Homogenisation and Extraction

Extensive investigations of lipid extraction are being currently undertaken. The standard procedures given below, which were introduced many years ago, have been tested thoroughly by TLC. It is known that these methods can lead to artifacts and that they possess other shortcomings [34, 202] improved instructions have, however, not been published so far. 

Procedure The main part of the oil is first squeezed out of seeds and other plant material of high lipid content the residue is then ground further and squeezed out once more. The residues from this process are finally extracted for 4—6 h in a Soxhlet apparatus, using petrol ether, B. P. 60—70 C or benzene chloroform, carbon tetrachloride, trichloroethylene or diethyl ether are also suitable. 

Whereas seeds often contain up to 60% fat, leaves and stems of green plants contain only about 1—5% lipids, principally phospholipids, sulpholipids and glycolipids. These compounds have to be extracted with relatively polar solvents. Since aliphatic ethers, ketones and esters 

Procedure Leaves or stems (1 g) are treated for 3—4 min at room temperature in a kitchen mixer with 100 ml isopropanol. The pulpy mass is centrifuged and the residue extracted once with 100 ml isopropanol and once with 100 ml chloroform-isopropanol (2 + 1). The combined extracts are concentrated in vacuo at ca. 35° C and purified by partition between chloroform-methanol (2 + 1) and 0.7% aqueous sodium chloride solution (see procedure of Folch, Lees and Sloane Stanley [42], p. 370). 

The total lipids thus obtained usually still contain free amino acids and peptides, sugars and other hydrophilic, naturally occurring material which are carried into the extract through the action of solubilisers like lecithin. Column chromatography on cellulose or Sephadex is suitable for removal of these contaminants. 

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Figure 5.1.2 Matrix solid-phase dispersion (MSPD) extraction as a micro-preparative extraction technique for an on-flow LC-NMR-MS screening. Since the latter requires only sample amounts in the 0.5-2 mg range, the sample preparation can be achieved by fast small-scale extraction procedures, such as MSPD. This is a sample preparation technique that combines both sample homogenisation and extraction of compounds of interest in one single step starting from the intact sample material. Thus, it simplifies the extraction and clean-up steps, reduces the sample manipulation and is much faster than conventional techniques. It is therefore very well suited for a rough separation of extracts into classes of compounds of similar polarities, which can then be submitted to LC-NMR-MS analysis...

Preparation of AIR and extraction of pectic fractions For the preparation of the alcohol-insoluble residue (AIR) the apples were peeled, cut into small pieces and boiled in 96% ethanol for lOmin. After this enzyme inactivation step, the sample material was blended, homogenised and filtered through a G3 sintered glass niter funnel. The residue was washed with 96% ethanol, followed by acetone and diethyl-ether, dried overnight at 40°C under vacuum and stored at -20°C in the dark. Portions of about lOg of AIR were fractionated according to the method of Selvendran et al. [10] as shown in figure 1. 

Sediment obtained from several sites in Lake Flumendosa, Italy, was collected, homogenised and, following a certification campaign, became available as BCR CRM 601 lake sediment certified for its extractable trace metal contents -sequential extraction (Quevauviller et d., 1997). In sediment CRM 601, concentrations of extractable Cd, Cr, Ni, Pb and Zn are certified in Step 1, but only Cd, Ni and Zn in Step 2, and Cd, Ni and Pb in Step 3. Indicative values are also given for extractable Cu in Step 1 and Pb in Step 2 (European Commission, 1997). The long-term stability of the extractable trace metal content of the reference material was recently demonstrated in a European intercomparison exercise (Lopez-Sanchez, 1998). 

To 15 ml of the sample of body fluid or homogenised tissue extract, add 3 ml of hydrochloric acid, and immerse in the liquid a 5 mm x 10 mm copper strip or a spiral of copper wire formed by winding the wire around a glass rod ten times heat gently for 2 hours. Remove the metal, wash gently with water and examine the surface. 

Fig. 5. Detection of amphetamine in homogenised liver extract. Standard zero order (A), and second derivative (B) spectra of the liver extract, compared with the standard zero order (C) and second derivative (D) spectra of amphetamine solutions were in 0.1 M...

Most soft materials such as tomatoes, grapes or bananas are homogenised and then solvent extracted before further sample processing is carried out. However, material such as potatoes or apples have to be chopped first and the initial step can be Soxhlet extraction or homogenisation. Some materials (for example oranges or potatoes) may have to be peeled depending on whether the aim is to analyse the peel, the inner part of the food or the whole sample. 

Some doubts were expressed on the procedure used by one laboratory which filtered the suspension and recovered TriML from the filtrate. Indeed, it was suspected that losses could have occurred by e.g, adsorption on the filter. The extraction recovery was not necessary as standard additions were performed prior to extraction recovery values of 66 and lT/ were obtained. At this stage, it was not possible to confirm the doubts expressed over the extraction recovery of TriML in this material. The participants recommended that emphasis be put on the verification of extraction recovery in a further exercise, i.e. that a small batch of candidate reference material of urban dust be spiked with a known amount of TriML, left to equilibrate, homogenised and made available to the participants so that the extraction recovery may be verified. Recommendation was made to spike the dust in a slurry which should be freeze-dried, rather than oven-dried, in order to avoid losses of TriML. 

Approximately 4 kg of flounder was purchased at Sletten Havn located in Niva Bay, 30 km north of Copenhagen, where high levels of total Hg in fish tissues had been reported previously. The fish sample was mixed with redistilled water, homogenised and stored at — 20°C. Six sub-samples of homogenate each of 0.2 g were analysed for total Hg by RNAA. The total content was found to be (191 20) pg kg (as Hg) on wet mass basis. Extracts were then prepared by the Danish Isotope Centre and the stability of... 

Figure 6.1 Overview on PHA recovery pretreatment, extraction and purification. DAF dissolved-air flotation HPH high-pressure homogenisation and NPCM non-polyhydroxyalkanoate cell mass...

Blood and tissue are brought to pH 5 with hydrochloric acid and the barbituric acids and their metabolites extracted with methylene dichloride [34] prior homogenisation of tissue is carried out with isotonic potassium chloride solution. Barbituric acids can be isolated from serum by adding 0.1 ml concentrated hydrochloric acid and 2 g anhydrous sodium sulphate to 3 ml serum and extracting the mixture with 15 ml chloroform. 10 ml of this extract are evaporated down, the residue dissolved in 0.2 ml 70% ethanol and an aliquot applied to the thin layer [93]. 

Rcnsl dipcptidflsc (from porcine kidney cortex) [9031-96-3] Mr 47,000 [EC 3.4.13.11]. Purified by homogenising the tissue, extracting with Triton X-100, elimination of insoluble material, and ion-exchange, size exclusion and affinity chromatography. [Hitchcock et al. Anal Biochem 163 219 7957.]... 

Pretreatment of biological samples for surfactant analysis is usually straightforward and includes either homogenising with anhydrous sodium sulphate or freeze-drying. Below, the sample treatment methods for extraction and clean-up of non-ionic and anionic surfactants in biota that have been encountered in the literature are reviewed. 

Wahlberg et al. [20] determined NPEO in Blue mussels by GC-ECD after derivatisation of the phenols with PFBC1. To this end, samples were homogenised in acetone/hexane (5 2), followed by extraction with hexane/diethylether (9 1). Lipids were removed by L/L extraction with acetonitrile in 0.1 M NaOH followed by L/L extraction in TMP/sulphuric acid. Recoveries of 93, 34, 65 and 100% were reported for NP, NPEOi, NPEO2 and NPEO3, respectively. 

Fish. Solid biota samples, mainly fish, should be quickly killed by liquid N2 [28] or cervical dislocation [32] and kept at low temperatures (— 20°C). Some authors preferred desiccation of the sample at high temperature (70°C) [33,34] or lyophylisation [28]. The extraction and isolation steps would be combined when using lyophylisation and homogenisation, followed by a Soxhlet extraction, usually with MeOH, and a subsequent solid-phase extraction (SPE) clean-up, prior to the quantification. 

Biological tissues Add water to tissue sample (at 50 C) and homogenise extract with carbon disulfide and analyze Gas chromatography flame ionization detector 0.5 ag/g No data Letz et al. 1984... 

The HbHnl is obtained from the leaves of the rubber tree plant and a crude extract is easily prepared by homogenisation of the frozen leaves, followed by centrifugation [38-40]. A 5-step purification procedure of this crude extract (with over a 100-fold purification factor) to yield a homogenous HbHnl has... 

Wet the lyophilized sample with 80 pi ddH20. After that, add 300 pi of Soln. A and homogenize the sample with a glass-Teflon homogenisator. After addition of 100 pi chloroform, centrifuge the mixture for phase separation. This extraction is repeated three to four times. 

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