Reducing Sugar Content Assay Kit (Fehling, Micro Method)

Cat. No.: R1509444

GRADE & PURITY BioReagent

Overview

  Fehling's Reagent (also known as Fehling's solution or Fehling's test) was invented by the German chemist Hermann von Fehling in 1849. Similar to Benedict's Reagent, it is used to detect the presence of reducing sugars. The principle is that soluble reducing sugars (such as glucose, fructose, and maltose) react with the reagent under heating conditions to form a brick-red precipitate of cuprous oxide (Cu₂O).

Assay Principle

Reducing sugars possess aldehyde or ketone groups. When boiled in an alkaline solution, they can reduce Cu²⁺ in Fehling's Reagent to Cu⁺, causing the blue color of the reagent to fade. The degree of decolorization is proportional to the concentration of reducing sugars in the solution. Absorbance can be measured colorimetrically at 590 nm, and the reducing sugar content in the sample can be calculated by referring to a standard curve. This kit is primarily used for the quantitative detection of reducing sugars in samples such as starch-containing foods, alcoholic beverages, carbonated drinks, meat products, preserved fruits, and plant materials. Total sugar content can also be measured but requires prior hydrolysis. It can also be used for qualitative tests of reducing sugars. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.

Applicable Samples: Starch-containing foods, alcoholic beverages, carbonated drinks, meat products, preserved fruits, plant materials, etc.

Reagents, consumables and Equipments not provided

• Microplate reader (capable of measuring absorbance at 590 nm)
• Test tubes or centrifuge tubes, conical flasks, volumetric flasks, glass beads, water bath or alcohol burner, 96-well plate
• Reducing sugar standards (1 mg/mL) such as fructose or invert sugar, hydrochloric acid solution, sodium hydroxide solution, 10% lead acetate solution, saturated sodium sulfate solution, distilled water, iodine solution.

Operating Steps (For Reference Only)

1. Preparation of Fehling's Reagent

Mix equal volumes of Fehling's Reagent A and B immediately before use. Do not store the mixture.

2. Preparation of pH Neutralization Solution (1×)

Dilute the pH Neutralization Solution (10×) with water at a 1:9 ratio.

3. Extraction of Reducing Sugars from Samples

3.1 Solid Samples (e.g., starch-containing foods, plant materials)

Weigh 10–20 g (accurate to 0.01 g) of pulverized or homogenized sample into a 250 mL volumetric flask. When the volume approaches 150 mL, add 1–3 drops of Methyl Red Indicator. If the solution turns red, adjust it to a faint yellow color using the pH Neutralization Solution (1×). For air-dried samples, weigh about 3 g directly into the flask, moisten with a small amount of water, then add water to about 150 mL before adding the indicator and neutralization solution. Place the flask in an 80°C constant temperature water bath for 30 minutes, shaking several times to fully extract reducing sugars.

For samples rich in protein, add lead acetate solution at this stage to precipitate proteins until no more white flocculent precipitate forms. Then add saturated sodium sulfate solution to remove excess lead ions. After 30 minutes, remove, cool, dilute to the mark with water, mix well, and filter. Collect the filtrate for use.

3.2 Alcoholic Beverages

Weigh 100 g (accurate to 0.1 g) of well-mixed sample into an evaporating dish. Add 1–3 drops of Methyl Red Indicator and adjust to a faint yellow color with the pH Neutralization Solution. Evaporate on a water bath to one-quarter of the original volume. Transfer to a 250 mL volumetric flask. Proceed with incubation in an 80°C water bath for 30 minutes (shaking occasionally) as described in 3.1. For protein-rich samples, follow the corresponding steps. Collect the filtrate for use.

3.3 Carbonated Beverages

Weigh 100 g (accurate to 0.1 g) of well-mixed sample into an evaporating dish. Gently stir on a water bath to remove carbon dioxide. Transfer to a 250 mL volumetric flask. Rinse the dish with water and combine the rinses into the flask. Dilute to the mark with water and mix well for use.

3.4 Hydrolysis and Extraction of Total Sugars

Weigh 0.5–3 g of plant sample, cut into pieces, add about 3 mL of distilled water, and homogenize. Transfer to a conical flask. Rinse the homogenizer 2–3 times with 12 mL of distilled water and combine the rinses into the flask. Add 10 mL of 6 M hydrochloric acid solution to the flask, mix, and boil for 30 minutes with occasional stirring. Check for complete hydrolysis by placing 2 drops of the hydrolysate in a small tube and adding 1 drop of iodine solution. No blue color indicates complete hydrolysis. After hydrolysis, cool to room temperature. Add 6 M sodium hydroxide solution dropwise to bring the pH close to neutral. For protein-rich samples, follow the corresponding steps. Filter or centrifuge, dilute the filtrate/supernatant to 100 mL with distilled water, mix well. Take 10 mL of this solution and dilute to 100 mL with distilled water, mix well for use.

4. Preparation of Reducing Sugar Standard Curve

Set up blank tube (No. 0) and gradient standard tubes (Nos. 1–6) according to the table below, adding reagents in order.

Tube No.	0 (Blank)	1	2	3	4	5	6
Glu Standard (1 mg/mL) Volume (µL)	0	100	200	300	400	500	600
Distilled Water Volume (µL)	600	500	400	300	200	100	0
Fehling's Reagent Volume (µL)	400	400	400	400	400	400	400
Theoretical Glucose Content (mg)	0	0.1	0.2	0.3	0.4	0.5	0.6

Mix each tube well, then heat in a boiling water bath for 15 minutes. Cool with cold or tap water. Centrifuge at 2500 rpm for 5–10 minutes. Transfer 280 µL of each supernatant in order to a 96-well plate. Measure the absorbance at 590 nm using a microplate reader. Plot the standard curve using the difference in absorbance between each standard tube and the blank tube as the y-axis and the corresponding glucose content as the x-axis.

5. Measurement of Sample Reducing Sugars

Pipette 3 mL of the prepared reducing sugar extract, add 2 mL of Fehling's Reagent, and mix well. Follow the same procedures as for the standard curve: heating in a boiling water bath, cooling, centrifuging, taking the supernatant, and measuring absorbance. Use the difference between the sample tube absorbance and the blank tube absorbance in the regression equation to calculate the reducing sugar content in the sample.

6. Result Calculation

Sample Reducing Sugar Content (%) = (m × V_T × N) / (m₀ × V_S × 1000) × 100 

Parameter Explanation: 

m: Reducing sugar content in the sample (mg) obtained from the standard curve. 

V_T: Total volume of the extract (mL). 

N: Dilution factor. m₀: Sample mass (g).

V_S: Volume of extract used for the assay (mL). 

1000: Unit conversion factor.

Qualitative Test for Reducing Sugars

1. Prepare Fehling's Reagent Working Solution: Immediately before use, mix equal volumes of Fehling's Reagent A and B.

2. Add 1–2 mL of the test sample to a clean test tube.

3. Add 1 mL of the Fehling's Reagent working solution to the tube and mix thoroughly.

4. Place the mixture in a boiling water bath for 1–3 minutes.

5. Observe the color change of the mixture. The color should change in the order: light blue -> brown -> brick red (precipitate).

Interpretation of Results

Reducing Sugar (e.g., ribose, glucose, fructose)	Brick red precipitate
Non-reducing Sugar (e.g., sucrose, starch)	No color change

Precautions

1. If the reducing sugar concentration in the sample extract is too high, dilute appropriately before measurement.

2. If the concentration is too low, increase the sample concentration or the volume of extract used.

3. The volumes of extract and Fehling's Reagent can be reasonably decreased or increased as needed.

4. Fehling's Reagent A and B must be stored separately and mixed as required immediately before use.

5. Fehling's Reagent B is strongly alkaline. Handle with care.

6. Preparation of 6 M HCl: Mix equal volumes of commercially available concentrated hydrochloric acid and distilled/deionized water. This process is exothermic. Handle with care to avoid injury.

7. Preparation of 6 M NaOH: Dissolve 24 g of sodium hydroxide in distilled water and make up to 100 mL. Dissolving NaOH is exothermic. Handle with care to avoid injury.

8. Pigments can interfere with sugar measurement. If the extract is deeply colored, decolorize it before assay. Decolorization methods and amounts of decolorizing agents vary for different samples; please consult relevant literature. For example, 5% activated charcoal can be used for decolorizing red wine.

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

10. Please use reagents as soon as possible after opening to avoid affecting subsequent experimental results.

Storage and Shipping

Storage

Store at 2-8°C,Room temperature

Shipped In

Wet ice

Stability And Storage

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

Contents & Storage

R1509444	Component	50T	Storage
R1509444A	Glu Standard (1 mg/mL)	5 mL	2-8℃.
R1509444B	Fehling's Reagent A	10 mL	RT.
R1509444C	Fehling's Reagent B	10 mL	RT.
R1509444D	Methyl Red Indicator	5 mL	RT.
R1509444E	pH Neutralization Solution (10×)	25 mL	RT.