Steps Following Extraction

The extract obtained may contain a mixture of conjugated and free bile acids, sometimes in different salt forms, along with numerous other lipids and compounds of similar polarity. The analysis can continue in two different ways (1) direct purification and separation of the mixture, and (2) hydrolysis of conjugated bile acids and subsequent purification and separation. 

Purification and Separation Without Previous Hydrolysis of Conjugated 

The first step should preferably lead to a group separation of bile acids and to elimination of non-bile acid contaminants. In quantitative work it may be useful to add—at this stage or earlier—labeled compounds, which permit an estimation of recovery of bile acids of different polarities. Tauro-cholic and 3-ketocholanoic acids constitute suitable extremes in polarity. If specific bile acids are to be analyzed, one may add a suitable internal standard to the biological material. Thus, Rooversc/ r/. (36) used nordeoxy-cholic acid for gas chromatographic determination of bile acids in feces and plasma. 

A counter-current extraction is sometimes useful as a first step. For purification purposes a three-stage counter-current extraction between 70 % aqueous ethanol and petroleum ether is sufficient (1). Other systems have been used for the same purpose (11). Extensive separations of bile acids can be obtained with the counter-current distribution systems developed by Ahrens and Craig (37). 

Ion-exchange chromatography may be used for purification and group separation. As mentioned, Okishio et al. (24) purified a liver extract by applying it in 0.1 M NaOH to an Amberlyst A-26 column as described above for solid extraction. An extract can also be dissolved in chloroform-methanol, 1 1, saturated with water, and applied with a slow flow rate to an Amberlyst A-26 column in OH form in the same solvent. A 10-ml column should be used for 250 mg of sample. Neutral lipid contaminants are removed by washing with a suitable organic solvent and bile acids are eluted with 0.2 M ammonium carbonate in 80% ethanol as described above (34). 

---

The classical methods of solvent extraction of polymers can be conveniently divided into those for which heat is required (Soxhlet/Soxtec ), and those methods for which no heat is added, but which utilise some form of agitation, i.e. shaking or sonication (Table 3.3). Other LSE procedures consist in soaking the polymer in boiling solvents [84,85] and cold LSE [80,86]. These methods are also time-consuming, use large amounts of solvents which are scheduled to be restricted in the future, and exhibit other limitations when analytes are present in small quantities, where they may actually be lost in concentration steps following extraction. Many norms are still based on such standard procedures [87,88],... 

Cleanup a preparatory step following extraction of a sample medium designed to remove components that may interfere with subsequent analytical measurements. 

Because SFE is a sample extraction-separation technique, it must precede other steps of the analytical process if the type and content of the species of interest in the extract are to be accurately determined. The analytical equipment required to develop the steps following extraction can either be coupled on-line to it or performed off-line. The way the extract should be treated with a view to identilying or quantifying the target analytes depends on its complexity and the type of information required. Thus, the analytes may require clean-up, individual separation or some other treatment prior to reaching the detector. 

To reduce the quantity of ash in the extracts even further, steps were implemented using a sequential solids removal scheme that entailed a combination of centrifugation and filtration. Following extraction of the coal... 

Estrone methyl ether (100 g, 0.35 mole) is mixed with 100 ml of absolute ethanol, 100 ml of benzene and 200 ml of triethyl orthoformate. Concentrated sulfuric acid (1.55 ml) is added and the mixture is stirred at room temperature for 2 hr. The mixture is then made alkaline by the addition of excess tetra-methylguanidine (ca. 4 ml) and the organic solvents are removed. The residue is dissolved in heptane and the solution is filtered through Celite to prevent emulsions in the following extraction. The solution is then washed threetimes with 500 ml of 10 % sodium hydroxide solution in methanol to remove excess triethyl orthoformate, which would interfere with the Birch reduction solvent system. The heptane solution is dried over sodium sulfate and the solvent is removed. The residue is satisfactory for the Birch reduction step. Infrared analysis shows that the material contains 1.3-1.5% of estrone methyl ether. The pure ketal may be obtained by crystallization from anhydrous ethanol, mp 99-100°. Acidification of the methanolic sodium hydroxide washes affords 10-12 g of recovered estrone methyl ether. 

A sequential analysis protocol includes three steps (1) extraction in water or other appropriate solvent for the colorant, (2) purification or concentration of the colorant, and (3) separation coupled with detection of the target molecule. Different methods of extracting synthetic colorants from foods have been developed using organic solvents followed by SPE protocols using as adsorption support RP-C18, amino materials, or Amberlite XAD-2. Eor qualitative evaluations, the easiest option for separating colorant molecules from unwanted ingredients found in an extract is SPE on polyamide or wool. 

In order to validate the hypothesis mentioned above, the Ni retention capacity of the Lac Tio waste rock was estimated using a batch sorption test performed on a fresh (C1) and weathered (C4) sample, followed by a 3-step Sequential Extraction Procedure (or SEP). The batch sorption test was done using a 10 mg/L Ni solution with an initial pH of 6, an ionic force adjusted to 0.05 M with NaN03 and with a liquid/solid ratio of 25. Some of the batch sorption results are presented in Figure 3. 

A chemist from the Federal Bureau of Narcotics and Dangerous Drugs, who was in Bolivia to observe clandestine cocaine operations, related the following step-by-step procedure for manufacturing cocaine. The method can be conveniently divided into three major steps (1) extraction of cocaine from the leaf and chemical conversion to the sulfate (2) treatment of cocaine sulfate with potassium permanganate and conversion to the free base (aka paste) and (3) conversion of the paste or free base to cocaine hydrochloride. In general, steps (1) and (2) are carried out in sulfate labs while step (3) is performed in crystal labs. 

B.4.2.2 Nonbenzodlazeptnes GG-NPD was used by Kivisto et al. (1999) to quantitate the nonbenzodiazepine anxiolytic buspirone and its major metabolite, l-(2-pyrimidinyl)-piperazine, using separate extraction methods and separate assays. The hmit of quantification in plasma for both compounds was 0.2 ng/mL, which makes this assay useful for pharmacokinetic studies of this compound (Kivisto et al., 1999). A rapid, simple method for analysis of buspirone in rat brain requiring a single extraction step followed by GC-NPD has also been described (Lai. et al., 1997). 

Early in 1970, Few et al. [10] radiolabelled polystyrene particles for a mucociliaiy clearance study. The radiolabelled aerosols were produced by a spinning-disk generator. The technique involves the key steps of extracting sodium pertechnetate (Na " Tc04) into chloroform as tetraphenylarsonium pertechnetate, followed by evaporation of the chloroform. A solution of polystyrene is added to the radioactive residue and dispersed Scheme 1). This technique has subsequently been adopted by... 

Most existing methods are based on instrumental analysis involving exhaustive sample pretreatment and preconcentration steps, followed by purification and fractionation before final chromatographic separation and detection. For fat and oil samples, dissolving the lipids in an appropriate solvent is usually the first treatment. This has been achieved by melting the fat at 90°C followed by LLE or direct solid liquid extraction (SEE) with an apolar solvent [37], column extraction with a mixture of apolar solvents after drying of the sample with anhydrous Na2S04, Soxhlet extraction and/or sonication with apolar solvents. Typically, sample intake is between 0.5 g and 1 g and quantitative recoveries >60% have been reported. 

Wysocka and Przybyl presented an efficient extraction method of quinolizidine alkaloids. The authors themselves developed and investigated this method, in which includes the following steps of extraction ... 

Vanadium pentoxide is an intermediate in recovering vanadium from minerals (See Vanadium). Sodium polyvanadate, obtained as a red cake in one of the steps in extracting vanadium from its ores is calcined at 700°C in air to form a melt of vanadium pentoxide. Pentoxide is prepared in purified form by dissolving red cake in sodium carbonate solution followed by addition of an aqueous solution of ammonia and ammonium chloride. Ammonium metavanadate is precipitated which on decomposition at 320 to 430°C forms vanadium pentoxide. 

Due to the low amounts in which PAHs are usually found in foodstuffs, their accurate quantitative determination is very difficult. Depending on the complexity of the matrix, sample preparation usually includes an extraction step followed by one or more purification steps. In few cases, extraction and purification can be performed in one single step. 

Analytical Methods. The samples of PAH were extracted with cyclohexane, and the extract was subjected to liquid-liquid extractions with N,N-dimethylformamide as reported elsewhere (26). Following a concentration step, the extract was analyzed by GCZ using a Carlo Erba Fractovap 2101 equipped with a flame ionization detector. The column was a 50 m x 0.32 mm i.d. persilanized glass capillary coated with 0V-73 according to the Grob method (27). 

Unlike clenbuterol, salbutamol is a difficult compound to analyze due to its particular chemical attributes. It is a basic compound subjected to protein binding poor recoveries are obtained especially when protein precipitation techniques are used to prepare the extracts (145). In addition, salbutamol is charged at all pH values and does not readily lend itself to simple, specific back-extracting procedures. This severely restricts the options of sample cleanup. However, a Subtilisin protease digestion step followed by acid clarification and solid-phase extraction has been suggested (146) as an adequate extraction and cleanup procedure prior to the end-point determination of salbutamol by an enzyme immunoassay (139) based on the cross-reactivity of anticlenbuterol antibodies. 

The advantage of this extraction method is that the parameters pressure, temperature and solvent to feed ratio can be varied in each extraction step. By this way a very accurate fractionation of the different compounds included in the feed can be achieved. The solubility of the compounds in the supercritical fluid, depending on pressure and temperature, can be changed in each extraction step. The highly soluble substances are extracted in the first step at low fluid density. Increasing the density in the following extraction steps leads to the removal of the less soluble substances. Further, the flow rate of the supercritical fluid can be adjusted in each extraction step, either constant flow for each step or different flow rates, depending on the separation to be achieved. 

One advantage of HPLC is that the analysis of unstable pesticides may be performed directly in aqueous medium without the extraction step or following extraction and concentration. Although the direct approach is quite useful for formulations or for kinetic studies to monitor the parent compounds in the presence of degradation products, its usefulness is limited in the case of environmental samples, where the concentration is usually in the parts-per-billion range (31). 

Why are salting out procedures often used as an initial purification step following the production of a crude extract by centrifugation ... 

---