Limit of nitrilotriacetic acid—
Cupric acetate solution—
Dissolve 20 g of cupric acetate in a mixture of 800 mL of water and 10 mL of glacial acetic acid. Adjust with 1 N sodium hydroxide to a pH of 4.2, dilute with water to obtain 1000 mL of solution, and filter.
Mobile phase—
Prepare a mixture of 1600 mL of water, 40 mL of glacial acetic acid, 30.4 mL of 0.5
M dodecyltriethylammonium phosphate, and 20 mL of
Cupric acetate solution. Adjust with 1 N sodium hydroxide to a pH of 4.0, dilute with water to obtain 2000 mL of solution, filter through a filter having a 0.5-µm or finer porosity, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Stock standard solution—
Transfer about 50 mg of nitrilotriacetic acid, accurately weighed, to a 100-mL volumetric flask, dilute with Cupric acetate solution to volume, and mix.
Standard solution—
Transfer 1.0 mL of the
Stock standard solution to a 25-mL volumetric flask, dilute with
Cupric acetate solution to volume, and mix. This solution contains about 0.02 mg of nitrilotriacetic acid per mL.
Test solution—
Transfer about 2 g of Pentetic Acid, accurately weighed, to a 100-mL volumetric flask. Add about 70 mL of Cupric acetate solution, and swirl to dissolve. Sonicate, if necessary, to dissolve. Dilute with Cupric acetate solution to volume, and mix.
Resolution solution—
Transfer 1.0 mL of the
Stock standard solution to a 25-mL volumetric flask, dilute with
Test solution to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 290-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1 that has been highly deactivated (carbon loading of about 30%). The flow rate is about 1 mL per minute. Equilibrate the column by passing, in sequence, water, methanol, and water for about 15 minutes each, and then
Mobile phase for about 45 minutes. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the resolution,
R, between pentetic acid and nitrilotriacetic acid is not less than 2.0, and the relative retention times are about 0.6 for pentetic acid and 1.0 for nitrilotriacetic acid. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 5.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard solution and the
Test solution into the chromatograph, and measure the responses for the major peaks. Calculate the percentage of nitrilotriacetic acid in the portion of Pentetic Acid taken by the formula:
10,000(C / W)(rU / rS),
of which
C is the concentration, in mg per mL, of nitrilotriacetic acid in the
Standard solution,
W is the weight, in mg, of Pentetic Acid taken to prepare the
Test solution, and
rU and
rS are the nitrilotriacetic acid peak responses obtained from the
Test solution and the
Standard solution, respectively. The limit is 0.1%.
;)
