Extraction of Amino Acids

Vanous techniques have been used to extract amino acids from biological tissues prior to analysis by GC-MF Unfortunately, there have been no systematic studies comparing the various procedures. The methods currently being used include extraction by trichloroacetic acid, perchloric acid, sodium acetate, zinc sulfate, ethyl alcohol, and hydrochloric acid. 

Ammo acids can be extracted from biological tissue or tissue powder with 5-7% trichloroacetic acid (Freeman et al, 1980 Berl and Frigyesi, 1969). The trichloroacetic acid has to be removed prior to preparation for GC-MF analysis by successive washes with ether and the washed extract reduced to dryness. 

Biological tissues can be homogenized in 0 4r-0.5N perchloric aad and the supernatants neutralized with KHCO3 or KOH to precipitate the perchlorate (Okada et al., 1971 Balcom et al, 1975). Prior to GC-MF, the supernatants must be freeze dried or evaporated under nitrogen. 

Biological tissue can be homogenized in 0.3N ZnS04 in the proportions of 0.6 g of tissue to 1 mL of reagent. Barium hydroxide (1 mL of 0.3N) IS then added and the mixture is again homogenized and centrifuged The use of barium instead of sodium hydroxide to neutralize the zinc sulfate results in an extract free of sulfate ions, but may be attended by additional loss of some constituents by absorption on the barium sulfate precipitate (Blass, 1960). The 

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Some recovery problems encountered during hydrolytic extraction of amino acids from environmental samples were discussed. A way was proposed for compensating for differential losses of neutral, acidic and basic amino acids, consisting of adding various nonprotein amino acids before the hydrolysis, that act as charge-matched recovery standards131. 

Application of reverse micelles for the extraction of amino acids and proteins. Chimia, 44, 270-82. 

The distribution ratios obtained were compared to the corresponding distribution ratios between water and Toctanol. The authors also calculated solvent parameters of [C4Cilm][PFg] (Section 9.3). It was shown that phenolate-ion associates with [C4CiIm][PFg] more strongly than other ions. The authors also mention the possibility of extraction of amino acids into [C4CiIm][PPg] in the presence of crown ether dibenzo-18-crown-6, though at rather moderate efficiency. 

The above consideration of the similarity and dissimilarity of ILs and conventional extraction solvents ignores one particularly striking feature of ILs. In sharp contrast to common solvents immiscible with water, ILs are capable of ion exchange. We exemplify this very important ability by considering the extraction of amino acids on the basis of our work [24],... 

Figure 9.7 Extraction of amino acids (1 lO M) with dicyclohexano-18-crown-6 (1 10 into [C4QIm][PF5]. (From Smirnova, S.V., Torocheshnikova, I.I., Formanovsky, A. A., and Pletnev, I.V., Anal. Bioanal. Chem., 378, 1369-1375, 2004. With permission.)...

The most interesting point is that extraction of amino acids into IL occurs without addition of a counterion. Typically, for amino acid extraction into conventional solvents (including extraction with crown ether), a hydrophobic counterion is required. Moreover, in most cases, even the presence of such a counteranion does not provide an efficient recovery. 

Interestingly, authors of a subsequent paper [25] studied the extraction of amino acids (Trp, Phe, Tyr, Leu, Val) info four imidazolium-based ILs ([C4CiIm][PFJ, [CgCiIm][PFg], [CgCiIm][BF4], and [C8CiIm][BFJ) in the absence of crown ether and observed the same pH-profiles of extraction (though, naturally, distribution ratios were lower). 

Svetlana V. Smirnova was born in Yartsevo, Smolensk region, Russia. She graduated from MSU Chemistry Department, having specialization in analytical chemistry. She holds MS and PhD degrees from MSU. Her PhD thesis was devoted to the solvent extraction of amino acids. She works at the MSU Chemistry Department as an assistant professor (since 2002) and an associate professor (since 2005) giving the lectures and practice courses in analytical chemistry. Her scientific interests lie in the area of application of ionic liquids in extraction processes. She is the coauthor of more than 20 scientific publications. 

Extraction is an essential step when analyzing solid samples. In some cases homogenization with a solvent suffices, but in others the sample must first be coimninuted. Water, solutions of acetic acid or sodium chloride, or more complex saline solutions are used as solvents. Mixtures of water and methanol or water and ethanol are also employed. The choice of solvent depends on the degree of selectivity desired in the extraction and whether the extraction yield is intended for quantitative analysis. Optimization of the extraction procedure is required in all cases, to fit the nature of the sample to be analyzed and the range of molecular weights of the peptides to be separated. For example, water has been used as the extraction solvent for cheese (33) and legumes (34). Saline solutions have been utilized to extract peptides from meat (35-38) and flour (39,40). Benedito de Barber et al. (41) examined differences in the extractability of amino acids and short peptides in various solvents (1M acetic acid, 70% ethanol, and distilled water) they concluded that extraction with 1M acetic acid yielded the maximum amino acid and peptide contents. 

Picric acid Suitable for extraction of amino acids and small peptides. Interferes with Kjeldahl or spectrophotometric methods Fox (1989), Salji and Kroger (1981), and Reville and Fox (1978)... 

Lipophilic lanthanide complexes of fluorinated 3-diketonate ligands were demonstrated to bind unprotected amino acids under neutral conditions. It is not clear whether amino acids are bound as anions or zwitterions. Chiral ligands 63-66 have been prepared and tested for extraction of amino acids from water into dichloromethane [84] (Table 7). NMR and CD spectroscopic... 

Dzygiel P and Wieczorek P. Extraction of amino acids with emulsion hquid membranes using industrial surfactants and lecithin as stabilisers. J Membr Sci 2000 172 223-232. 

Carriers for cation transport in LMs have been demonstrated only very recently. Yagodin elal (22) has proposed the use of di-2-ethylhexylphosphoric acid (D2EHPA) for the LM extraction of amino acids. Lorbach et (23) and Draxler et aL (24) have recommended bis-(2-ethylhexylthio)phosphoric acid for LM extraction of metals including zinc. These diesters of phosphoric acid contain a single ionizable proton which can exchange with other cations as shown in Figure 2(b). 

The extraction of amino acids (Fig. 6,8) from medical plant extracts is again the use of cation exchange to remove organic bases (amino acids) from extracts of plants. Homogenize and blend the plant material with methanol. Filter the extract and evaporate to dryness. Reconstitute the sample with 10% methanol/water and filter. The extract is prepared for cation exchange. 

Teramoto, M., Yamashiro, T., Inoue, A., Yamamoto, A., Matsuyama, H. and Miyake, Y. (1991). Extraction of amino acids by emulsion hquid membranes containing di(2-ethylhexyl)phosphoric acid as a carrier biotechnology coupled, facilitated transport diffusion. J. Membr. Sci., 58, 11-32. 

Isolation of the amino acids from such wastewaters might be of commercial value. For example, L-phenylalanine has been used as a precursor in the synthesis of a variety of industrial products, for example, the artificial sweetener Aspartame [117]. Purified amino acids can, on the other hand, be used as nutritional supplements in the diet of livestock [118], as weU as in human nutrition [119]. Therefore, there is potential for the extraction of amino acids from dairy and proteinaceous wastewaters for additional income of the producing industries. Application of LMs in treatment of such wastewaters could be of substantial benefit. 

A much more challenging and interesting problem is the polytopic binding and extraction of amino acids, particularly in their preferred neutral... 

The extraction method depends on the extraction of amino acids from the acid hyrolysates of proteins. Although the method was commercialized for amino acid production (proline, serine, tyrosine, etc), the availability of raw materials like hair, feather, keratin, blood meal, etc is usually a limiting factor. Moreover, the yield of amino acids depends on the amino acid composition of the raw material. 

Table 177. Literature on the extraction of amino acids from biological material ...

The extraction of amino acids from protein is the oldest method for their manufacture. L-Cysteine is still obtained in this way, being isolated from an acid hydrolysate of the protein keratin, the principal component of hair and feathers. Chemical routes are successful where the racemate is a satisfactory product as it is with methionine (Figure 6.11), and there are several effective methods which link a chemical synthesis with a biochemical resolution of the... 

The shell of living rhynchonellate brachiopods typically consists of an organic perio-stracum succeeded by a thin calcitic primary layer and a secondary layer composed of alternately stacked calcitic fibres sheathed in membranes (Williams, 1966). The only significant variations in this sequence are the virtual suppression of secondary shell secretion in thecideidines and the deposition of a prismatic tertiary layer in some terebratulides (Williams, 1968). In 1965 Jope reported the extraction of amino acids, lipids and carbohydrates from living (and fossil) rhynchonellates. These organic residues were mainly intercrystalline components of the shell. However, organic traces. 

M. Adachi, M. Harada, A. Shioi, and Y. Sato 1991 Extraction of amino acids to microemulsion, J. Phys. Chem. 95,7925-7931. 

Wang, W, Weber, M.E., and Vera, J.H. 1995. Reverse micellar extraction of amino acids using dioctyldimethylammonium chloride. Ind. Eng. Chem. Res., 34,599-606. 

Two preliminary operations are necessary (o) hydrolysis, and (5) extraction of amino acids freed by hydrolysis. Chemical treatments, still used in many laboratories, are to be avoided because they lead to deiodi-nation which can be quite extensive in some cases (40,62). Hydrolysis by proteolytic enzymes is actually the best method (40). Proteolytic hydrolysis involves small losses of iodinated amino acids because it is never complete, but it has the great advantage of avoiding formation of partially deiodinated substances. Inorganic acids cause deiodination which is much more extensive than that produced by alkaline reagents. 

As expected for lipophilic guanidinium cations/ both 13 and 14 extract carboxylates from aqueous into organic phases, presumably exchanging for chloride. As hoped, this can happen with significant enantioselectivity for amino acid derivatives. Table 4 summarises the results from a series of experiments employing A -acetyl a-amino carboxylates 15. The selectivities are not exceptional, but are close to the best previously reported for extractions of amino acid derivatives. Considering the variations possible on the theme of 12, we can reasonably hope for improved performance in future, more refined versions of the system. 

Micellization of R4N" A is also a problem in the organic phase. Such anion exchange is also a powerful technique used in the solvent extraction of amino acids under appropriate pH conditions so that the amino acid (Am) is in a form (Am ) (see Section 5.2.3.1.2) ready for anion exchange, for example using a quaternary ammonium compound ... 

Smirnova, S. V. Torocheshnikova, II Formanovsky, A. A. Pletnev, I. V. (2004). Solvent extraction of amino acids into a room temperature ionic liquid with dicydohexano-18-crown-6. Anal. Bioanal. Chem., 378,1369-1375. 

Fig. 9. Enantiomer-selective extraction of amino acids with chiral rare earth tris(/ -diketonates) 9. Redrawn, with...

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