in Biological Science Research, 20196.7 Enzyme inhibitor studiesConventionally, spectrophotometry (UV and fluorescence), chromatography (HPLC), and electrochemical methods have been used for screening enzyme inhibitors. However, more recently, microscale enzyme reactors that utilize capillary electrophoresis and enzyme immobilization techniques for in-house enzyme assays, determination of enzyme kinetics, and enzyme inhibitor studies have been developed [38,39].In-line and precapillary enzyme assays based on capillary electrophoresis have been popularized in inhibitor screening studies because of fewer requirements of the samples and solvent systems, efficient and faster separation, and the ease to couple with automated detection systems. The in-line capillary electrophoretic method uses a single capillary for the entire length of time starting with sample loading and ending with detection [38]. This is different from precapillary enzyme assays wherein the capillary is used only to separate molecules. The enzyme reaction is carried out off-line, i.e., outside the capillary system and later injected into the capillary electrophoresis system. Precapillary detection has been used to determine the activity of membrane enzymes such as chlorophyllase (which is involved in chlorophyll metabolism), enzyme reaction rates, and inhibitors [39].In-line capillary enzyme assays are divided into two types: electrophoretically mediated microanalysis (EMMA) and immobilized enzyme microreactor (IMER) [38,39]. EMMA uses the difference in electrophoretic mobility of molecules for separation of constituents in the mixture. EMMA can also be used for enzyme activity determination, stereospecificity, enzyme-mediated metabolic reactions, etc. IMERs use immobilized enzymes in the first part of the capillary while the rest of the capillary is used for separation of the metabolites [39] and are excellent tools to study enzyme assays. Scientists have prepared such IMERs to study the inhibitors of tyrosinase and trypsin from natural extracts as well as for enhancement of the loading capacity of cytochrome P-450 and glucose-6-phosphate dehydrogenase during immobilization procedures [40,41]. The same technology could be used for screening inhibitors of other enzymes of industrial significance.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780128174975000069Recommended publicationsInfo iconProtein Expression and PurificationJournalFood MicrobiologyJournalExperimental ParasitologyJournalResearch in MicrobiologyJournalBrowse books and journalsFeatured AuthorsBetaInfo iconWing Tak WongHong Kong Polytechnic University, Kowloon, Hong KongCitations14,258h-index56Publications157Kewen TangHunan Institute of Science and Technology, Yueyang, ChinaCitations1,419h-index18Publications62About ScienceDirectRemote accessShopping cartAdvertiseContact and supportTerms and conditionsPrivacy policyWe use cookies to help provide and enhance our service and tailor content and ads. By continuing you agree to the use of cookies.Copyright © 2021 Elsevier B.V. or its licensors or contributors. 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