Plant extracts chromatography applications

Preparative planar chromatography is a very important step in the complicated procedures of isolation of group of compounds or pure substances from complex matrices. The method gives additional possibilities of using various adsorbents and eluent systems to achieve complete separation of stracmral analogs. The method also enables combining the various methods of sample application, plate development, and derivatization to achieve satisfactory separation of isolated plant extracts components. 

In earlier times, thin-layer chromatography (TLC), polyamide chromatography, and paper electrophoresis were the major separation techniques for phenolics. Of these methods, TLC is still the workhorse of flavonoid analysis. It is used as a rapid, simple, and versatile method for following polyphenolics in plant extracts and in fractionation work. However, the majority of published work now refers to qualitative and quantitative applications of high-performance liquid chromatography (HPLC) for analysis. Llavonoids can be separated. 

Afton, S., K. Kubachka, B. Catron, et al. 2008. Simultaneous characterization of selenium and arsenic analytes via ion-pairing reversed phase chromatography with inductively coupled plasma and electrospray ionization ion trap mass spectrometry for detection. Applications to river water, plant extract and urine matrices. J. Chromatogr. A 1208 156-163. 

Application of Prep-HPLC to PGS Analysis. Reverse phase liquid chromatography has proven to be well suited for cleanup of plant extracts by prep-HPLC (4, 46, 47, 48). When the mobile phase is initially an aqueous buffer at pH 2.8, all but the highly charged (e.g., zeatin ribotide with 5 AMP used as a representative compound for zeatin ribotide) plant hormones are retained at the head of the column (Fig. 1). Since the PGS are retained, samples can be injected onto the column in a dilute form. In-... 

The third category of drugs are phytotherapeutical preparations 80% of the world population use exclusively plants for the treatment of illnesses [11]. Chromatography is relied on to guarantee preparations contain therapeutically effective doses of active drug and maintain constant batch composition. A quantitative determination of active principles is performed when possible, using pure reference standards. In many phytotherapeutic preparations, the active constituents are not known, so marker substances or typical constituents of the extract are used for the quantitative determination [11]. The Applications chapter of this book (Chapter 8) contains numerous references to the use of chromatographic methods in the control of plant extracts. 

Pomponio, R., Gotti, R., Hudaib, M., and Cavrini, V. 2002. Analysis of phenolic acids by micellar electrokinetic chromatography application of Echinacea purpurea plant extracts. J. Chromatogr. A 945, 239-247. 

Figure 7.1 Two dimension paper chromatography of plant extracts. The methanol-water extract is deposited in the application point (origin), dried, and developed with BAW (M-Butanol/acetic acid/water 4 1.5, upper phase) and 15% or 30% HO Ac (acetic acid). (A) Flavonoid aglycones with different hydroxylation patterns (B) Flavone monoglycosides (C) Flavone di- and tri-glycosides (D) Flavone di- and triglycosides acylated with hydroxycinnamic acids).

The story of the discovery of chromatography is classical [16,17]. A most lucid analysis of Tswett s work from the point of view of the preparative applications of chromatography, has been written by Verzele and Dewaele [18]. The Russian botanist Tswett discovered arormd 1902 that plant pigments could be separated by eluting a sample of plant extract with a proper solvent on a column packed with a suitable adsorbent [1]. Did he name the technique chromatography because it separates pigment mixtures into a rainbow of colored bands, or because "tswett" means color in Russian, or both Nobody knows. What is remarkable, however, is the extreme care with which Tswett selected the adsorbents he used [19-21]. For the famous separation of a- and j3-carotenes, he tried 110 different adsorbents and selected inulin (a water-soluble polyfructose plant reserve material) as the... 

Abscisic acid (ABA) levels in rice plants, 308,31Or levels in squash hypocotyls, 315/.316 Active component of brassins identification, 9,lQf pilot plant extraction, 6,7/,8 solvent partition and column chromatography, 8 Adventitious root(s) development, 233,234r,235 formation, 247 Agriculture, application of 24-epibrassinolide, 280-290 22-Aldehydes, synthesis of brassinosteroids, 47-50f a hormone function, description for brassins, 4... 

A comparison of various methods for the study of noradrenaline fixation to various proteins showed that the most useful results were obtained by gel chromatography on Sephadex G-25 [147]. Croizer et al. [148] included GPC in the purification process for plant hormones (gibberelins, indole-3-acetic acid, abscisic acid). Chromatography was carried out on Sephadex columns in the environment of tetrahydrofuran (THF) or a mixture of 100 mM acetic acid in THF. The efficient partial purification of the plant extracts thus achieved allowed the application of high resolution procedures. 

Immunoaffinity chromatography can provide extensive purification of endogenous hormones in plant extracts [60] (see Figs. 6 and 7 in Section 6.2). Both monoclonal and polyclonal antibodies have been used to produce immunoaffinity supports for lAA [60,61], GAs [62,63] and cytokinins [64,65]. Despite the enormous potential of the procedure, it has as yet not found widespread application in plant hormone purification protocols. The situation is unlikely to change until a range of immunoaffinity supports are available from commercial sources at affordable prices. The raising of antibodies against plant hormones, the preparation of a variety of immunoaffinity supports and their application in plant hormone analysis are discussed and evaluated in Chapter 3. 

The main fields of applications for SFA systems are, in order of current importance, water, soil and plant extracts, tobacco, food and beverages, chemical production, and clinical analysis. The reasons for the decline of the formerly dominant clinical market have been described pharmaceutical applications, also previously important, have declined because standard methods developed for new products are generally based on separative techniques, especially liquid chromatography (LC). Table 1 shows the most common applications of SFA. 

TLC has been used to determine ascorbic acid in foods, pharmaceutical preparations, and biological fluids. Paper chromatography was used to separate ascorbic and dehydroascorbic acid in plant extracts, and the spots were visualized using 2,5-dichlorophenol indophenol (Bui-Nguyen, 1985). This procedure is probably applicable to TLC on cellulose. 

In the period 1910-1930 several distinguished chemists — Emil Fischer, Karl Freudenberg and Paul Karrer — made substantial contributions to early ideas on the gallotannins . But progress was severely hampered by the slow realisation that all the plant extracts consisted of mixtures of closely related metabolites and other phenols whose presence made separation and purification of the desired materials extremely difficult (97). Many of these problems have been relieved in recent years by application of various forms of chromatography and by concomitant use of spectroscopic methods for identification. 

Crude chloroform-methanol-water (30 60 8, v/v) extracts of immunostainedTLC bands were analyzed without further purification by nanoelectrospray low-energy mass spectrometry. The authors showed that this effective PLC/MS-joined procedure offers a wide range of applications for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Phenyl-boronic acid (PBA) immobilized on stationary support phases can be put to similar applications. This technology, named boronate affinity chromatography (BAC), consists of a chemical reaction of 1,2- and 1,3-diols with the bonded-phase PBA to form a stable... 

Sample extracts are cleaned up with a cartridge column before the acetylation of E-2 to E-16 and of E-1 to E-15. The final cleanup, plant material and soil samples are analyzed by gas chromatography (GC)/MSD. The GC/NPD method is applicable to water samples. 

A brilliant example for the industrial-scale application of plant cell fermentation is the new process for the production of the anticancer drug paclitaxel developed by Bristol-Myers Squibb (see Figure 15.1). It starts with clusters of paclitaxel producing cells from the needles of the Chinese yew, T. chinensis, and was introduced in 2002. The API is isolated from the fermentation broth and is purified by chromatography and crystallization. The new process substitutes the previously used semisynthetic route. It started with lO-deacetylbaccatin(III), a compound that contains most of the structural complexity of paclitaxel and can be extracted from leaves and twigs of the European yew, T. baccata. The chemical process to convert 10-deacetylbaccatin(III) to paclitaxel is complex. It includes 11 synthetic steps and has a modest yield. 

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