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斐林试剂（Fehling's Reagent）又称菲林试剂或裴林试剂，是德国化学家Hermann von Fehling 1849年所发明，斐林试剂与班氏试剂（Benedict's Reagent）相似，均是用来检测还原糖的存在，其原理是与可溶性的还原性糖（葡萄糖、果糖和麦芽糖）在加热的条件下，能够生成砖红色的氧化亚铜沉淀。
测定原理：还原糖具有醛基和酮基，在碱性溶液中煮沸，能把斐林试剂中的Cu²⁺还原成Cu⁺，使蓝色的斐林试剂脱色，脱色程度与溶液中还原糖含量成正比，在590nm下可用比色法测定吸光度，查标准曲线即可计算出样品中还原糖的含量，主要用于含淀粉食品、酒精饮料、碳酸饮料、肉制品、蜜饯等食品和植物等样品中还原糖的定量检测；总糖的含量也可以测定，但需要提前水解后才能检测，也可用于还原糖的定性试验。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
适用样本：含淀粉食品、酒精饮料、碳酸饮料、肉制品、蜜饯等食品和植物等
自备仪器和试剂
操作步骤（仅供参考）
1、配制斐林试剂
Fehling's Reagent A液和B液等比例混合即成，不可久置。
2、配制pH中和液 (1×)
pH中和液 (10×) 和水按1:9比例混合即成。
3、样品中还原糖的提取
3.1 固体样品（如含淀粉食品、植物样品等）
称取粉碎或混匀后的试样10~20g（精确到0.01g），置250mL容量瓶中，当体积接近150mL时，滴加1~3滴甲基红指示剂，如呈红色，可用pH中和液调至微黄色；若用风干样品，可称取3g，直接加入容量瓶，加入少量水湿润后，再加水至150mL左右加入指示剂和中和液；将容量瓶置于80℃的恒温水浴中保温30min，期间摇动数次，以便将还原糖充分提取出来。
对于含蛋白质较多的样品，此时可加入乙酸铅溶液除去蛋白质，至不再产生白色絮状沉淀时，加入饱和的硫酸钠溶液除去多余的铅离子。30min后取出冷却并定容至刻度，摇匀后取滤液备用。
3.2 酒精饮料
称取混匀后的试样100g（精确到0.1g），置于蒸发皿上，滴加1~3滴甲基红指示剂，用pH中和液调至微黄色，在水浴上蒸发至原体积的1/4后，移入250mL容量瓶中，置于80℃的恒温水浴中保温30min，期间摇动数次，含蛋白质较多的样品参考上述方法，滤液备用。
3.3 碳酸饮料
称取混匀后的试样100g（精确到0.1g），置于蒸发皿上，在水浴上微微搅拌除去二氧化碳后，移入250mL容量瓶中，用水洗涤蒸发皿，并入容量瓶，加水至刻度，混匀后备用。
3.4 总糖的水解和提取
称取植物样品0.5~3g，剪碎，加入蒸馏水约3mL匀浆，转移至三角烧瓶中，用12mL蒸馏水冲洗研磨器2~3次，冲洗液也转移至烧瓶中；向三角烧瓶中加入10mL 6M盐酸溶液，摇匀，煮沸30min，并不时搅拌；取2滴加于小离心管中，再滴加1滴碘液，检查水解是否完全，如已经水解完全，则不显示蓝色；水解完毕后，冷却至室温，滴加6M氢氧化钠溶液，使溶液pH接近中性；含蛋白质较多的样品参考上述方法；滤液或上清液用蒸馏水定容至100mL，混匀，取10mL，用蒸馏水定容至100mL，摇匀备用。
4、制作还原糖标准曲线
按下表设置空白管（0号）、梯度标准管（1~6号），按顺序依次加入。
| 管号 | 0（空白管） | 1 | 2 | 3 | 4 | 5 | 6 |
| Glu标准 (1 mg/mL) 加入量 (µL) | 0 | 100 | 200 | 300 | 400 | 500 | 600 |
| 蒸馏水加入量 (µL) | 600 | 500 | 400 | 300 | 200 | 100 | 0 |
| 斐林试剂加入量 (µL) | 400 | 400 | 400 | 400 | 400 | 400 | 400 |
| 理论葡萄糖含量 (mg) | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 | 0.6 |
将各管混匀后，沸水浴加热15min。冷水或自来水冷却，2500r/min离心5~10min。取上清280μL，按顺序依次加入到96孔板中，酶标仪测定590nm的吸光度。以各标准管吸光度减去空白管吸光度的差值为纵坐标、对应葡萄糖含量为横坐标，绘制标准曲线。
5、样品还原糖的测定
吸取准备好的还原糖提取液3mL，加入2mL斐林试剂，混匀。沸水浴加热、冷却、离心、取上清、测定等操作方法同标准曲线。用样品管的吸光度减去空白管吸光度的差值带入回归方程即可计算出样品中还原糖的含量。
6、结果计算
样品还原糖含量 (%) =m×VT×N/(m0×VS×1000)×100
参数说明：
m：样品中还原糖的含量 (mg)。
VT：提取液总体积 (ml)。
N：稀释倍数。
m0：样品质量 (g)。
VS：测定时取用的提取液体积 (ml)。
1000：单位换算系数。
还原糖的定性试验
1、配制斐林试剂工作液
临用前，取适量Fehling's Reagent A和B等量混合即成，即配即用。
2、向洁净试管中加入1~2mL待测样品。
3、向该试管中加入1mL斐林试剂工作液，充分摇匀。
4、将上述混合液置于沸水浴中，并持续1～3min。
5、观察试管内混合液颜色是否发生变化，其颜色变化顺序应为浅蓝色-棕色-砖红色（沉淀）。
鉴定结果
| 还原性糖（如核糖、葡萄糖、果糖等） | 砖红色沉淀 |
| 非还原性糖（蔗糖、淀粉等） | 无颜色变化 |
注意事项
1、样品提取液中还原糖浓度过高时，应适当稀释后再行测定。
2、样品提取液中还原糖浓度过低时，可提高样品的浓度或增加提取液的用量。
3、可合理减少或增加提取液和斐林试剂的用量。
4、斐林试剂的A、B液须分开储存，临用前按要求混合使用。
5、斐林试剂B液呈强碱性，需小心操作。
6、6M盐酸配制：用市售盐酸和蒸馏水或去离子水等比例混合即成6M盐酸，该过程会放热，应小心操作，避免伤人。
7、6M氢氧化钠配制：称取氢氧化钠24g溶解于蒸馏水，补至100mL即成；氢氧化钠溶于水会放热，应小心操作，避免伤人。
8、色素对糖类的测定存在干扰，当提取液颜色较深时，应事先脱色后再进行测定；不同样品脱色方法和脱色剂用量不同，需自行查找文献资料，5%活性炭可用于红葡萄酒的脱色。
9、为了您的安全和健康，请穿实验服并戴一次性手套操作。
10、试剂开封后请尽快使用，以防影响后续实验效果。
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
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 × VT × N) / (m0 × VS × 1000) × 100
Parameter Explanation:
m: Reducing sugar content in the sample (mg) obtained from the standard curve.
VT: Total volume of the extract (mL).
N: Dilution factor. m0: Sample mass (g).
VS: 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
If the reducing sugar concentration in the sample extract is too high, dilute appropriately before measurement.
If the concentration is too low, increase the sample concentration or the volume of extract used.
The volumes of extract and Fehling's Reagent can be reasonably decreased or increased as needed.
Fehling's Reagent A and B must be stored separately and mixed as required immediately before use.
Fehling's Reagent B is strongly alkaline. Handle with care.
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.
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.
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.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Please use reagents as soon as possible after opening to avoid affecting subsequent experimental results.
| R1509444 | Component | 50T | Storage |
| R1509444A | Glu标准 (1mg/mL) | 5 mL | 2-8℃. |
| R1509444B | Fehling's Reagent A | 10 mL | RT. |
| R1509444C | Fehling's Reagent B | 10 mL | RT. |
| R1509444D | 甲基红指示剂 | 5 mL | RT. |
| R1509444E | pH 中和液 (10×) | 25 mL | RT. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 G2601312 |
分析证书 | R1509444 |
| ZJ26F0332412 |
分析证书 | R1509444 |
| ZJ26F0332413 |
分析证书 | R1509444 |
| ZJ26F0332414 |
分析证书 | R1509444 |
| ZJ26F0332415 |
分析证书 | R1509444 |
| ZJ26F0332416 |
分析证书 | R1509444 |
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