Plant tissue purification methods

Pectin extraction is a multiple stage physicochemical process which involves hydrolysis and extraction of pectin macromolecules from plant tissue, purification of the liquid extract and isolation of the extracted pectin from the liquid. These processes are influenced by various factors, mainly temperature, pH, and time (Pagan et al., 2001). The most commonly used methods for the extraction of pectin include direct boiling (Guo et al., 2014 Guo et al.,... 

Similar GC-MS methods have been developed for quantification of GAs [136] and ABA [Moritz, unpublished data] in small amounts of plant tissues without recourse to extensive sample purification. For analysis of GAs, high resolution selected ion monitoring (HR-SIM), SRM and four sector MS-MS were compared. The best selectivity was found with four-sector MS-MS, but the sensitivity was too low for the analysis of extracts from mg amounts of tissue. HR-SIM and SRM had similarly low limits of detection, but SRM provided the best balance of sensitivity and selectivity. This method has been used successfully for investigating GA levels in the apical zone of Salix pentandra [140]. However, analysis of GAs without extensive purification has to be performed with great care, as highly abundant GAs with similar retention times and mass spectra to the GAs of interest may interfere with the analysis. For example, GC-MS-SRM of a plant extract from Arabidopsis thaliana, without prior HPLC separation of some of the GAs of interest, resulted in inaccurate data being obtained [Moritz, unpublished data]. 

Extraction of Sodium Channel Blockers. A review of published reports shows that methods for purification of sodium channel blockers from bacterial cultures are similar to techniques for isolation of TTX and STX from pufferfish and dinoflagellates (30, 31, 38, 39). Typically, cell pellets of bacterial cultures are extracted with hot 0.1% acetic acid, the resulting supernatant ultra-filtered, lyo-philized, and reconstituted in a minimal volume of 0.1% acetic acid. Culture media can also be extracted for TTX by a similar procedure (Ji). Both cell and supernatant extracts are analyzed further by gel filtration chromatography and other biological, chemical, and immunological methods. Few reports describe purification schemes that include extraction of control samples of bacteriological media (e.g., broths and agars) which may be derived from marine plant and animal tissues. 

One method that has been used to acquire carbohydrates is isolation and purification from natural sources such as human or animal tissue, milk, urine, plants, and bacteria (see cross reference Isolation of glycans). Access to homogeneous carbohydrate stmctures can be challenging due to the difficulties in separation of complex mixtures, identification of carbohy-drate(s) contained within each fraction, and preparation of sufficient quantities from the limited amounts present in a particular sample. Alternatively, mixtures of unknown composition can be used to survey a broad repertoire of the glycome. On identification of a mixture containing one or more members with interesting receptor-binding properties, the mixture can then be deconvoluted by further fractionation and separation by routine analytical techniques (13). 

The classical column procedures for the preparation of lysozyme are difficult to use with high viscosity solutions such as serum. A batch method was developed based on affinity chromatography using deami-nated chitin [82]. The method was found to give a one-step purification of nearly theoretical amounts in tissue homogenates tested. These included tissue homogenates from humans, primates, avian egg white and plants. The use of a chitin-coated cellulose affinity column for purification of lysozyme has also been described [83]. 

Details of numerous HPLC methods that can be used for the purification of plant hormones are presented in Rivier and Crozier [1]. With some tissues, partitioning, cartridge systems and/or immunoaffinity chromatography can provide adequate sample purification prior to ABA and lAA analysis. However, HPLC fractionation is almost always required before GC-SIM analysis of individual cytokinins and GAs. As illustrated in Figs 2 and 3, good separations of free GAs and cytokinins of wide ranging polarity can be obtained by gradient elution, reverse phase HPLC. 

Which is better, GC-MS or immunoassay This is a question often asked about plant hormone quantification. GC-MS, which is now more widely available since the Introduction of bench-top instruments, has the advantage that it not only provides quantification of the hormone by the isotope dilution method, but also confirms the identity of the compound concerned by comparison of its spectrum with that of a standard. However, once validated for a particular tissue, immunoassay has the advantage that many samples can be analysed very quickly. Both techniques require sample pre-purification, often by the same methods. A more recent development is a powerful combination of the two technologies which uses the antibody immobilised on a polymer support as a method of affinity-purifying the hormones (together with interfering substances) from plant extracts prior to analysis by GC-MS. Immunoaffinity chromatography is discussed in the next section. 

Succinic acid plays an important role in the Krebs cycle and can deliver electrons to the electron transport chain. Succinic acid has been found in a very wide range of plant and animal tissues, though its purification challenged chemists for a long time before it was fincdly successfully purified from tissues. Today succinic acid can be produced readily in the laboratory, and one method even can use corn as a feedstock. In addition to its role in the Krebs cycle, succinic acid has been used as an intermediate in dyes, perfumes, paints, inks, and fibers. 

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