Plant extracts sampling

The plant extract samples can be applied directly onto the plate, or a specific class of compounds is extracted in a suitable solvent before TLC. The polarity of the solvent used for extraction should be similar to that of the desired compounds. The samples can be applied onto the plates manually, by using calibrated micropipettes or automated applicators. The applied samples can be spots or bands, and the mobile-phase migration distances vary between 8 and 15 cm. 

Table II. Intraassay Variability of Picloram in Four Fortified Plant Extract Samples from Enzyme Immunoassay Standard Curve using Polyclonal or Monoclonal Antibodies...

A practical example for the behavior of a bitumen B80 (sample 6) during manufacmre is shown in Fig. 4-70 and 4-71. Fig. 4-70 shows the plot of weight loss versus time at three different isothermal temperatures, for virgin material. Fig. 4-71 shows a similar plot for the bitumen after passage through the asphalt mixing plant (extracted sample). 

Figure 16. Separation of RNAs from plant extracts. Sample, viroid (PSTV) infected plant RNA extract column, Nucleogen -DEAE 500-7 size, 6 mm i.d. x 125 mm eluant A, 250 mM KCl, 20 mM phosphate buffer, pH 6.6, 5 M urea eluant B, 1 M KQ. 20 mM phosphate buffer, pH 6.6.5 M urea gradient, 100% A (0% Bj —100% B (0% A), linear flow-rate, 1 ml/min, 36 bar temperamre, ambient.

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). 

Rycroft, D. S. 1996. Fingerprinting of plant extract using NMR spectroscopy application to small samples of liverworts. Chem. Comm. (18) 2187-2188. 

Various extraction methods for phenolic compounds in plant material have been published (Ayres and Loike, 1990 Arts and Hollman, 1998 Andreasen et ah, 2000 Fernandez et al., 2000). In this case phenolic compounds were an important part of the plant material and all the published methods were optimised to remove those analytes from the matrix. Our interest was to find the solvents to modily the taste, but not to extract the phenolic compounds of interest. In each test the technical treatment of the sample was similar. Extraction was carried out at room temperature (approximately 23 °C) for 30 minutes in a horizontal shaker with 200 rpm. Samples were weighed into extraction vials and solvent was added. The vials were closed with caps to minimise the evaporation of the extraction solvent. After 30 minutes the samples were filtered to separate the solvent from the solid. Filter papers were placed on aluminium foil and, after the solvent evaporahon, were removed. Extracted samples were dried at 100°C for 30 minutes to evaporate all the solvent traces. The solvents tested were chloroform, ethanol, diethylether, butanol, ethylacetate, heptane, n-hexane and cyclohexane and they were tested with different solvent/solid ratios. Methanol (MeOH) and acetonitrile (ACN) were not considered because of the high solubility of catechins and lignans to MeOH and ACN. The extracted phloem samples were tasted in the same way as the heated ones. Detailed results from each extraction experiment are presented in Table 14.2. 

PLC of plant extracts is presented in Chapter 11, with sections on the choice of systems, sampling, choice of the sample solvent, detection, and development modes. These applications in the field of pharmacognosy play a key role in the investigation and understanding of the healing potential of the constituents of medicinal plants. 

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 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. 

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. 

Fig. 8.8 Macrophage cytotoxic assays of plant extracts. Supernatant samples were tested from Trgeneration of chloroplast transgenic line pLD-JWl (proteins were extracted in buffer containing no detergent and MTTwas added after 5 hours). pLD-JWl (extract stored for 2 days) pLD-JWl (extract stored for 7 days) -> PA 5 pg ml-1 —x— Control wild type (extract stored for 2 days) ...

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

In both cases, the samples from contaminated sites were rinsed with a solvent to obtain an extract of contaminated transformer oil. The effects of biological degradation were investigated by using a commercial mixture of microorganisms and pure strain under aerobic and anaerobic condition. In the thermal method, a laboratory plasma system was used to decompose the contaminated transformer oil by a direct injection of the oil extract into the plasma system or by melting the extract samples with power plant fly ash in the plasma reactor. For the contaminated transformer oil both methods showed a destruction efficiency of 99.99% and the products of destruction were environmentally friendly. 

The method is more sensitive than the biuret method and has an analytical range from 10 ju,g to 1.0 mg of protein. Using the method outlined below this is equivalent to sample concentrations of between 20 mg l-1 and 2.0 g l-1. The relationship between absorbance and protein concentration deviates from a straight line and a calibration curve is necessary. The method is also subject to interference from simple ions, such as potassium and magnesium, as well as by various organic compounds, such as Tris buffer and EDTA (ethylenediamine-tetraacetic acid). Phenolic compounds present in the sample will also react and this may be of particular significance in the analysis of plant extracts. 

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