BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
植物体内的可溶性糖主要是指能溶于水及乙醇的单糖和寡聚糖，植物体内的碳素营养状况以及农产品的品质、性状，常以糖含量作为重要指标，植物为了适应逆境条件如干旱、低温等条件会主动积累一些可溶性糖，降低渗透势和冰点，以适应外界环境条件的变化，测定植物体内可溶性糖的方法有：蒽酮比色法、3,5-二硝基水杨酸比色法、苯酚比色法、斐林试剂比色法等化学方法。
| 组分货号 | 组分名称 | 物理外观 | 50T | 储存温度 | 单次测试使用量 |
| P1507806A | 蔗糖标准溶液 (10mg/mL) | 无色透明液体 | 1 mL | 2-8℃ | 按需求使用 |
| P1507806B | 苯酚溶液 (10×) | 无色透明液体 | 6 mL | RT. 避光. | 按需求使用 |
注：整套试剂盒未拆封时于2-8℃避光保存，自生产之日起有效期 1 年；拆封后各组分稳定性不同，部分组分可转至RT保存（有效期仍为 1 年），其余需继续2-8℃保存。请严格按照组分表标注温度分类存放，保障试剂性能。
自备仪器和试剂
1、蒸馏水、浓硫酸
2、电子天平、剪刀、研钵或匀浆器、水浴锅或电磁炉、分光光度计、比色杯
3、50ml烧杯或三角瓶、容量瓶、20ml刻度试管或10ml螺旋盖离心管
操作步骤
1、可溶性糖的提取
1.1 称取新鲜的植物样品（干样品亦可）0.5~1g，剪碎，加入蒸馏水约3ml匀浆，转移至刻度试管中，用12ml蒸馏水冲洗研磨器2~3次，洗出液也转移至该容器。
1.2 塑料薄膜封口，于沸水浴中提取30min，待冷却后过滤，将滤液转入50ml容量瓶。
1.3 收集残渣再次匀浆、加水提取、合并滤液，定容。
2、配制苯酚工作液
取1份苯酚溶液 (10×)，再加入9份蒸馏水，混匀即可，现用现配。
3、稀释蔗糖标准：取1ml蔗糖标准溶液 (10mg/ml) 加入100ml容量瓶中，用蒸馏水定容至刻度，即为蔗糖标准 (100μg/ml)；取干净离心管或试管，按下表操作，依次获得系列质量的蔗糖标准。
加入物质 (ml) | 1 | 2 | 3 | 4 | 5 |
蔗糖标准 (100μg/ml) | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
蒸馏水 | 1.8 | 1.6 | 1.4 | 1.2 | 1 |
相当于蔗糖质量 (μg) | 20 | 40 | 60 | 80 | 100 |
蔗糖标准浓度 (μg/ml) | 10 | 20 | 30 | 40 | 50 |
4、加样
取10ml螺旋盖离心管，按照下表设置空白管、标准管、测定管，溶液应按照顺序依次加入，并注意避免产生气泡，小心混匀；如果样品中的糖浓度过高，可以减少样品用量或适当稀释后再进行测定，样品的检测最好能设置2~3平行管，求平均值（各种试剂的加入量可以等比例的缩小，但应保证最小的所需量）。
加入物 (ml) | 空白管 | 标准管 | 测定管 |
蒸馏水 | 2 | － | － |
系列蔗糖标准 (1~5号) | － | 2 | － |
提取液 | － | － | 2 |
苯酚工作液 | 1 | 1 | 1 |
浓硫酸 | 5 | 5 | 5 |
注：加浓硫酸时请参考注意事项3-5。
5、可溶性糖测定
加入浓硫酸后充分振荡，室温下反应30min。以空白管调零，比色杯光径1cm，分光光度计测定485nm处标准管、测定管的吸光度。
6、结果计算
以系列蔗糖标准(1~5号)的质量(μg)为横坐标，以相应的吸光度为纵坐标，绘制标准曲线并求出线性回归方程，根据测定管的吸光度计算出相应的可溶性糖的质量及含量；亦可根据蔗糖标准浓度(μg/ml)与吸光度值绘制标准曲线，并求出样品的可溶性糖浓度。可溶性糖的含量，以质量分数(%)表示：
可溶性糖含量(%)=(m1×VT×N)/(m0×VS×1000000)×100% =(c×VT×N)/(m0×1000000)×100%
参数说明：
m1：从标准曲线查得的可溶性糖的质量(μg)
VT：提取液的总体积(ml)
N：样品提取液的稀释倍数
m0：植物样品的质量(g)
VS：测定时所取样品提取液体积(ml)
c：样品的可溶性糖浓度(μg/ml)
1000000：μg与g的换算关系
注意事项
1、实验前建议选择2~3个预期差异大的样本做预实验，如果样本吸光值不在建议测量范围内应稀释或者增加样本量进行检测。
2、如果样品可溶性糖浓度过高，应用蒸馏水稀释，糖的浓度在10~50μg/ml时线性梯度良好，最低检出限为1μg/ml，并且产生的颜色可稳定160min以上。
3、浓硫酸(相对密度1.84)有强氧化性、强腐蚀性，危险性极大，操作应十分小心。
4、加浓硫酸时应缓慢加入，以免产生大量热量而爆沸，灼伤皮肤和衣服，如出现此类现象，应迅速用自来水冲洗，如有必要应及时就医。
5、浓硫酸的纯度、滴加方式和速度，如直接加在液面还是慢加等，都会对实验结果产生影响。因此，操作方式一定要一致才能获得较好的重现性。一般建议加浓硫酸时应从管液正面在5~20s内加完，并摇匀。
6、如果样品中葡萄糖含量较多时，显色时间可相应延长至45min。
7、样品中可溶性糖含量的测定过程和时间必须与标准曲线的时间一致。
8、苯酚溶液在低温条件下可能会结晶，应在45~70℃水浴或温箱中充分溶解后使用。
9、为了您的安全和健康，请穿实验服并戴一次性手套操作。
附录
标准曲线制作：按说明书操作，通过分光光度计485nm对系列标准进行吸光度的测定，其数值及标准曲线如下（仅供参考）：
蔗糖标准 (μg/ml) | 0 | 10 | 20 | 30 | 40 | 50 |
吸光度 | 0.266 | 0.462 | 0.635 | 0.802 | 0.961 | 1.122 |
由结果可知，蔗糖标准在10~50μg/ml以内线性梯度良好，蔗糖浓度超过50μg/ml时偏差较大。
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.
| Component No. | Component Name | Appearance | 50T | Storage | Quantity Per Test |
| P1507806A | Sucrose Standard Solution (10 mg/mL) | Colourless clear liquid | 1 mL | 2-8℃ | Use as per requirements |
| P1507806B | Phenol Solution (10×) | Colourless clear liquid | 6 mL | RT. Store in the dark. | Use as per requirements |
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 × VT × N) / (m₀ × VS × 1,000,000) × 100% = (c × VT × N) / (m₀ × 1,000,000) × 100%
Parameter Description:
m₁: Mass of soluble sugar obtained from the standard curve (µg)
VT: Total volume of the extraction solution (ml)
N: Dilution factor of the sample extraction solution
m₀: Mass of the plant sample (g)
VS: 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
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.
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.
Concentrated sulfuric acid (specific gravity 1.84) is highly oxidizing and corrosive, posing significant hazards. Handle with extreme care.
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.
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.
If the sample contains a high amount of glucose, the color development time can be extended to 45 minutes accordingly.
The process and timing for measuring soluble sugar content in samples must be consistent with those used for the standard curve.
Phenol Solution may crystallize at low temperatures. It should be fully dissolved in a 45~70°C water bath or incubator before use.
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.
| 组分货号 | 组分名称 | 物理外观 | 50T | 储存温度 | 单次测试使用量 |
| P1507806A | 蔗糖标准溶液 (10mg/mL) | 无色透明液体 | 1 mL | 2-8℃ | 按需求使用 |
| P1507806B | 苯酚溶液 (10×) | 无色透明液体 | 6 mL | RT. 避光. | 按需求使用 |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131499 |
分析证书 | P1507806 |
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