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维生素C (Vitamin C) 是一种具有抗氧化性质的有机化合物，又称抗坏血酸，是高等灵长类动物与其他少数生物的必需营养素，在生物体内，维生素C是一种抗氧化剂，同时也是一种辅酶，为酸性己糖衍生物，是烯醇式己糖酸内酯，可保护身体免于自由基的威胁，其广泛的食物来源为新鲜的蔬菜和水果，VC有L-抗坏血酸和D-抗坏血酸，而且两种都具有强还原性，但只有L-抗坏血酸对人体具有生物活性。L-抗坏血酸有还原型抗坏血酸和氧化型抗坏血酸两种，前者含量较高，在一般蔬菜和水果中占90%以上，L-还原型抗坏血酸极易被氧化为L-脱氢抗坏血酸；L-脱氢抗坏血酸亦可被还原为L-还原型抗坏血酸，通常也称作氧化型抗坏血酸。
该试剂盒检测原理是还原型维生素C易被氧化型的2,6-二氯酚靛酚氧化成氧化型维生素C，2,6-二氯酚靛酚不仅是一种氧化剂，而且是一种酸碱指示剂，在中性和碱性溶液中呈蓝色，在酸性溶液中为红色，而被还原的2,6-二氯酚靛酚为无色，当用2,6-二氯酚靛酚滴定含有维生素C的酸性溶液时维生素C尚未完全被氧化前，滴下的2,6-二氯酚靛酚立即被还原成无色；一旦维生素C全部被氧化时，滴下的2,6-二氯酚靛酚立即被氧化成粉红色，因此当溶液从无色转变为微红色时即表示维生素C刚刚全部被氧化，此时即为滴定终点，如果无其他杂质干扰，待测样品所还原的标准2,6-二氯酚靛酚量与样品中所含的还原型维生素C量成正比，常用于蔬菜、水果极其制品中还原型维生素C含量的测定。该试剂盒仅用于科研领域，不用于临床诊断或其他用途。
自备材料：
1、组织捣碎器、匀浆器、滤纸、漏斗、50 ml容量瓶
2、100 ml三角烧瓶、10 ml吸管、5 ml微量滴定管
操作步骤 (仅供参考)：
1、配制1×组织匀浆液：按组织匀浆液 (12×): 蒸馏水=1: 11的比例稀释，即得。
2、制备样品提取液：清洗干净新鲜蔬菜或水果，用布或吸水纸吸干表面水分或自然晾干。称取5g，加入5 ml的1×组织匀浆液，置于组织捣碎器中打成浆状，滤纸过滤，滤液倒入50 ml容量瓶内，滤渣可用1×组织匀浆液重复匀浆1~2次，合并滤液倒入容量瓶内，定容滤液总体积至50 ml。
3、配制Vitamin C标准液：取少量1×组织匀浆液加入10 mg Vitamin C标准品中，充分混匀，然后完全转移至1×组织匀浆液，即为Vitamin C标准液 (0.1 mg/ml)。注意：溶解应完全、准确，否则有可能导致标准液不准确，配制好的Vitamin C标准液4℃避光保存，4周有效 (最好临用前配制)。
4、配制二氯酚靛酚标准液：取少量二氯酚靛酚稀释液加入50 mg二氯酚靛酚中，充分混匀，然后完全转移至二氯酚靛酚稀释液中，并定容至250 ml，即为二氯酚靛酚标准液 (0.2 mg/ml)。注意：溶解应完全、准确，否则有可能导致标准液不准确，配制好的二氯酚靛酚标准液4℃避光保存，1周有效。
5、标定二氯酚靛酚标准液：准确吸取1 ml Vitamin C标准液 (0.1 mg/ml)，置于三角烧瓶中，加入9 ml 1×组织匀浆液，用微量滴定管以二氯酚靛酚标准液进行滴定，直至呈现淡红色，并保持15s不褪色，即为滴定终点，记录染料的用量 (V₁)；另取一个三角烧瓶，用1×组织匀浆液10 ml做空白对照，按相同方法滴定，记录染料的用量 (V₂)，由于1 ml Vitamin C标准液含量是已知的，即0.1 mg；由所用二氯酚靛酚标准液的体积可计算出1 ml二氯酚靛酚相当于Vitamin C的毫克数，即滴定度T (重复3次，取平均值)。
6、维生素C样品滴定：准确吸取滤液10 ml置于100 ml三角烧瓶内，按相同方法滴定，记录染料的用量 (V₃) (重复3次，取平均值)。
计算：
100g样品中维生素C的质量(mg)=(V₃-V₂)×Vₜ/(Vₛ×m)×T×100
T (mg/ml)=0.1×1/(V₁-V₂)
式中：Vₜ=样品提取液的总体积 (ml)
Vₛ=滴定时所取的样品提取液体积 (ml)
V₁=染料标定所用的二氯酚靛酚标准液的体积 (ml)
V₂=空白标定所用的二氯酚靛酚标准液的体积 (ml)
V₃=样品提取液标定所用的二氯酚靛酚标准液的体积 (ml)
T=滴定度，即1 ml二氯酚靛酚相当于Vitamin C的毫克数 (mg/ml)
m=制备样品提取液所用的样品质量 (g)
注意事项：
1、组织匀浆液 (12×)久置或低温保存，容易产生乳白色浑浊；如果白色浑浊不明显，可以直接使用，不影响效果；如果白色浑浊较多，应弃用。
2、配好的Vitamin C标准液-20℃避光保存，4周有效 (最好临用前配制)。
3、配好的二氯酚靛酚标准液4℃避光保存，1周有效；如有条件可称取二氯酚靛酚10 mg，加入二氯酚靛酚稀释液并定容至50 ml后使用，可避免浪费试剂。
4、本产品提供的组织匀浆液可用于大部分的蔬菜和水果等样品，可抑制抗环血酸氧化酶活性。
5、松针类样品宜用1%盐酸溶液提取。
6、若样品中含大量Fe²⁺，可用8%醋酸溶液提取， Fe²⁺不会很快与染料起作用。
7、待测样本如不能及时测定，应置于2~8℃避光保存，3天内稳定。
8、样品切碎的过程中，尽量避免与铁器、铜器接触，并且尽量迅速。
9、某些水果、蔬菜浆状物泡沫太多，可滴加数滴丁醇或辛醇等。
10、如用干滤纸过滤，应弃去起初的数毫升滤液，也可离心取上清。
11、如果浆状物不易过滤，可低速离心，留取上清液进行滴定。
12、样品提取液要避免日光直射，否则会加速抗坏血酸的氧化。
13、如果提取物中含有较多色素，可用白陶土脱色过滤后再滴定，或加入1 ml氯仿，到达滴定终点时，氯仿层呈现淡红色。也可采用二甲苯萃取比色法进行测定。
14、如果样品是流质 (如橙汁等)，则无需研磨，直接取5 ml汁液，加入等量的1×组织匀浆液混匀后滴定即可。
15、为了防止还原型维生素C被氧化，滴定应迅速，一般不超过2 min。
16、滴定所用的二氯酚靛酚标准液一般控制在1~4 ml之间，2 ml左右最好。
17、滴定开始时二氯酚靛酚标准液应迅速加入，随后尽量一滴一滴的加入，并不断摇动三角烧瓶，直至呈粉红色，且保持15s不褪色，这样可以减少其他杂质的干扰。
18、如果样品浓度过高，应用蒸馏水稀释后重测，结果乘以稀释倍数。
19、为了您的安全和健康，请穿实验服并戴一次性手套操作。
20、试剂开封后请尽快使用，以防影响后续实验效果。
附录：该试剂盒提供的试剂均经严格称量或容量瓶定容，如果试剂盒开封使用后或可考虑Vitamin C标准液不再标准时，尤其是配制Vitamin C标准液后，放置过久，可以考虑重新标定Vitamin C标准液含量。其方法如下：
将Vitamin C标准液 (0.1 mg/ml)用1×组织匀浆液稀释至0.02 mg/ml，取Vitamin C标准液 (0.02 mg/ml) 5 ml置于三角烧瓶中，加入6%碘化钾溶液0.5 ml，1%淀粉指示剂3滴，再以0.001 mol/L碘酸钾标准液进行滴定，终点为蓝色。
维生素C浓度 (mg/ml)= 0.088×V₁/V₂
式中：V₁=滴定时所消耗的碘酸钾标准液的量 (ml)
V₂=滴定时所取的Vitamin C标准液的量 (ml)
0.088=1 ml碘酸钾标准液相对于Vitamin C的量 (mg)
Vitamin C is an organic compound with antioxidant properties, also known as ascorbic acid. It is an essential nutrient for higher primates and a small number of other organisms. In organisms, vitamin C acts as both an antioxidant and a coenzyme. It is an acidic hexose derivative and an enol hexonolactone, protecting the body from free radical damage. It is widely abundant in fresh vegetables and fruits.There are L-ascorbic acid and D-ascorbic acid, both possessing strong reducibility, yet only L-ascorbic acid exhibits biological activity in humans.L-ascorbic acid exists in reduced and oxidized forms. The reduced form accounts for over 90% in ordinary vegetables and fruits. Reduced L-ascorbic acid is easily oxidized into L-dehydroascorbic acid, which can also be reduced back to reduced L-ascorbic acid, commonly referred to as oxidized ascorbic acid.
The detection principle of this kit: Reduced vitamin C is readily oxidized to oxidized vitamin C by oxidized 2,6-dichlorophenolindophenol (DCPIP). DCPIP serves as both an oxidant and an acid-base indicator. It appears blue in neutral and alkaline solutions, red in acidic solutions, and colorless when reduced.When titrating acidic vitamin C solution with DCPIP, the dropped DCPIP is immediately reduced and decolorized before vitamin C is fully oxidized. Once vitamin C is completely oxidized, excess DCPIP turns pink. The titration endpoint is reached when the solution changes from colorless to pale red.Without impurity interference, the amount of standard DCPIP reduced by the sample is proportional to the reduced vitamin C content in the sample. This kit is widely applied to determine reduced vitamin C in vegetables, fruits and their products.This kit is for scientific research only, not for clinical diagnosis or other applications.
Self-Prepared Materials
1. Tissue homogenizer, blender, filter paper, funnel, 50 mL volumetric flask
2. 100 mL Erlenmeyer flask, 10 mL pipette, 5 mL micro burette
Procedure (For Reference Only)
1. Prepare 1× tissue homogenate: Dilute 12× tissue homogenate with distilled water at a ratio of 1:11.
2. Prepare sample extract: Wash fresh vegetables/fruits thoroughly, blot surface moisture or air-dry naturally. Weigh 5 g sample, add 5 mL 1× tissue homogenate, homogenize into slurry. Filter with filter paper, transfer filtrate into a 50 mL volumetric flask. Re-homogenize residue 1–2 times with 1× homogenate, combine all filtrates and dilute to a total volume of 50 mL.
3. Prepare vitamin C standard solution: Add a small amount of 1× tissue homogenate to 10 mg vitamin C standard, mix well, then transfer completely into homogenate to obtain 0.1 mg/mL vitamin C standard solution.
Note: Complete and accurate dissolution is required to avoid inaccurate standard concentration. Store prepared solution at 4℃ away from light, valid for 4 weeks; freshly prepared solution is recomended.
4. Prepare DCPIP standard solution: Add a small amount of DCPIP diluent to 50 mg DCPIP powder, mix uniformly, transfer fully into diluent and fix volume to 250 mL to obtain 0.2 mg/mL DCPIP standard solution.
Note: Ensure full dissolution. Store at 4℃ protected from light, valid for 1 week.
5. Calibrate DCPIP standard solution: Accurately pipette 1 mL 0.1 mg/mL vitamin C standard into an Erlenmeyer flask, add 9 mL 1× tissue homogenate. Titrate with DCPIP solution until pale red color persists for 15 seconds, record dye consumption (V₁).
Take another flask with 10 mL 1× homogenate as blank control, titrate identically and record blank consumption (V₂). Calculate titration factor T (mg vitamin C per mL DCPIP). Repeat 3 times and take average value.
6. Sample titration: Pipette 10 mL sample filtrate into a 100 mL Erlenmeyer flask, titrate following the same steps and record dye consumption (V₃). Repeat 3 times for average result.
Calculation
Vitamin C content in 100 g sample (mg)= (V₃-V₂)×Vₜ/(Vₛ×m)×T×100
T (mg/mL)= 0.1×1/(V₁-V₂)
Where: Vₜ= Total volume of sample extract (mL)
Vₛ= Volume of sample extract used for titration (mL)
V₁= DCPIP volume for standard calibration (mL)
V₂= DCPIP volume for blank titration (mL)
V₃= DCPIP volume for sample titration (mL)
T= Titration titer, mass of vitamin C equivalent to 1 mL DCPIP (mg/mL)
m= Mass of raw sample used for extraction (g)
Precautions
1. 12× tissue homogenate may develop milky turbidity after long storage or low-temperature preservation. Slight turbidity does not affect detection and can be used directly; discard severely turbid reagent.
2. Store prepared vitamin C standard at -20℃ away from light, valid for 4 weeks. Fresh preparation is preferred.
3. Store DCPIP standard at 4℃ protected from light, valid for 1 week. To save reagent, weigh 10 mg DCPIP, dilute to 50 mL with special diluent for use.
4. The supplied tissue homogenate is suitable for most vegetable and fruit samples and inhibits ascorbate oxidase activity.
5. Pine needle samples shall be extracted with 1% hydrochloric acid solution.
6. Samples rich in Fe²⁺ shall be extracted with 8% acetic acid, as Fe²⁺ reacts slowly with DCPIP dye.
7. Untested samples can be stored at 2–8℃ in dark and remain stable within 3 days.
8. Avoid contact with iron and copper utensils during sample cutting, and operate quickly.
9. Add a few drops of butanol or octanol to reduce excessive foam from fruit and vegetable pulp.
10. Discard initial several milliliters of filtrate when using dry filter paper, or collect supernatant after centrifugation.
11. Centrifuge at low speed and use supernatant for titration if filtration is difficult.
12. Keep sample extract away from direct sunlight to prevent accelerated oxidation of ascorbic acid.
13. For highly pigmented extracts, decolorize with kaolin before titration, or add 1 mL chloroform. The chloroform layer turns pale red at endpoint. Xylene extraction colorimetry is also applicable.
14. Liquid samples such as orange juice do not require grinding. Mix 5 mL juice with equal volume of 1× homogenate directly for titration.
15. Complete titration within 2 minutes to prevent oxidation of reduced vitamin C.
16. DCPIP consumption is recommended between 1–4 mL, optimally around 2 mL.
17. Add DCPIP rapidly at first, then dropwise with continuous shaking until stable pale red lasts 15 s, to minimize impurity interference.
18. Dilute high-concentration samples with distilled water before retesting, and multiply final result by dilution factor.
19. Wear lab coat and disposable gloves for safe operation.
20. Use opened reagents promptly to guarantee experimental accuracy.
Appendix
All reagents in this kit are accurately weighed or volumetrically fixed. If vitamin C standard solution deviates after long storage, recalibrate its concentration as follows:
Dilute 0.1 mg/mL vitamin C standard to 0.02 mg/mL with 1× homogenate. Pipette 5 mL diluted standard into flask, add 0.5 mL 6% potassium iodide solution and 3 drops 1% starch indicator. Titrate with 0.001 mol/L potassium iodate standard solution until stable blue color appears.
Vitamin C concentration (mg/mL) = 0.088×V₁÷V₂
Where: V₁= Consumed potassium iodate volume (mL)
V₂= Volume of vitamin C standard used (mL)
0.088= Vitamin C equivalent mass of 1 mL potassium iodate solution (mg)
| 项目号 | 物理外观 | 组件 | 50T | Storage |
| R1515897A | 固体 | Vitamin C标准品 | 10 mg | RT. Store in the dark. |
| R1515897B | 液体 | 组织匀浆液 (12×) | 250 ml | RT. |
| R1515897C | 固体 | 二氯酚靛酚 (DCPIP) | 50 mg | 2-8℃. Store in the dark. |
| R1515897D | 液体 | 二氯酚靛酚稀释液 | 250 ml | RT. |
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 |
|---|---|---|
 ZJ26F0838264 |
分析证书 | R1515897 |
| E2618501 |
分析证书 | R1515897 |
| E2614465 |
分析证书 | R1515897 |
| ZJ26F0535158 |
分析证书 | R1515897 |
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