Extraction with Solids

These extraction techniques are relatively new in the bile acid field, and improvements and simplifications of such methods are likely to appear. They offer extraction and purification in one step and conditions can often be kept very mild. 

An alternative procedure to extract bile acids from biological fluids can be based on the properties of a neutral polymer, Amberlite XAD-2 (Rohm and Haas, Co.), to adsorb detergent-like compounds. The use of this resin to extract steroids from urine was reported by Bradlow (33). Preliminary experiments with rat bile have indicated that 80-100% of the bile acids are recovered if the bile is first diluted with 50 volumes of 1 % NaCl in water and this solution is filtered through a 100-ml column of the resin in water at 4°C. After rinsing with 2 bed volumes of water, bile acids are eluted at room temperature with methanol and chloroform/methanol, 1 1. 

Used as described for the analysis of steroids (33, 34) the method may be particularly useful for urine analyses. Norman obtained a quantitative yield of different types of labeled bile acids in urine and the purification was tenfold (35). The method may also be useful for separation of inorganic salts from bile salts. However, several factors influence the results of XAD-2 extractions, e.g., flow rate, temperature, pH, protein and electrolyte concentrations. Further studies of the method are needed. 

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Fig. 2.3. Rapid extraction with solid-phase columns.

Supercritical fluid extractions with solid feed stocks are industrially carried out batchwise because of lack of equipment for feeding solid materials to a pressurized extractor. 

Hartonen K, Bowadt S, Hawthorne SB, et al. 1997. Supercritical fluid extraction with solid-phase trapping of chlorinated and brominated pollutants from sediment samples. J Chromatogr A 774(l-2) 229-242. 

A fourth conclusion, based on the advantages of the use of solid adsorbents, is the gradual replacement of solvent extractions with solid phase extractions. The movement toward this replacement is already evidenced by the commercial availability of several different cartridges of bonded phases and high-surface-area synthetic polymers. 

An effective combination of focused microwave-assisted extraction with solid-phase microextraction (FMAE-SPME) was carried out for the extraction of cocaine... 

Carbon Dioxide Extraction with Solid Absorbents... 

Hageman, K. J., Mazeas, L., Grabanski, C. B., Miller, D. J., and Hawthorne, S. B., Coupled subcritical water extraction with solid-phase microextraction for determining semivolatile organics in environmental solids. Anal. Chem., 68, 3892-3898, 1996. 

Pro-Phe-NH2, in human and rabbit plasma. In this procedure, after the addition of deuterated IS, the sample was desalted, proteins were precipitated, and the drug was extracted with solid-phase extraction (SPE). The HPLC separation was performed in the isocratic mode with a mobile phase that consists of 1% methanol and 20% acetonitrile in 10 mM ammonium formate (pH 5.2). A flow splitter was used to allow only 5% of the flow to enter the ESI source. 

There are many different ways to extract bile acids from different organs and fluids. They may be divided into two types extraction with solvents and extraction with solids. 

Batch extraction with solid-phase sorbents has been widely used for preffactiona-tion of biological mixtures containing small proteins and peptides [4]. The technique is inexpensive, can be applied to laige volumes, and is amenable to handling viscous samples that would plug solid-phase cartridges or analytical HPLC columns. The steps in batch extraction are listed in Table 3. 

FIGURE 8.28 Basic flowsheet of Scandium extraction with solid polymeric extractants ( TVEX-Ss process ). 

NMR spectra were measured with a Varian Unity 300 FT-NMR spectrometer. Liquid chromatography-atmospheric chemical ionization-mass spectrometry (LC-APCl-MS) in positive and negative ion modes was performed using a Hitachi M-1000 spectrometer. Unknown metabolites were isolated and purified from the grape fruits extracts with solid phase extraction method (Porapak Q) and HPLC. Isolated metabolites were analyzed by free form or derivatized (methylated, acetylated) form for identification. All of the non-radiolabelled reference standards 1-6 are synthesized in our laboratory and their chemical structures are shown in Figure 2. 

Suspend 1 g of the substance in 10 ml of concentrated hydrochloric acid, and add 2 ml of alcohol together with about 3 g of granulated tin. Cool the mixture until the initial reaction has subsided, and then heat under reflux for 20-30 minutes. Decant the liquor, cool it, and to it add carefully suifident 20% aqueous sodium hydroxide solution to dissolve the initial precipitate of stannous hydroxide. Extract the free amine with ether (or separate by steam distillation), and dry the extract with solid potassium hydroxide. Evaporate the solvent and identify the amine. 

If no solid precipitate is obtained but the solution becomes cloudy, a low-melting or liquid phenol is indicated this will, of course, be revealed also by the characteristic phenolic odour. Transfer to a separating-funnel and extract with an equal volume of ether. Separate and dry with anhydrous sodium sulphate. Distil off the ether and identify the residue. 

If no solid precipitate is obtained, an oil or an oily suspension, may be produced. Allow to stand, and then, if possible, separate the oil directly in a separating Tunnel and dry with solid KOH. If the volume of the oil is too small for such separation, extract with ether and then separate the ethereal solution, dry as before, filter, and distil off the ether. Distil the amine (if considered necessary) and identify. 

The process of extraction with solvents is generally employed either for the isolation of dissolved substances from solutions or from solid mixtures or for the removal of undesired soluble impurities from mixtures. The latter process is usually termed washing. 

It is marketed as a 35-40 per cent, solution in water (formalin). The rpactions of formaldehyde are partly typical of aldehydes and partly peculiar to itself. By evaporating an aqueous solution paraformaldehyde or paraform (CHjO), an amorphous white solid is produced it is insoluble in most solvents. When formaldehyde is distilled from a 60 per cent, solution containing 2 per cent, of sulphuric acid, it pol5unerises to a crystalline trimeride, trioxane, which can be extracted with methylene chloride this is crystalline (m.p. 62°, b.p. 115°), readily soluble in water, alcohol and ether, and devoid of aldehydic properties ... 

Into a 500 ml. three-necked flask, provided with a mechanical stirrer, a gas inlet tube and a reflux condenser, place 57 g. of anhydrous stannous chloride (Section 11,50,11) and 200 ml. of anhydrous ether. Pass in dry hydrogen chloride gas (Section 11,48,1) until the mixture is saturated and separates into two layers the lower viscous layer consists of stannous chloride dissolved in ethereal hydrogen chloride. Set the stirrer in motion and add 19 5 g. of n-amyl cyanide (Sections III,112 and III,113) through the separatory funnel. Separation of the crystalline aldimine hydrochloride commences after a few minutes continue the stirring for 15 minutes. Filter oflF the crystalline solid, suspend it in about 50 ml. of water and heat under reflux until it is completely hydrolysed. Allow to cool and extract with ether dry the ethereal extract with anhydrous magnesium or calcium sulphate and remove the ether slowly (Fig. II, 13, 4, but with the distilling flask replaced by a Claisen flask with fractionating side arm). Finally, distil the residue and collect the n-hexaldehyde at 127-129°. The yield is 19 g. 

To obtain a maximum yield of the acid it is necessary to hydrolyse the by-product, iaoamyl iaovalerate this is most economically effected with methyl alcoholic sodium hydroxide. Place a mixture of 20 g. of sodium hydroxide pellets, 25 ml. of water and 225 ml. of methyl alcohol in a 500 ml. round-bottomed flask fitted with a reflux (double surface) condenser, warm until the sodium hydroxide dissolves, add the ester layer and reflux the mixture for a period of 15 minutes. Rearrange the flask for distillation (Fig. II, 13, 3) and distil off the methyl alcohol until the residue becomes pasty. Then add about 200 ml. of water and continue the distfllation until the temperature reaches 98-100°. Pour the residue in the flask, consisting of an aqueous solution of sodium iaovalerate, into a 600 ml. beaker and add sufficient water to dissolve any solid which separates. Add slowly, with stirring, a solution of 15 ml. of concentrated sulphuric acid in 50 ml. of water, and extract the hberated acid with 25 ml. of carbon tetrachloride. Combine this extract with extract (A), dry with a httle anhydrous magnesium or calcium sulphate, and distil off the carbon tetrachloride (Fig. II, 13, 4 150 ml. distiUing or Claisen flask), and then distil the residue. Collect the wovaleric acid 172-176°. The yield is 56 g. 

Benzoates. Dissolve 0-5 g. of the amino acid in 10 ml. of 10 per cent, sodium bicarbonate solution and add 1 g. of benzoyl chloride. Shake the mixture vigorously in a stoppered test-tube remove the stopper from time to time since carbon dioxide is evolved. When the odour of benzoyl chloride has disappeared, acidify with dilute hydrochloric acid to Congo red and filter. Extract the solid with a little cold ether to remove any benzoic acid which may be present. RecrystaUise the benzoyl derivative which remains from hot water or from dilute alcohol. 

The experimental conditions for conducting the above reaction in the presence of dimethylformamide as a solvent are as follows. In a 250 ml. three-necked flask, equipped with a reflux condenser and a tantalum wire Hershberg-type stirrer, place 20 g. of o-chloronitrobenzene and 100 ml. of diinethylform-amide (dried over anhydrous calcium sulphate). Heat the solution to reflux and add 20 g. of activated copper bronze in one portion. Heat under reflux for 4 hours, add another 20 g. portion of copper powder, and continue refluxing for a second 4-hour period. Allow to cool, pour the reaction mixture into 2 litres of water, and filter with suction. Extract the solids with three 200 ml. portions of boiling ethanol alternatively, use 300 ml. of ethanol in a Soxhlet apparatus. Isolate the 2 2- dinitrodiphenyl from the alcoholic extracts as described above the 3ueld of product, m.p. 124-125°, is 11 - 5 g. 

Dissolve the solid in 700 ml. of water in a 1500 ml. round-bottomed flask, and add a solution of 88 ml. of concentrated sulphuric acid in about 200 ml. of water until the liquid has a distinct odour of sulphur dioxide sufficient heat will be liberated in the neutralisation to cause the solution to boil. Immediately steam distil the liquid (Fig. II, 40, 1 it is better to use the apparatus shown in Fig. II, 41, 3) until a sample of the distillate gives only a slight precipitate with bromine water. About 700 ml. of distillate should be collected. Saturate the steam distillate with salt, extract the dl with ether, dry the extract with a little anhydrous magnesium or calcium sulphate, distil oflF the ether (compare Fig. II, 13, 4, but with a 50 ml. Claisen flask replacing the distilling flask) and distil the residue under diminished pressure. Collect the p-cresol at 95-96°/15 mm. the colourless liquid solidifies to a white crystalline solid, m.p. 31°. The yield is 24 g. 

Amino-5-methylthiazole. Suspend 76 g. of thiourea in 200 ml. of water in a 500 ml. three-necked flask equipped as in the preceding pre paration. Stir and add 92 -5 g. (80 ml.) of monochloroacetone (1) over a period of 30 minutes. The thiourea dissolves as the reaction proceeds and the temperature rises. Reflux the yellow solution for 2 hours. To the cold solution immersed in an ice bath add, with stirring, 200 g. of solid sodium hydroxide. Transfer to a separatory funnel, add a little ice water, separate the upper oil layer and extract the aqueous layer with three 100 ml. portions of ether. Dry the combined oil and ether extracts with anhydrous magnesium sulphate, remove the ether by distillation from a steam bath, and distil the residual oil under diminished pressure. Collect the 2-amino-5-methylthiazole at 130-133°/18 mm. it solidifies on coohng in ice to a solid, m.p. 44-45°. The yield is 84 g. 

Neutralise the cold contents of the flask with 500-600 ml. of 40 per cent, aqueous sodium hydroxide solution, equip the flask for steam distillation and steam distil until about 1 litre of distillate is collected. The steam distillate separates into two layers. Add solid sodium hydroxide (< 100 g.) to complete the separation of the two layers as far as possible. Remove the upper (organic) layer and extract the aqueous layer with three 50 ml. portions of chloroform. Dry the combined organic layer and chloroform extracts with anhydrous potassium carbonate and distil the mixture through a short fractionating column (e.g., an 8 Dufton column) after a fore run of chloroform, followed by pyridine, collect the crude 4-ethylpyridine at 150-166° (49 g.). Redistil through a Fenske-... 

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