Plant extracts preparative layer chromatography

Goals of Preparative Layer Chromatography of Plant Extracts.252... 

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. 

The methods employed for isolation of the alkaloids depend on the nature of the compounds, and specific conditions have frequently been devised for the selective isolation of particular types of compounds. Usually, fresh or dried plant material is extracted with dilute acid solution or with alcohol, and the extract obtained is further fractionated by extraction into organic solvents with variation of pH. Extraction columns (288), membrane processes (425), and ion-exchange materials (288-290) may be particularly useful for subfractionation or isolation procedures. For further identification and isolation of separate compounds, preparative thin-layer chromatography, (288, 291, 292, 426), liquid chromatography (293, 294), or gas chromatography may be used (202, 296, 297). Because some of the products reviewed in this chapter occur naturally in very small amounts, they have not been isolated in crystalline form. Gas chromatography-mass spectrometry (87, 213, 299), mass fragmentography (192), and mass spectrometry-mass spectrometry (301, 359) have proved to be particularly useful techniques for identification of trace alkaloids in complex mixtures. 

Use of the cELISA to determine methoprene content of tobacco residues prepared as described above, showed that the extracts contained materials which interfered with the assay (Figure 8). Extracts of tobacco not only contained methoprene, but also plant substances that interfered with the assay. Thin-layer chromatography (TLC) was then used to clean up crude extracts from tobacco samples to reduce and/or eliminate interference. 

It was of interest to examine how the vegetation which grows in polluted wetlands contributes to their additional purification. For that purpose plant samples, after preliminary preparation, were extracted in an Soxlet s apparatus using a mixture of methanol/chloroform. After that the extracts were let through a column filled with aluminum oxide and by Thin Layer Chromatography were separated to three fractions sterols, terpenic and aliphatic alcohols and hydrocarbons. The hydrocarbon fractions were analyzed by Gas Chromatography and Mass Spectrometry. 

Rotation planar chromatography (RPC), as with OPLC, is another thin-layer technique with forced eluent flow, employing a centrifugal force of a revolving rotor to move the mobile phase and separate chemical compounds. The RPC equipment can vary in chamber size, operative mode (analytical or preparative), separation type (circular, anticircular, or linear), and detection mode (off-line or online). The described technique was applied in analytical and micropreparative separation of coumarin compounds from plant extracts. 

It is sometimes especially difficult to separate chlorophyll and other plant colouring matter. A procedure which has proved helpful for prior purification of methanolic extracts, very rich in chlorophyll, is given here the extract is concentrated to dryness under reduced pressure in a rotary evaporator, the residue taken up in a little dilute HCl (pH ca. 5) and this solution filtered through a short (1—2 cm) silica gel column [133], Pre-purification by chromatography with water in an atmosphere of acetic acid ([3], p. 508), if necessary on thicker (preparative) layers, is a promising but so far scarcely used procedure. 

Compared to paper chromatography, thin layer is more versatile, faster and more reproducible. It is often used as pilot technique to quickly determine the complexity of a mixture. It may otherwise be used as an aid in order to find out the best conditions for large-scale chromatography. Because of its speed and simplicity, it is often used to follow the course of reactions. Thin layer technique has often been used to identify drugs, contaminants and adulterants. It has also been widely used to resolve plant extracts and many other biochemical preparations. 

In pharmaceutical preparations and fruit juices, ascorbic acid is readily separated from other compounds by TLC on silica gel and quantified directly by absorption at 254 nm. Serum and plasma may be deproteinized with twice its volume of methanol or ethanol. Various ascorbic acid compounds in plant extracts and foods have been separated on cellulose layers and detected by spraying with 2,S dichlorophenol indophenol (36). Heulandite, a natural zeolite (particle size 45 p) has successfully been employed as an adsorbent and ascorbic acid and other hydrophilic vitamins have separated within 5 cm by ascending chromatography in dimethylformamide (37). HPTLC and OPLC methods have been developed to improve the separation of ascorbic acid from other water soluble vitamins, with mixed success (11). 

The formulation of any biosynthetic pathway depends on the isolation or at least on the detection of all the presumed intermediates. Polyacetylenic compounds are often unstable molecules whose successful isolation depends on the techniques used in handling both the plants and their extracts. The possible influence of work-up procedures on the yields of isolated components has been recently investigated with thin-layer chromatography over silica gel, a standard analytical and preparative procedure in natural products analysis. The recovery of 1 and a-terthienyl (Tl) placed on silica gel plates ranged between 77% and 97%... 

---