The phenolic substances in the soil are mainly released by plants and produced by the decomposition of plant residues and fallen leaves. Due to the difficulty in their degradation, the accumulation of phenolic substances in the soil can affect the carbon-nitrogen transformation and greenhouse gas emissions of the soil, thereby causing a decline in soil fertility. Back. In addition, phenolic substances also have significant effects on the mineralization of soil organic matter and the nutrient cycle. This kit uses the Folin phenol method to determine the content of phenolic substances in the soil. Under alkaline conditions, phenolic substances will reduce tungsten molybdate, generating a blue compound. There is a characteristic absorption peak at 750 nm. By detecting the absorbance value at 750 nm, the content of phenolic substances in the soil can be calculated.
Components：
| S1521999 | Components | Appearance | 48T | Storage | Quantity Per Test | | S1521999A | Extraction Solution
| Colorless clear liquid | 60 mL | 2-8℃. Store in the dark | 1 mL for 1 reaction | | S1521999B | Reagent 1 | Yellow liquid | 25 mL | 2-8℃. Store in the dark | 450 μL for 1 reaction | | S1521999C | Reagent 2 | Colorless clear liquid | 5 mL | 2-8℃ | 90 μL for 1 reaction | | S1521999D | Standard Sample | White powder | 10 mg | 2-8℃ | Use as per requirements |
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Required instruments and supplies:
Visible spectrophotometer, 1 mL glass cuvette (with a path length of 0.4 cm), adjustable pipette, balance, centrifuge.
Determination of total phenolic content in soil:
It is recommended to select 2 samples for prediction before the formal experiment. This will help us understand the situation of this batch of samples, familiarize ourselves with the experimental process, and avoid waste of experimental samples and reagents!
1. Sample preparation:
Take approximately 0.5 g of soil sample, add 1 mL of extraction solution, and shake at room temperature for 30 minutes. Centrifuge at 25℃ and 8000 rpm for 10 minutes, then take the supernatant for testing.
2. Machine testing:
(1) The spectrophotometer was preheated for 30 minutes, the wavelength was adjusted to 750 nm, and distilled water was used for zero adjustment.
(2) Add the following substances in sequence to the EP tube:
| Reagent Name (μL) | Testing tube | Blank tube (used only once) | | Sample | 180 | | Distilled water | - | 180 | | Reagent 1 | 450 | 450 | | Reagent 2 | 90 | 90 | | Mix well, let it stand at room temperature (25℃) for 30 minutes. Transfer all the liquid to a 1 mL glass cuvette (with a path length of 0.4 cm). Measure the absorbance at 750 nm as A, and calculate △A = A<sub>(measured)</sub> - A<sub>(blank)</sub> |
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[Note]:
1. If absorbance ΔA > 1.2, dilute the supernatant with distilled water before re-testing; multiply the final result by dilution factor D during calculation.
2. If ΔA is close to zero, increase soil sample mass W or raise sample loading volume V₁ (e.g., increase to 300 μL; reduce Reagent I volume accordingly to maintain total reaction volume). Substitute the adjusted W and V₁ into the calculation formula for result computation.
Result Calculation:
1. Standard curve: Y = 0.07740* X + 0.008000, where X represents the mass of the standard sample (in μg) and Y represents ΔA.
2. Total phenol content in soil (μg/g soil) = (∆A - 0.008000) ÷ 0.07740 ÷ (V1 ÷ V × W) × D
V---Volume of the extracted liquid, 1 mL;
D---Dilution factor, 1 for undiluted sample;
V1---Volume of the sample in the reaction, 0.18 mL;
W---Mass of soil sample, g.
Appendix Procedure for preparing the standard curve:
1. Prepare standard stock solution (10 mg/mL): Add 1 mL distilled water to the standard powder vial, sonicate until fully dissolved.
2. Dilute the standard stock solution with distilled water to obtain a 1 mg/mL standard dilution. Then, follow the procedures in the table below to dilute it to the corresponding standard dilution. When 180 μL is used for sample loading, this will be the loading volume for the corresponding standard curve.
Operation Table：
| ① | ② | ③ | ④ | | Target loading amount (μg) | 5 | 10 | 15 | 20 | | 1 mg/mL standard dilution solution (μL) | 20 | 40 | 60 | 80 | | Distilled water (μL) | 700 | 680 | 660 | 640 |
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3. Operate following the pipetting protocol for test tubes above, then plot the standard curve using recorded absorbance values.
