Metaphosphoric acid protein precipitation

Bergstrom et al. [63] used HPLC for determination of penicillamine in body fluids. Proteins were precipitated from plasma and hemolyzed blood with trichloroacetic acid and metaphosphoric acid, respectively, and, after centrifugation, the supernatant solution was injected into the HPLC system via a 20-pL loop valve. Urine samples were directly injected after dilution with 0.4 M citric acid. Two columns (5 cm x 0.41 cm and 30 cm x 0.41 cm) packed with Zipax SCX (30 pm) were used as the guard and analytical columns, respectively. The mobile phase (2.5 mL/min) was deoxygenated 0.03 M citric acid-0.01 M Na2HP04 buffer, and use was made of an electrochemical detector equipped with a three-electrode thin-layer cell. The method was selective and sensitive for mercapto-compounds. Recoveries of penicillamine averaged 101% from plasma and 107% from urine, with coefficients of variation equal to 3.68 and 4.25%, respectively. The limits of detection for penicillamine were 0.5 pm and 3 pm in plasma and in urine, respectively. This method is selective and sensitive for sulfhydryl compounds. 

The most important point during sample preparation is to prevent oxidation of ascorbic acid. Indeed, it is easily oxidized by an alkaline pH, heavy metal ions (Cu and Fe ), the presence of halogens compounds, and hydrogen peroxide. The most suitable solvent for this purpose is metaphosphoric acid, which inhibits L-ascorbic oxidase and metal catalysis, and it causes the precipitation of proteins. However, it can cause serious analytical interactions with silica-based column, e.g., C18 or amino bonded-phases [542] and it could co-elute with AA. 

Sample extraction/deproteinization is usually accomplished with mild acidic solvents to free the noncovalently bound tetracyclines from macromolecules. Mcllvaine buffer, pH 4.0 (286, 287), Mcllvaine/EDTA buffer, pH 4.0 (283, 287-293), succinate buffer, pH 4,0 (278-281,294-296), acidic acetonitrile (297-299), and acidic methanol (14, 199, 300) have all been used successfully. Moreover, trichloroacetic acid, pH 2.0 (301, 302), metaphosphoric acid (303), acetate buffer (126, 280), citrate buffer, pH 4.0 (304), citrate buffer/ethyl acetate, pH 4-5 (305), and hydrochloric acid/glycine buffer (306, 307) have all been employed with varying success to precipitate proteins from the sample homogenates. 

Narkis and Henfield-Furie [578] have described a direct method for the identification and determination of volatile water-soluble Ci C5 acids in municipal waste water and raw sewage. The method involves direct injection of the sewage into a gas chromatograph equipped with a Carbowax 20m on acid-washed Chromosorb W column and a flame ionisation detector. Preliminary preparation of the sample is limited to the addition of solid metaphosphoric acid to the sewage and removal of precipitated proteins and suspended solids by centrifuging. 

Prompt stabilization of ascorbic acid is especially important in the case of plasma or serum samples. Metaphosphoric acid is often used for this purpose because it also serves as a protein precipitant. Such properties are desirable in the inactivation of oxidase and the catalytic eflFect of copper. Oxalic acid is an attractive stabilizer for ascorbic acid analysis because of its lower cost and greater stability however, it is not a protein precipitant, therefore, it has a limited use for the extraction of animal tissues. The use of ethylenediaminetetraacetic acid (EDTA) in addition to the metaphosphoric acid has been recommended (96). EDTA would chelate divalent cations, and a study has shown it will stabilize ascorbic acid in the presence of copper for several days (96). Perchloric acid has been used also but because of its inherent dangerous properties its use is generally avoided. Trichloroacetic acid and EDTA also seem appropriate extractants for ascorbate in plant materials (97). 

The nse of a high ionic strength acidic extraction solvent is required to suppress metabolic activity upon disrnption of the cell and to precipitate proteins. A metal chelator snch as ethylenediaminetetraacetic acid (EDTA) is also usually required. The use of metaphosphoric acid is the best way to extract and stabilize L-ascorbic acid, as suggested by various authors [72,78,80,81]. For this reason, the samples were extracted with 3% metaphosphoric acid-8% acetic acid-1 mM EDTA solution, which is known to limit L-ascorbic acid degradation to less than 5% [82]. 

An ascorbic acid standard solution of 5 mg/mL has a pH value of 3. Thus, the stock standard can be made in distilled water. For dilute standard solutions, it is advisable to make the standard in acid, such as 5% metaphosphoric acid (MPA). For extracting biological samples, acids are used to precipitate proteins, to stabilize ascorbic acid and DHA and to remove interfering substances. MPA (62-64), trichloroacetic acid (TCA) (27,67), or perchloric acid (PCA) (53,68,69)... 

Direct titration of protein with an acid, the anions of which are bound firmly to the precipitated protein, such as metaphosphoric (188) and trichloroacetic (189) acids, offers interesting possibilities for determination of protein. Without doubt the method is more useful for determining the acid binding groups of the protein than for determining the protein itself, but under some circumstances, where the protein is of uniform composition and of calibrated acid binding capacity, it may have significant possibilities. 

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