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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。
本试剂盒根据国家标准（GB/T 5009.7-2016食品中还原糖的测定）推荐的方法（直接滴定法）而成，主要由酒石酸钠钾、硫酸铜、亚甲蓝、亚铁氰化钾、葡萄糖标准等组成，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，该方法的检测原理是试样经去蛋白处理后，以亚甲蓝为指示剂，在加热条件下滴定标定过的斐林试剂（A液与B液等量混合生成的可溶性蓝色的酒石酸钾钠铜络合物，也称作碱性酒石酸铜溶液），样品中的还原糖将酒石酸钾钠铜中的二价铜还原成红色的氧化亚铜沉淀，氧化亚铜沉淀又与亚铁氰化钾反应生成可溶性的无色络合物，当二价铜全部被还原，稍过量的还原糖把亚甲蓝还原，溶液就由蓝色变为无色，即为滴定终点，根据样品液的消耗体积计算还原糖含量，本产品未经标定，需用户自行标定。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
自备仪器和试剂
操作步骤（仅供参考）
1、Fehling's Reagent的标定
25mL的酸式滴定管中加入还原糖标准溶液 (1mg/mL) 约15mL，向150mL锥形瓶中依次加入Fehling's Reagent A液和B液各5mL，再向其中加入10mL水和2~4粒玻璃珠，加热锥形瓶，控制时间在2min内沸腾，保持沸腾以每2秒1滴的速度滴加还原糖标准溶液，直到溶液蓝色刚好褪去为终点，记录消耗标准溶液的总体积，平行操作3次，取平均值，计算每10ml Fehling's Reagent (A、B液各5mL) 相当于还原糖的质量 (mg)。
2、试样准备
2.1 含淀粉食品
称取粉碎或混匀后的试样10~20g（精确到0.001g），置于250mL容量瓶中加水200mL，45℃水浴中加热1h，并时时振摇，冷却后加水至刻度，混匀，静置，沉淀，吸取200mL上清液置于另一250mL容量瓶中，缓慢加入乙酸锌溶液和亚铁氰化钾溶液各5mL，加水至刻度，混匀静置30min，用干燥滤纸过滤，弃去初滤液，后续滤液备用。
2.2 酒精饮料
称取混匀后的试样100g（精确到0.01g），置于蒸发皿上，用氢氧化钠溶液中和至中性，在水浴上蒸发至原体积的1/4后移入250mL容量瓶中，缓慢加入乙酸锌溶液和亚铁氰化钾溶液各5mL，加水至刻度，混匀静置30min，用干燥滤纸过滤，弃去初滤液，后续滤液备用。
2.3 碳酸饮料
称取混匀后的试样100g（精确到0.01g），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。
2.4 其他食品
称取粉碎后的固体试样2.5~5g（精确到0.001g）或混匀后的液体试样5~25g（精确到0.001g），置于250mL容量瓶中，加水50mL，缓慢加入乙酸锌溶液和亚铁氰化钾溶液各5mL，加水至刻度，混匀静置30min，用干燥滤纸过滤，弃去初滤液，后续滤液备用。
3、试样溶液预测
25mL的酸式滴定管中加入试样溶液约15mL，向150mL锥形瓶中依次加入Fehling's Reagent A液和B液各5mL，再向其中加入10mL水和2~4粒玻璃珠，加热锥形瓶，控制时间在2min内沸腾，保持沸腾以先快后慢的速度滴加试样溶液，待溶液颜色变浅时，以每2秒1滴的速度滴加，直到溶液蓝色刚好褪去为终点，记录消耗试样溶液的总体积。(注：试样溶液中还原糖浓度过高时，应适当稀释后再行测定。尽量使每次滴定消耗样液的体积和标定斐林试剂所消耗的还原糖标准溶液的体积相近，约10mL。当浓度过低时，可直接加入10mL试样溶液，不再加10mL水，再用还原糖标准溶液滴定至终点，记录消耗的体积与标定时消耗的还原糖体积之差相当于10mL样液中所含还原糖的质量。)
5、结果计算
试样中还原糖的含量（以某种还原糖计）按下边公式计算：
X=m1×100/(m×F×V/250×1000)
当浓度过低时，试样中还原糖的含量（以某种还原糖计）按下边公式计算：
X=m2×100/(m×F×10/250×1000)
参数说明：
X=试样中还原糖的含量（以某种还原糖计）（g/100g）；
m1=斐林试剂（A、B液各半）相当于某种还原糖的质量（mg）；
m2=标定时体积与加入样品后消耗的还原糖标准溶液体积之差相当于某种还原糖的质量（mg）；
m=试样质量（g）；
F=样品系数，含淀粉食品为0.8，其他为1；
V=测定时平均消耗试样溶液体积（ml）；
10=样液体积（ml）；
250=定容体积（ml）；
1000=单位换算系数。
还原糖含量≥10g/100g时，计算结果保留三位有效数字；
还原糖含量＜10g/100g时，计算结果保留两位有效数字。
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，颜色变化顺序应为浅蓝色-棕色-砖红色（沉淀）。
附录2：总糖的水解和提取
1、称取植物样品0.5~3g，剪碎，加入蒸馏水约3mL匀浆，转移至烧杯或三角瓶中，用12mL蒸馏水冲洗研磨器2~3次，洗出液也转移至该容器。
2、向容器中加入10mL 6M盐酸溶液，搅拌均匀，煮沸30min，并不时搅拌。
3、取2滴滴加于载玻片上，滴加1滴显色液（约50μL），检查水解是否完全，如已经水解完全，则不显示蓝色。
4、水解完毕后，冷却至室温，加入6M氢氧化钠溶液，使溶液pH值至7.4，用蒸馏水定容至100mL，混匀，4000g离心5min或过滤。
5、取上清或滤液10mL，用蒸馏水定容至100mL，成稀释10倍的总糖水解液（提取液），取0.5mL总糖水解液，测定其还原糖的含量。
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注意事项
1、测样品总糖含量时应先水解成还原糖后参考还原糖的测定方法即可。
2、试样溶液中还原糖浓度过高时，应适当稀释后再行测定，尽量使每次滴定消耗样液的体积和标定斐林试剂所消耗的还原糖标准溶液的体积相近。
3、试样溶液中还原糖浓度过低时直接加入10mL试样溶液，不再加10mL水，再用还原糖标准溶液滴定至终点，记录消耗的体积与标定时消耗的还原糖体积之差相当于10mL样液中所含还原糖的质量。
4、也可按上述方法标定4~20mL斐林试剂（A、B液各半）来适应试样中还原糖浓度的变化。
5、斐林试剂的A、B液须分开储存，临用前按比例混合使用。
6、斐林试剂B液呈强碱性，需小心操作，B液含有亚铁氰化钾，可消除氧化亚铜沉淀对滴定终点的干扰。
7、该试剂盒适用于各类食品中还原糖的测定，但在分析酱油等深色样品时会受色素干扰，滴定终点模糊不清，影响准确性，应脱色后再进行测定，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。
8、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) 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).
This kit is based on the method (direct titration) recommended by the National Standard of China (GB/T 5009.7‑2016 – Determination of Reducing Sugars in Foods). It is mainly composed of potassium sodium tartrate, copper sulfate, methylene blue, potassium ferrocyanide, glucose standard, etc. It is primarily used for the quantitative determination of reducing sugars in foods such as starch‑containing foods, alcoholic beverages, carbonated drinks, meat products, and preserved fruits. Total sugar content can also be measured but requires prior hydrolysis. It can also be used for qualitative tests of reducing sugars.
The detection principle of this method is as follows: after deproteinization of the sample, using methylene blue as an indicator, a standardized Fehling's reagent (equal volumes of Solution A and B are mixed to form a soluble blue complex of potassium sodium tartrate‑copper, also called alkaline copper tartrate solution) is titrated under heating conditions. The reducing sugars in the sample reduce the divalent copper in the potassium sodium tartrate‑copper complex to red cuprous oxide precipitate. The cuprous oxide precipitate then reacts with potassium ferrocyanide to form a soluble colorless complex. When all divalent copper is reduced, a slight excess of reducing sugar reduces the methylene blue, changing the solution from blue to colorless, which is the titration endpoint. The reducing sugar content is calculated based on the volume of sample solution consumed. This product is not pre‑standardized; users must standardize it themselves. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.
Reagents, consumables and Equipments not provided
Operating Steps (For Reference Only)
1. Standardization of Fehling's Reagent
Fill an acid burette (25 mL) with about 15 mL of reducing sugar standard solution (1 mg/mL). Into a 150 mL conical flask, add 5 mL each of Fehling's Reagent A and B, then add 10 mL of water and 2–4 glass beads. Heat the flask to boiling within 2 minutes. Maintain boiling and titrate with the reducing sugar standard solution at a rate of about 1 drop every 2 seconds until the blue color just disappears as the endpoint. Record the total volume of standard solution consumed. Perform in triplicate and take the average. Calculate the mass (mg) of reducing sugar equivalent to 10 mL of Fehling's Reagent (5 mL each of A and B).
2. Sample Preparation
2.1 Starch‑containing Foods
Weigh 10–20 g (accurate to 0.001 g) of pulverized or homogenized sample into a 250 mL volumetric flask, add 200 mL of water, and heat in a 45°C water bath for 1 hour with occasional shaking. Cool, dilute to the mark with water, mix well, and let stand. After settling, pipette 200 mL of the supernatant into another 250 mL volumetric flask. Slowly add 5 mL each of Zinc Acetate Solution and Potassium Ferrocyanide Solution. Dilute to the mark with water, mix well, and let stand for 30 minutes. Filter through dry filter paper, discard the initial filtrate, and keep the subsequent filtrate for use.
2.2 Alcoholic Beverages
Weigh 100 g (accurate to 0.01 g) of well‑mixed sample into an evaporating dish. Neutralize with Alkaline Solution, then evaporate on a water bath to one‑quarter of the original volume. Transfer to a 250 mL volumetric flask. Slowly add 5 mL each of Zinc Acetate Solution and Potassium Ferrocyanide Solution. Dilute to the mark with water, mix well, let stand for 30 minutes, filter through dry filter paper, discard the initial filtrate, and keep the subsequent filtrate for use.
2.3 Carbonated Beverages
Weigh 100 g (accurate to 0.01 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.
2.4 Other Foods
Weigh 2.5–5 g (accurate to 0.001 g) of pulverized solid sample or 5–25 g (accurate to 0.001 g) of homogenized liquid sample into a 250 mL volumetric flask. Add 50 mL of water, then slowly add 5 mL each of Zinc Acetate Solution and Potassium Ferrocyanide Solution. Dilute to the mark with water, mix well, let stand for 30 minutes, filter through dry filter paper, discard the initial filtrate, and keep the subsequent filtrate for use.
3. Preliminary Titration of Sample Solution
Fill an acid burette (25 mL) with about 15 mL of the sample solution. Into a 150 mL conical flask, add 5 mL each of Fehling's Reagent A and B, then add 10 mL of water and 2–4 glass beads. Heat the flask to boiling within 2 minutes. Maintain boiling and titrate with the sample solution, starting quickly and then slowing down. When the color becomes lighter, titrate at about 1 drop every 2 seconds until the blue color just disappears as the endpoint. Record the total volume of sample solution consumed. (Note: If the reducing sugar concentration in the sample solution is too high, dilute appropriately before measurement. Try to make the volume of sample solution consumed in each titration similar to the volume of reducing sugar standard solution consumed during standardization, approximately 10 mL. When the concentration is too low, directly add 10 mL of sample solution (without the extra 10 mL water) and then titrate with the reducing sugar standard solution to the endpoint. The difference between the volume consumed and the volume consumed during standardization corresponds to the mass of reducing sugar in 10 mL of sample solution.)
4. Formal Titration of Sample Solution
Fill an acid burette (25 mL) with about 20 mL of the sample solution. Into a 150 mL conical flask, add 5 mL each of Fehling's Reagent A and B, then add 10 mL of water and 2–4 glass beads. Heat the flask to boiling within 2 minutes. Maintain boiling and titrate with the sample solution at a rate of about 1 drop every 2 seconds until the blue color just disappears as the endpoint. Record the total volume of sample solution consumed. Perform in triplicate and take the average.
5. Result Calculation
The reducing sugar content in the sample (expressed as a specific reducing sugar) is calculated using the formula below:
X=m1×100/(m×F×V/250×1000)
When the concentration is too low, use the following formula:
X=m2×100/(m×F×10/250×1000)
Parameter Explanation:
X: Reducing sugar content in the sample (expressed as a specific reducing sugar) (g/100 g).
m<sub>1</sub>: Mass (mg) of a specific reducing sugar equivalent to Fehling's Reagent (half each of A and B).
m<sub>2</sub>: Mass (mg) of a specific reducing sugar corresponding to the difference between the volume consumed during standardization and the volume of reducing sugar standard solution consumed after adding the sample.
m: Sample mass (g).
F: Sample coefficient: 0.8 for starch‑containing foods, 1 for others.
V: Average volume of sample solution consumed during measurement (mL).
10: Volume of sample solution (mL) used in the low‑concentration method.
250: Volumetric flask volume (mL).
1000: Unit conversion factor.
Reporting of results
When reducing sugar content ≥10 g/100 g, report with three significant figures.
When reducing sugar content <10 g/100 g, report with two significant figures.
Appendix 1: 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).
Appendix 2: Hydrolysis and Extraction of Total Sugars
1. Weigh 0.5–3 g of plant sample, cut into pieces, add about 3 mL of distilled water, and homogenize. Transfer to a beaker or conical flask. Rinse the homogenizer 2–3 times with 12 mL of distilled water and combine the rinses into the container.
2. Add 10 mL of 6 M hydrochloric acid solution to the container, stir well, and boil for 30 minutes with occasional stirring.
3. Place 2 drops of the hydrolysate on a glass slide and add 1 drop (about 50 µL) of chromogenic solution to check for complete hydrolysis. No blue color indicates complete hydrolysis.
4. After hydrolysis, cool to room temperature. Add 6 M sodium hydroxide solution to adjust the pH to 7.4. Dilute to 100 mL with distilled water, mix well, and centrifuge at 4000 g for 5 minutes or filter.
5. Take 10 mL of the supernatant or filtrate and dilute to 100 mL with distilled water to obtain a 10‑fold diluted total sugar hydrolysate (extract). Take 0.5 mL of this total sugar hydrolysate to measure its reducing sugar content.
| Sample Type | Observation |
| Reducing Sugar (e.g., ribose, glucose, fructose) | Brick red precipitate |
| Non‑reducing Sugar (e.g., sucrose, starch) | No color change |
Precautions
1. To measure total sugar content, hydrolyze the sample to reducing sugars first and then refer to the method for reducing sugar determination.
2. If the reducing sugar concentration in the sample solution is too high, dilute appropriately before measurement. Try to keep the volume of sample solution consumed in each titration similar to the volume of reducing sugar standard solution consumed during standardization.
3. When the reducing sugar concentration in the sample solution is too low, directly add 10 mL of sample solution (without the extra 10 mL water) and titrate with the reducing sugar standard solution to the endpoint. The difference between the volume consumed and the volume consumed during standardization corresponds to the mass of reducing sugar in 10 mL of sample solution.
4. You may also standardize 4–20 mL of Fehling's Reagent (half each of A and B) according to the above method to adapt to variations in reducing sugar concentration in samples.
5. Fehling's Reagent A and B must be stored separately and mixed in proportion immediately before use.
6. Fehling's Reagent B is strongly alkaline; handle with care. It contains potassium ferrocyanide, which eliminates interference from cuprous oxide precipitate at the titration endpoint.
7. This kit is suitable for the determination of reducing sugars in various foods. However, when analyzing dark‑colored samples such as soy sauce, pigments may interfere, making the titration endpoint unclear and affecting accuracy. Decolorize before measurement. 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.
8. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
| R151034 | Component | 40T | Storage |
| R151034A | 葡萄糖标准 (1mg/mL) | 50 mL | 2-8℃. |
| R151034B | Fehling's Reagent A | 250 mL | RT. |
| R151034C | Fehling's Reagent B | 250 mL | RT. |
| R151034D | 乙酸锌溶液 | 200 mL | RT. |
| R151034E | 亚铁氰化钾溶液 | 200 mL | RT. |
| R151034F | 碱性溶液 | 50 mL | RT. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 E2618500 |
分析证书 | R151034 |
| ZJ26F0332964 |
分析证书 | R151034 |
| ZJ26F0332965 |
分析证书 | R151034 |
| ZJ26F0332963 |
分析证书 | R151034 |
| ZJ26F0332966 |
分析证书 | R151034 |
| ZJ26F0332962 |
分析证书 | R151034 |
| ZJ26F0332961 |
分析证书 | R151034 |
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