Plant Soluble Sugar Content Assay Kit (Phenol, Colorimetric Method)

Cat. No.: P1507806

GRADE & PURITY BioReagent

Overview

  Soluble sugars in plants mainly refer to monosaccharides and oligosaccharides soluble in water and ethanol. The carbon nutritional status in plants and the quality and characteristics of agricultural products are often evaluated based on their sugar content. To adapt to adverse conditions such as drought and low temperature, plants actively accumulate soluble sugars to lower osmotic potential and freezing point, thereby adjusting to changes in the external environment. Methods for determining soluble sugars in plants include chemical approaches such as the anthrone colorimetric method, 3,5-dinitrosalicylic acid colorimetric method, phenol-sulfuric acid method, and Fehling's reagent colorimetric method.

Detection Principle

  Under the action of concentrated sulfuric acid, reducing sugars undergo dehydration to form furfural or hydroxymethylfurfural. These products react with phenol to form an orange-red compound. Within a certain range, the color intensity is proportional to the reducing sugar content, with a maximum absorption peak at 485 nm. This method can be used for the determination of methylated sugars, pentoses (xylose, ribose, arabinose), and polysaccharides. It is simple, uses inexpensive reagents, offers high sensitivity, is largely unaffected by the presence of proteins during the experiment, and the developed color remains stable for over 160 minutes. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.

P1507806	Component	50T	Storage
P1507806A	Sucrose Standard Solution (10 mg/mL)	1 mL	2-8℃
P1507806B	Phenol Solution (10×)	6 mL	RT. Store in the dark.

Reagents, consumables and Equipments not provided

1. Distilled water, Concentrated sulfuric acid

2. Electronic balance, Scissors, Mortar or homogenizer, Water bath or hot plate, Spectrophotometer, Cuvette

3. 50 ml beaker or conical flask, Volumetric flask, 20 ml graduated test tube or 10 ml screw-cap centrifuge tube

Procedure

1. Extraction of Soluble Sugars

1.1 Weigh 0.5~1 g of fresh plant sample (dry sample is also acceptable), cut into small pieces, add about 3 ml of distilled water for homogenization, transfer to a graduated test tube, rinse the homogenizer 2~3 times with 12 ml of distilled water, and transfer the rinsate to the same container.

1.2 Seal the opening with plastic film and extract in a boiling water bath for 30 minutes. After cooling, filter and transfer the filtrate to a 50 ml volumetric flask.

1.3 Collect the residue, repeat homogenization and extraction with water, combine all filtrates, and adjust to volume.

2. Preparation of Phenol Working Solution

Take 1 volume of Phenol Solution (10X) and add 9 volumes of distilled water. Mix well. Prepare fresh for each use.

3. Dilution of Sucrose Standard

Take 1 ml of Sucrose Standard Solution (10 mg/mL) and add to a 100 ml volumetric flask. Adjust to volume with distilled water to obtain the Sucrose Standard (100 µg/mL). Using clean centrifuge tubes or test tubes, prepare a series of sucrose standards with different masses according to the table below.

Tube No.	1	2	3	4	5
Sucrose Standard (100 µg/mL) (ml)	0.2	0.4	0.6	0.8	1
Distilled Water (ml)	1.8	1.6	1.4	1.2	1
Equivalent Sucrose Mass (µg)	20	40	60	80	100
Sucrose Standard Concentration (µg/ml)	10	20	30	40	50

4. Sample Loading

Take 10 ml screw-cap centrifuge tubes and set up Blank, Standard, and Test tubes according to the table below. Add reagents in the specified order, avoid bubble formation, and mix carefully. If the sugar concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up 2~3 replicate tubes for each sample and use the average value (volumes of all reagents can be scaled down proportionally, but ensure the minimum required amount is met).

Additive (ml)	Blank Tube	Standard Tube	Test Tube
Distilled Water	2	－	－
Series Sucrose Standards (No. 1~5)	－	2	－
Extraction Solution	－	－	2
Phenol Working Solution	1	1	1
Concentrated Sulfuric Acid	5	5	5

Note: Please refer to Notes 3-5 when adding concentrated sulfuric acid.

5. Determination of Soluble Sugars

After adding concentrated sulfuric acid, mix thoroughly and let the reaction proceed at room temperature for 30 minutes. Use the Blank tube to zero the instrument. Measure the absorbance at 485 nm for the Standard and Test tubes using a spectrophotometer with a 1 cm light path cuvette.

6. Calculation of Results

Plot a standard curve using the mass (µg) of the series sucrose standards (No. 1~5) as the x-axis and the corresponding absorbance as the y-axis, and determine the linear regression equation. Calculate the mass of soluble sugar in the Test tube based on its absorbance, then compute the content. Alternatively, plot the standard curve using sucrose standard concentration (µg/ml) versus absorbance to determine the sample's soluble sugar concentration. The soluble sugar content, expressed as a mass percentage (%), is calculated as follows:

Soluble Sugar Content (%) = (m₁ × V_T × N) / (m₀ × V_S × 1,000,000) × 100% = (c × V_T × N) / (m₀ × 1,000,000) × 100%

Parameter Description:

m₁: Mass of soluble sugar obtained from the standard curve (µg) 

V_T: Total volume of the extraction solution (ml) 

N: Dilution factor of the sample extraction solution 

m₀: Mass of the plant sample (g) 

V_S: Volume of the sample extraction solution taken for measurement (ml) 

c: Soluble sugar concentration of the sample (µg/ml) 

1,000,000: Conversion factor between µg and g

Notes

1. It is recommended to perform a preliminary experiment with 2~3 samples expected to have significant differences before formal testing. If the sample absorbance is outside the recommended measurement range, dilute the sample or increase the sample amount for detection.

2. If the soluble sugar concentration in the sample is too high, dilute with distilled water. The linear gradient is good when the sugar concentration is between 10~50 µg/ml. The minimum detection limit is 1 µg/ml, and the developed color remains stable for over 160 minutes.

3. Concentrated sulfuric acid (specific gravity 1.84) is highly oxidizing and corrosive, posing significant hazards. Handle with extreme care.

4. Add concentrated sulfuric acid slowly to avoid violent boiling due to heat generation, which may cause skin or clothing burns. In case of contact, rinse immediately with running water and seek medical attention if necessary.

5. The purity of concentrated sulfuric acid, as well as the method and speed of addition (e.g., adding directly to the liquid surface or adding slowly), can affect the experimental results. Therefore, consistent operation is essential for good reproducibility. It is generally recommended to add concentrated sulfuric acid directly to the liquid surface within 5~20 seconds and mix well.

6. If the sample contains a high amount of glucose, the color development time can be extended to 45 minutes accordingly.

7. The process and timing for measuring soluble sugar content in samples must be consistent with those used for the standard curve.

8. Phenol Solution may crystallize at low temperatures. It should be fully dissolved in a 45~70°C water bath or incubator before use.

9. For your safety and health, please wear a lab coat and disposable gloves during operation.

Appendix

Standard Curve Preparation: Following the instructions, measure the absorbance of the series standards at 485 nm using a spectrophotometer. Example data and standard curve are as follows (for reference only):

Sucrose Standard (µg/ml)	0	10	20	30	40	50
Absorbance	0.266	0.462	0.635	0.802	0.961	1.122

The results indicate a good linear gradient for sucrose standards within the range of 10~50 µg/ml. Significant deviation occurs when sucrose concentration exceeds 50 µg/ml.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.