Sample preparation from plant material

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

Therefore, a mild and quick extraction technique is necessary to exclude the preparation of artifacts. The carotenoid stereoisomers can be quantitatively analysed, employing MSPD extraction, from plant material, as well as from serum samples, using on-line SPE without any isomerisation or oxidation of the carotenoids. The extraction step is coupled to the separation and identification steps. Here, LC-NMR hyphenation, employing C30 stationary phases, is suitable for unambiguous distinction between all of these stereoisomers. 

Alternatively, to reduce interference from any iron and tungsten present in the sample, the powdered plant material is boiled under reflux with 5.5 N hydrochloric acid. An ethonolic solution of 2% a-monoxime is added and a chloroform extract prepared. To the chloroform phase is added nitric acid per-chloric acid 3 1 v/v, and the mixture is heated until perchloric acid fumes are evolved. 

More knowledge about the separation conditions for the HPLC analysis of phenolic acids in plant materials is available in the cited references. Some of them represent chapters in books with abundant additional information about the above topic, or review article, with details from more than 60 papers published in the last 5 years, dealing with the choice of stationary phases, mobile phases, detection, sample preparation of plants prior to phenolic acid HPLC analyses, etc. 

Sample preparation scheme is presented at Figure 24.4. Grain (wheat, oats, barley, 100 g) should be ground in a laboratory mill for 3 min 25-g portion of the milled grain should be transferred into a 250-mL Erlenmayer flask with a glass stopper and extracted with 100 mL of the extraction solution acetonitrile-methanol mixture (84 16 v/v).This mixture is the most frequently used for the trichothecenes extraction from plant material prior to GC and also LC analyses and provide satisfactory recoveries of target compounds [40,44]. 

Fatty alcohol ethoxylates were extracted from plant materials. The C 2 portion was preparatively purified into individual n = 8 to n = 13 oligomeric fractions. A silica coluiim (RI detector) and a 98/2 butanone/water mobile phase were used [875]. A 0.5 mL injection of a 40% sample solution (using the mobile phase as the solvent) produced excellent peak resolution and good peak shapes. Elution was complete in 22 min. 

Gamoh K, Yamaguchi I, Takatsuto S (1994) Rapid and selective sample preparation for the chromatographic detmminatirai of brassinosteroids from plant material using solid-phase extraction method. Anal Sd 10 913-917... 

Sample preparation techniques vary depending on the analyte and the matrix. An advantage of immunoassays is that less sample preparation is often needed prior to analysis. Because the ELISA is conducted in an aqueous system, aqueous samples such as groundwater may be analyzed directly in the immunoassay or following dilution in a buffer solution. For soil, plant material or complex water samples (e.g., sewage effluent), the analyte must be extracted from the matrix. The extraction method must meet performance criteria such as recovery, reproducibility and ruggedness, and ultimately the analyte must be in a solution that is aqueous or in a water-miscible solvent. For chemical analytes such as pesticides, a simple extraction with methanol may be suitable. At the other extreme, multiple extractions, column cleanup and finally solvent exchange may be necessary to extract the analyte into a solution that is free of matrix interference. 

The determination of volatile elemental species in biological or environmental samples, such as body fluids, tissues, soils, plants or water, generally requires a careful preconcentration and clean-up procedure in order to separate the analytes from matrix material. Several existing sample preparation procedures and applied measurement techniques (especially GC-ICP-MS in combination with... 

Fahmy, T.M., Pulaitis, M.E., Johnson, D.M., McNally, M.E.P., Modifier effects in the supercritical fluid extraction of solutes from clay, soil, and plant materials. Anal. Chem., 65 (10), 1462-1469,1993. Langenfeld, J.J., Hawthorne, S.B., Miller, D.J., Pawliszyn, J., Role of modifiers for analytical scale supercritical fluid extraction of environmental samples. Anal. Chem., 66(6), 909-916,1994. Hawthorne, S.B., Methodology for off-line supercritical fluid extraction. In Supercritical Fluid Extraction and Its Use in Chromatographic Sample Preparation, Westwood S.A. (Ed.), Blackie Academic and Professional, 39-64, 1993. 

This is a rapid technique which can be used to determine whether mescaline might be present in a sample [2]. An extract is prepared from the plant material by powdering the latter and then repeatedly extracting this into methanol/880... 

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