Plant extracts separation

General analytical. General analytical applications include, analysis of polymers, detection and estimation of metals in soils and geological specimens, investigation of phenolic materials in plant extracts, separation of alkaloids and separation of radioisotopically labelled compounds. 

Until separation techniques such as chromatography (28,29) and counter-current extraction had advanced sufficientiy to be of widespread use, the principal alkaloids were isolated from plant extracts and the minor constituents were either discarded or remained uninvestigated. With the advent of, first, column, then preparative thin layer, and now high pressure Hquid chromatography, even very low concentrations of materials of physiological significance can be obtained in commercial quantities. The alkaloid leurocristine (vincristine, 22, R = CHO), one of the more than 90 alkaloids found in Catharanthus roseus G. Don, from which it is isolated and then used in chemotherapy, occurs in concentrations of about 2 mg/100 kg of plant material. 

Lube oil extraction plants often use phenol as solvent. Phenol is used because of its solvent power with a wide range of feed stocks and its ease of recovery. Phenol preferentially dissolves aromatic-type hydrocarbons from the feed stock and improves its oxidation stability and to some extent its color. Phenol extraction can be used over the entire viscosity range of lube distillates and deasphalted oils. The phenol solvent extraction separation is primarily by molecular type or composition. In order to accomplish a separation by solvent extraction, it is necessary that two liquid phases be present. In phenol solvent extraction of lubricating oils these two phases are an oil-rich phase and a phenol-rich phase. Tne oil-rich phase or raffinate solution consists of the "treated" oil from which undesirable naphthenic and aromatic components have been removed plus some dissolved phenol. The phenol-rich phase or extract solution consists mainly of the bulk of the phenol plus the undesirable components removed from the oil feed. The oil materials remaining... 

Indeed, great emphasis was placed on the presentation of compounds in crystalline form for many years, early chromatographic procedures for the separation of natural substances were criticized because the products were not crystalline. None the less, the invention by Tswett (3) of chromatographic separation by continuous adsorption/desorption on open columns as applied to plant extracts was taken up by a number of natural product researchers in the 1930s, notably by Karrer (4) and by Swab and lockers (5). An early example (6) of hyphenation was the use of fluorescence spectroscopy to identify benzo[a]pyrene separated from shale oil by adsorption chromatography on alumina. 

A further thirty years were to pass before Kuhn and his co-workers (3) successfully repeated Tswetf s original work and separated lutein and xanthine from a plant extract. Nevertheless, despite the success of Kuhn et al and the validation of Tswett s experiments, the new technique attracted little interest and progress continued to be slow and desultory. In 1941 Martin and Synge (4) introduced liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica in the form of a packed bed and used it to separate some acetyl amino acids. 

Separation of lutein esters from complex plant extract mixture... 

Chemical Studies of Plant Extract Components (Separations on Silica Gel)... 

In the chromatography of plant extracts on an enlarged scale, there are a few main problems general elution because of the differentiated polarity of complex mixture components being separated the structural and chemical analogy of compounds and resolution decrease due to band broadening. 

As plant extracts mainly comprise large amonnts of ballast substances (e.g., lipids and chlorophylls), their purification is often a priority in the analysis. Such purification can be expensive in terms of both time and solvent consumed and can lead to losses of sample components. Online purification and separation of extracts contaminated with plant oil, can be readily performed by TLC in equilibrium chambers [1] that enable the use of continuous elution. 

The choice of the chromatographic system depends on the chemical character of the extracts being separated. The mobile phase should accomplish all requirements for PLC determined by volatility and low viscosity, because nonvolatile components (e.g., ion association reagents and most buffers) should be avoided. It means that, for PLC of plant extracts, normal phase chromatography is much more preferable than reversed-phase systems. In the latter situation, mixtures such as methanol-ace-tonitrile-water are mostly used. If buffers and acids have to be added to either the... 

SPECIAL MODES OF DEVELOPMENT AS A TOOL IN PREPARATIVE SEPARATION OF PLANT EXTRACTS... 

The problem of the separation of samples containing components of widely different polarities is difficult because of general elution. This can be solved by use of gradient elution. As has been observed, in TLC separation of plant extracts, gradient elution markedly improves the separation of spots owing to stronger displacement effects... 

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. 

The acetonitrile-n-hexane partitioning is an additionai procedure in the residue analysis of plant samples having high oil content (e.g., rice grain, bean, and corn). A 30-mL volume of acetonitrile is added to the above-mentioned n-hexane layer of plant extract and the mixed solution is shaken vigorously. The acetonitrile layer is separated, a further 30 mL of acetonitrile are added to the n-hexane layer, and the mixed solution is shaken vigorously. The combined acetonitrile layers are carefully concentrated to dryness. 

Chemical separations may first be accomplished by partitioning on the basis of polarity into a series of solvents from non-polar hexane to very polar compounds like methanol. Compounds may also be separated by molecular size, charge, or adsorptive characteristics, etc. Various chromatography methods are utilized, including columns, thin layer (TLC) gas-liquid (GLC), and more recently, high pressure liquid (HPLC) systems. HPLC has proven particularly useful for separations of water soluble compounds from relatively crude plant extracts. Previously, the major effort toward compound identification involved chemical tests to detect specific functional groups, whereas characterization is now usually accomplished by using a... 

Other kinds of bloassays have been used to detect the presence of specific allelochemical effects (8), effects on N2 fIxatlon (9), the presence of volatile compounds (10) and of Inhibitory substances produced by marine microalgae (11). Putnam and Duke (12) have summarized the extraction techniques and bioassay methods used In allelopathy research. Recent developments In high performance liquid chromatography (HPLC) separation of allelochemlcals from plant extracts dictates the need for bloassays with sensitivity to low concentrations of compounds contained In small volumes of eluent. Einhellig at al. (13) described a bloassay using Lemna minor L. growing In tissue culture cluster dish wells that maximizes sensitivity and minimizes sample requirements. 

The identification and structural characterization of biological materials, obtained for example from plants, was traditionally carried out via the classical sequence involving extraction, separation, isolation and characterization, a sequence which requires large amounts of substance and a great deal of time. Industrial problems, for example the search for small amounts of contaminants in industrial products or in waste water, also require intensive analytical studies. 

Two-dimensional techniques are usually employed if both phospho-glycerides and glycolipids are present, but it is possible to resolve members of both classes using a diisobutylketone-acetic acid-water mixture (40 25 5). A solvent composed of acetone, acetic acid and water (100 2 1) will separate the mono- and di-galactosyldiglycerides, which are particularly abundant in plant extracts, from phosphoglycerides, which remain at the origin. 

TLC as a rapid and easy-to-carry-out method has been frequently applied for the separation of the active ingredients in various medicinal plants. Its widespread application in this field can be partially explained by the fact that TLC requires a less cumbersome prepurification procedure of plant extracts than HPLC. 

A simple TLC method has been developed for the separation and identification of flavons and flavon glycosides in the extract of Phillyrea latifolia L. The leaves (100 g) were defatted in 11 of chloroform for 24 h and then extracted with 2 X 11 of ethanol-water (80 20, v/v). The collected extracts were concentrated and extracted again with n-hexane to remove chlorophylls and other apolar constituents. Analytes were extracted with ethyl acetate. Both normal phase and RP-TLC have been used for the separation of flavonoids. The results are compiled in Table 2.36. It was concluded from the data that TLC can be successfully applied for the quality control of plant extracts containing various flavone derivatives [124],... 

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