Trichloroacetic acid stock

Trichloroacetic acid (TCA [81.6 g]) and disodium hydrogen orthophosphate dodecahydrate Na2HP04.12H20 (89.6 g) are dissolved in 600 ml water. Paraquat dichloride (l,l-dimethyl-4,4 -bipyridinium dichloride) (70 ml 36%) is then added, and the solution is adjusted to 1,000 ml with distilled water. The mixture is then cooled on ice to prevent formation of large crystals that are slow to dissolve on thawing and is stored at -15 °C. Extractant A is prepared from 5 ml distilled water adjusted to 1,000 ml with stock solution. Extractant B is prepared by adding 10 ml stock solution (0.1 mM ATP) to approximately 950 ml Extractant A and adjusting to 1 1 with further Extractant A. Normally it is advisable to prepare 51 of stock solution so that the same batch of extractant is used for the complete analysis. 

The standard curve is set up from malonyldialdehyde prepared from freshly hydrolysed malonyldialdehydebisacetal the standard (1 g/ml) is diluted 1 1000 in phosphate-buffered saline and diluted again 1 1000 to a working concentration of 1 ng//d. Tubes containing a range of aliquots from the diluted stock, ranging from 100 fA up to 500 fA, were brought up to a volume of 0.8 ml and then the thiobarbituric acid and the trichloroacetic acid added as described above for the samples. 

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)... 

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