Soil Total Phosphate Assay Kit (MB, Micro Method)

Cat. No.: S1522127

GRADE & PURITY BioReagent

Overview

In acidic solution, under the action of decomposing agent and high temperature, inorganic phosphate and organic phosphate are hydrolyzed into orthophosphate. Orthophosphate reacts with ammonium molybdate to form phosphomolybdic acid, which is then reduced to phosphomolybdenum blue in the presence of a reducing agent, with a characteristic absorbance peak at 710 nm.

Item No.	Appearance	Components	48T	96T	Storage
S1522127A	Liquid	Reagent Ⅰ	10 mL	20 mL	2-8 ℃
S1522127B	Liquid	Reagent Ⅱ	15 mL	30 mL	2-8 ℃
S1522127C	Liquid	Reagent Ⅲ	2.8 mL	5.6 mL	2-8 ℃. Store in the dark.
S1522127D	Powder	Reagent Ⅳ	1 vial	1 vial	2-8 ℃
S1522127E	Liquid	Reagent V	44.8 µL	44.8 µL	2-8 ℃
S1522127F	Liquid	Standard	1 mL	1 mL	2-8 ℃

Notes: Before formal detection, it is recommended to conduct preliminary trials with 2–3 samples with expected large differences in phosphate content.

Self-Prepared Consumables and Instruments

1.Microplate reader or visible spectrophotometer (capable of absorbance detection at 710 nm)

2.96-well microplate or micro glass cuvettes, adjustable pipettes and pipette tips

3.Tabletop centrifuge, 30–50 mesh sieve, thermostatted water bath

4.Deionized water

Reagent Preparation

Reagent I: Ready-to-use; equilibrate to room temperature before use; store at  2-8 °C.

Reagent II: Ready-to-use; equilibrate to room temperature before use; store at 2-8 °C.

Reagent III: Ready-to-use; equilibrate to room temperature before use; store at 2-8 °C protected from light.

Working Reagent IV: Prepare immediately before use. Add 5.6 mL deionized water and dissolve completely, then add Reagent V and mix thoroughly. Working Reagent IV contains a small amount of insoluble precipitate; shake well before use. Stable for 1 week when stored at  2-8 °C.

Reagent V: Ready-to-use; equilibrate to room temperature before use; store at 2-8 °C.

Notes: Reagent I, Reagent II, Reagent III and Reagent V are irritant. Adequate personal protective equipment must be worn during all operations.

Standard Solution: Ready-to-use; equilibrate to room temperature before use; store at  2-8 °C.

Standard Solution Preparation: Dilute the 1 μmol/mL standard stock solution stepwise as shown in the table below.

No.	Standard Volume	Deionized Water Volume (μL)	Concentration (μmol/mL)
Std.1	50 μL of 100 μL 1 μmol/mL stock	150	0.25
Std.2	100 μL of Std.1 (0.25 μmol/mL)	100	0.125
Std.3	100 μL of Std.2 (0.125 μmol/mL)	100	0.063
Std.4	100 μL of Std.3 (0.063 μmol/mL)	100	0.031
Std.5	100 μL of Std.4 (0.031 μmol/mL)	100	0.016
Std.6	100 μL of Std.5 (0.016 μmol/mL)	100	0.008
Blank	0	100	0

Notes: A standard curve must be generated alongside each independent experiment. Diluted standard solutions are unstable and must be used within 4 hours of preparation.

Sample Preparation

Fresh samples are recommended for immediate detection. If testing cannot be performed promptly, samples can be stored at −80 °C for several weeks. Control thawing temperature and duration strictly; complete thawing within 4 hours at room temperature.

1.Air-dry fresh soil samples and sieve through a 30–50 mesh screen.

Mix soil mass (g) : deionized water volume (mL) : Reagent I volume (mL) : Reagent II volume (mL) = 1:10:1:2.

Practical operation: Weigh approximately 0.1 g soil sample, add 1 mL deionized water, followed by 100 μL Reagent I and 200 μL Reagent II. Shake in a 95 °C boiling water bath for 30 min, centrifuge at 10,000 g, 25 °C for 10 min, collect supernatant for subsequent detection.

Assay Procedure

1.Preheat the microplate reader or visible spectrophotometer for ≥30 min, set detection wavelength to 710 nm. Zero the spectrophotometer with deionized water if applicable.

2.Assay layout (operations performed in 96-well microplate or micro glass cuvettes):

Reagent	Blank Well (μL)	Standard Well (μL)	Test Well (μL)
Sample	0	0	40
Standard Solution	0	40	0
Reagent Ⅲ	40	40	40
Working Reagent Ⅳ	40	40	40
Deionized Water	120	80	80

3. Mix thoroughly and incubate at 25 °C for 10 min. Measure absorbance at 710 nm and record the absorbance values as A_blank, A_standard and A_sample respectively.

Calculation formulas: ΔA_sample = A_sample − A_blank ΔA_standard = A_standard − A_blank
Notes: Blank wells and standard curve only need to be tested once per experiment. It is recommended to conduct preliminary experiments with 2–3 samples with obvious expected differences before formal detection. 

If ΔA_sample is less than 0.01, appropriately increase the sample dosage. 

If ΔA_sample exceeds the ΔA_standard value corresponding to 0.25 μmol/mL, further dilute the sample with deionized water (multiply the final calculation result by the dilution factor), or reduce the amount of soil sample used for extraction.

Result Calculation

Notes: Two equivalent formulas are provided, namely the full derivation formula and the simplified formula.

1. Standard Curve Construction: Plot the standard curve with standard solution concentration as the x-axis and ΔA_standard as the y-axis to obtain the standard equation. Substitute ΔA_sample into the equation to get x (μmol/mL).2. Calculation of Total Phosphate Content: Total phosphate content (μmol/g) = x × V_sample ÷ (W × V_sample ÷ V_{total-extraction}) = 1.3 × x ÷ W 

V_sample: Volume of sample added, 0.04 mL 

V_{total-extraction}: Total extraction volume of sample, 1.3 mL 

W: Mass of soil sample, g

Results Presentation

Figure 1. Total phosphate content in soil detected by this kit

Disclaimer

This product is for research purposes only and is not suitable for clinical diagnosis. Please wear a lab coat and disposable gloves during operation to ensure your safety and health.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8 ℃ long term (12 months). Store in the dark.

Contents & Storage

Item No.	Appearance	Components	48T	96T	Storage
S1522127A	Liquid	Reagent Ⅰ	10 mL	20 mL	2-8 ℃
S1522127B	Liquid	Reagent Ⅱ	15 mL	30 mL	2-8 ℃
S1522127C	Liquid	Reagent Ⅲ	2.8 mL	5.6 mL	2-8 ℃. Store in the dark.
S1522127D	Powder	Reagent Ⅳ	1 vial	1 vial	2-8 ℃
S1522127E	Liquid	Reagent V	44.8 µL	44.8 µL	2-8 ℃
S1522127F	Liquid	Standard	1 mL	1 mL	2-8 ℃