Plant extracts chromatography

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. 

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. 

CALDWELL c R (2001) Oxygen radical absorbance capacity of the phenolic compounds in plant extracts fractionated by high-performance liquid chromatography, ,4Biochem, 293, 232-8. 

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

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. 

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... 

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. 

Residues of flumioxazin are extracted from plant matrices with aqueous acetone. The extracted residues are partitioned into dichloromethane. The dichloromethane is removed through rotary evaporation. Partitioning between hexane-acetonitrile followed by Florisil column chromatography purifles the plant extract. Residues of flumioxazin are quantitated by gas chromatography GC. 

A large amount of oily substance in the plant extract can vary the elution volume of fenothiocarb in silica gel column chromatography. 

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. 

Tarantilis, P. A., G. Tsoupras et al. (1995). Determination of saffron (Crocus sativa L.) components in crude plant extracts using hihg-performance liquid chromatography-UV-visible photodiode array detection-mass spectrometry. J. Chromat. A 699 107-118. 

Although relatively unknown, the instrumentation for 2DLC was conceived and implemented by Emi and Frei (1978). They reported the valve configuration presently used in most comprehensive 2DLC systems. However, they automated neither the valve nor the data conversion process to obtain a contour map or 2D peak display. They used a gel permeation chromatography (GPC) column in the first dimension and a reversed-phase liquid chromatography (RPLC) column in the second dimension and studied complex plant extracts. 

Stalcup, A.M., Faulkner, J.R., Jr, Tang, Y., Armstrong, D.W., Levy, L.W., Regalao, E. (1991). Determination of the enantiomericpurity of scopolamine isolated from plant extract using achiral/chiral coupled column chromatography. Biomed. Chromatogr. 5, 3-7. 

SUMNER, L., PATVA, N.L., DIXON, R.A., GENO, P.W., High-performance liquid chromatography/continuous-flow liquid secondary ion mass spectrometry of flavonoid glucosides in leguminous plant extracts, J. Mass Spectrom., 1996,31,472-485. 

C.W. Huck, M.R. Buchmeiser and G.K. Bonn, Fast analysis of flavonoids in plant extracts by liquid chromatography-ultraviolet absorbance detection on poly(carboxylic acid)coated silica and electrospray ionization tandem mass spectrometric detection. J. Chromatogr.A 943 (2002) 33-38. 

Cui, M., Song, F., Zhou, Y., Liu, Z., and Liu, S. (2000). Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 14, 1280-1286. 

Cherkaoui, S., Cahours, X., and Veuthey, J. L. (2003). Analysis of selected withanolides in plant extract by capillary electrochromatography and microemulsion electrokinetic chromatography. Electrophoresis 24, 336—342. 

Cataldi TRI, Rubino A, Lelario F, Bufo SA (2007) Naturally occurring glucosinolates in plant extracts of rocket salad (Eruca Sativa L.) identified by liquid chromatography coupled with negative ion electrospray ionization and quadrupole ion-trap mass spectrometry. Rapid Commun Mass Spectrom 21 2374-2388... 

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. 

Modifications of the method (dry-column chromatography, vacuum liquid chromatography, VLC, for example) are also of practical use for the rapid fractionation of plant extracts. VLC with a polyamide support has been reported for the separation of flavonol glycosides. ... 

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