Plant extracts purification

Conventional IRMS requires relatively large sample volumes in a purified gaseous form. Recently, an on-line GC-IRMS system has been developed which combines the high purification effect of GC with the utmost precision of IRMS. Sometimes this system may not be Sufficient to determine characteristic minor components from complex matrices, and therefore MDGC-IRMS systems have been developed for the analysis of complex plant extracts and flavour components (25-27). 

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. 

Pure xylan is not employed in industry. but crude xylan or pentosans are of industrial importance. Xylan has been proposed as a textile size but is not employed as yet for this purpose.130 Perhaps the largest use of pentosans is in their conversion to furfural, which has many applications and serves as the source of other furan derivatives. At the present time, large quantities of furfural are used in the extractive purification of petroleum products, and recently a large plant has been constructed to convert furfural by a series of reactions to adipic acid and hexamethylene-diamine, basic ingredients in the synthesis of nylon. In commercial furfural manufacture, rough ground corn cobs are subjected to steam distillation in the presence of hydrochloric acid. As mentioned above, direct preferential hydrolysis of the pentosan in cobs or other pentosan-bearing products could be used for the commercial manufacture of D-xylose. 

Alkaloids are found mainly in plants, and are nitrogenous bases, typically primary, secondary, or tertiary amines. The basic properties facilitate their isolation and purification. Water-soluble salts are formed in the presence of mineral acids (see Section 4.11.1), and this allows separation of the alkaloids from any other compounds that are neutral or acidic. It is a simple matter to take a plant extract in a water-immiscible organic solvent, and to extract this solution with aqueous acid. Salts of the alkaloids are formed, and, being water soluble, these transfer to the aqueous acid phase. On basifying the acid phase, the alkaloids revert back to an uncharged form, and may be extracted into fresh organic solvent. 

With the advent of HPLC, a new tool was born that revolutionized peptide/protein chemistry as a whole as it allowed not only purification of biologically active peptides and proteins from complex mixtures of tissue or plant extracts/231 but also allowed purification of synthetic unprotected peptide mixtures analytically and preparatively.124-261 This was particularly significant in that purity could be assessed of peptide intermediates made by the classical solution-phase methodology that promoted characterization of all intermediates, as well as the purity of final products made by the solid-phase approach of MerrifieldJ27 ... 

The extraction and purification of proteins from organisms or biological tissue can be a laborious and expensive process, and often represents the principal reason why vaccines and other therapeutic agents reach costs that become unattainable for many. Downstream processing also can be a major obstacle with respect to cost for large-scale protein manufacturing in plants. However, purification from plant tissues, while still costly, is in general less expensive than purification from their mammalian and bacterial counterparts. Indeed, some plant-derived biopharmaceuticals, such as topically applied monoclonal antibodies, may require only partial purification and thus be even less intensive in terms of labor and cost. 

Anthocyanin purification steps are important for anthocyanin characterization. Removal of interfering compounds allows for more reliable HPLC separation, spectral information, mass spectra, and NMR spectra during the identification of anthocyanins in plant extracts. 

This unit describes procedures for extraction, purification, and identification by MALDI-MS of fiavonol glycosides from a plant source. The extraction and purification protocols are not meant to be comprehensive, but rather to offer guidelines for sample preparation prior to a MALDI-MS analysis. The MALDI-MS technique is suggested as a complement to other analytical methods such as HPLC or NMR. Its strength lies in the ability to rapidly screen a number of samples for the presence of fiavonol glycosides, which can be identified on the basis of their molecular weights. 

Techniques of Purification. The purification techniques have included column and batch adsorption, extraction, dialysis, and column and paper chromatography. Liberal use was made of the fact that the gibberellate anion is not soluble in ethyl acetate, whereas the free acid is soluble, and that charcoal will adsorb GA3 from aqueous solutions but release it with acetone (26). As a rough rule, preliminary concentration by a factor of about 105 was necessary before significant use of paper chromatography could be made. For kudzu vine and pinto bean, a known amount of GA3 was added to an aliquot of the plant extract and taken through the same procedure as the initial extract. These controls are subsequently referred to as "spiked extracts, to differentiate them from the initial or "natural extract. 

Preliminary Purification of Dandelion and Sweet Corn. The plant extract in pH 7 buffer was sealed in a Visldng cellulose dialysis membrane bag, immersed in a volume of aqueous pH 7 buffer equal to twice the volume of its contents, and shaken for at least 2 hours. The outside solution was replaced, and die entire operation repeated twice. The three outside buffer solutions were combined. A dialysis blank was prepared in similar fashion. The dialyzate was washed once with ethyl acetate. The aqueous phase was adjusted to pH 2 with sulfuric acid and extracted several times with ethyl acetate. The combined ethyl acetate extracts were evaporated to dryness, and redissolved in a small volume of methanol. 

The use of GLC-EC has become a well accepted method for the analysis of ABA as described by Saunders (37). The purity of ABA (methyl ester) detected on this system may be confirmed by forming the trans-ABA isomer methyl ester in sunlight while in acetone and rerunning the sample. We have found that prep-HPLC is useful in the purification of plant extracts for ABA analysis by GLC-ED (45). Another unique identification method takes advantage of the extreme cotton effect that ABA exhibits. The degree of optical rotation can be used for quantification of ABA if the sample is highly purified (37). 

Analytical methods for PGS research have been greatly improved during this past decade. GLC-MS analysis has proven to be the method of choice, particularly when appropriate internal standards are used for accurate assessment of PGS recovery. HPLC, the most rapidly developing form of separation science, should substantially enhance present PGS analytical efforts. One advantage of HPLC is the substantial purification obtained for PGS compounds from crude plant extracts. For analytical identification by instrumentation, scrupulous purification is required, along with selective identification of the PGS. Preferably, two different analytical procedures should be utilized for positive identification of a given compound. 

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