Method for rapidly detecting ammonia nitrogen in water on large batches by microplate reader micro-colorimetric method

[0027] Example 1 Preparation of standard series

[0028] Spectrophotometrically pipette 0.00, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00 and 10.00mL ammonia nitrogen standard use solution into a 50mL graduated tube with stopper, and add water to 50mL. Measure according to standard method.

[0029] The micro-colorimetric method of the microplate reader corresponds to 0, 2, 4, 8, 16, 24, 32 and 40μL of ammonia nitrogen standard use solution in a 96-well microtiter plate, and add water to 200μL. The corresponding ammonia nitrogen content is consistent with the spectrophotometric method. When making the standard curve of different sample volumes, pipette the ammonia nitrogen standard solution in proportion.

[0030] Example 2 Experimental method

[0031] The Nessler's reagent spectrophotometric method is determined by the standard method (HJ 535-2009). Add 1.0mL 500g/L potassium sodium tartrate solution and shake well, then add 1.0mL Nessler's reagent and shake well. After placing it at room temperature for 10 minutes, use a cuvette with an optical path of 20 mm, and measure the absorbance with water at a wavelength of 420 nm as a reference.

[0032] Microplate reader micro-colorimetric method corresponds to adding 4μL potassium sodium tartrate solution and 4μL Nessler's reagent. After color development under optimized conditions, optical density (OD) is measured at 420nm wavelength. When making the standard curve of different sample volumes, pipette the potassium sodium tartrate solution and Nessler's reagent in proportion.

[0033] Use linear range, detection limit, minimum detection limit, precision and sensitivity analysis parameters optimized results; linear range is determined by measuring the minimum and maximum concentration of standard solution; detection limit is 3 times the standard deviation of the blank water sample ; The minimum detection limit is 4 times the detection limit; precision is expressed by relative standard deviation; sensitivity is expressed by the slope of the calibration curve; accuracy is expressed by the recovery rate of standard addition.

[0034] The sample volume, color development method, color development time, etc. are important parameters that affect the micro-assay of the microplate reader. This experiment will optimize them one by one. The optimum temperature for the determination of ammonia nitrogen by Nessler's reagent spectrophotometry is between 20 and 25℃. This study is operated at room temperature, and the optimization of the color temperature is no longer required.

[0035] Example 3 Sample volume

[0036] The light source of the microplate reader is transmitted from the bottom of the microwell plate, and the sample amount should cover the bottom of the microwell without overflowing the microwell. The experimental Thermo 96-well microplate has a working volume of 50-250μL, with 5 sample volume gradients of 50, 100, 150, 200, and 250μL. figure 1 It is the standard curve of the same concentration of ammonia nitrogen standard sequence samples under different sample volume gradients. The correlation coefficients of curves drawn with samples of different volumes can reach 0.999. With the increase of the sample amount, the optical density of the same concentration sample gradually increases, and the slope of the standard curve also gradually increases. The slope of the standard curve has a more obvious influence on the calculation of the detection value of the standard sample. The measurement results of the standard sample (BW085515 160957) with a known concentration of 0.540±0.027mg/L are shown in Table 1. When the sample volume is 50 μL, the measurement result is inaccurate; when the sample volume is 100 μL, the measurement result is qualified, but the relative error with the standard value is large; when the sample volume reaches 150 μL and above, the measurement result is better. Considering the sensitivity, accuracy, and convenience of operation, the sample volume should be 200μL.

[0037] Table 1 Determination error of micro-colorimetric method of microplate reader

[0038]