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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。
叶绿体是光合作用的细胞器，在光合作用研究中，常需要用提取的叶绿体展开下游研究工作，叶绿体中所含的色素主要有两大类，叶绿素（包括叶绿素a和叶绿素b）和类胡萝卜素（包括胡萝卜素和叶黄素），它们与类囊体膜上的蛋白质结合，成为色素蛋白复合体，其中叶绿素又称叶绿体色素（Chlorophyll）。
检测原理：叶绿素不溶解于水，而溶于有机溶剂，以有机溶剂提取叶绿素，根据朗伯-比尔定律，某有色溶液的吸光度（A）与其中溶质浓度（C）和液层厚度（L）成正比，即A=αCL，其中α为比例常数，当溶液浓度以百分比浓度为单位，层液厚度为1cm时，α为该物质的吸光系数，在该试剂盒情况下叶绿素a和叶绿素b在665nm、649nm处有最大吸收波，根据经验公式可计算出叶绿素a、叶绿素b及总叶绿素含量，主要用于植物组织中叶绿素的提取以及定量检测叶绿素a、叶绿素b及总叶绿素含量。
适用样本：植物
自备仪器和试剂
操作步骤
1、叶绿体提取
1.1 取菠菜或其他植物新鲜叶片，洗净，擦干，去中脉，称取剪碎的新鲜样品0.1g，置于研钵或匀浆器，加入少量提取粉剂（约50mg）和1ml Chlorophyll Assay Buffer，研磨或匀浆成液态。
2、滤纸或三层纱布过滤，留取滤液，也可采用4000r/min离心5min，取上清液待测。
3、测定
分光光度计开机预热30min以上，调节多波长至665nm和649nm；取叶绿素提取液加入光径为1cm的比色杯中，以Chlorophyll Assay Buffer调零，分别用分光光度计测定提取液在665nm、649nm处吸光度（A665、A649）。
4、结果计算
叶绿素a含量(mg/g)=Ca×V×N/(W×1000)
叶绿素b含量(mg/g)=Cb×V×N/(W×1000)
总叶绿素含量(mg/g)=CT×V×N/(W×1000)
参数说明：
Ca=13.95×A665-6.88×A649 (mg/L)
Cb=24.96×A649-7.32×A665 (mg/L)
CT=6.63×A665+18.08×A649 (mg/L)
V：叶绿素提取液体积(ml)=10(ml)
N：稀释倍数
W：样品鲜重或干重(g)
1000：ml与L的单位换算
注意事项
1、为了避免叶绿素见光分解，操作时应尽量避光，研磨或匀浆时应尽量缩短时间。
2、当样品吸光值大于1时，可适当稀释后再进行测定，计算时乘以稀释倍数。
3、Chlorophyll Assay Buffer易挥发，不用时需拧紧瓶盖。
4、叶绿素提取液不能出现浑浊现象，否则应重新过滤。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
6、试剂开封后请尽快使用，以防影响后续实验效果。
7、该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
Chloroplasts are organelles for photosynthesis. In photosynthesis research, extracted chloroplasts are often needed for downstream studies. The pigments contained in chloroplasts mainly fall into two categories: chlorophyll (including chlorophyll a and chlorophyll b) and carotenoids (including carotene and lutein). These pigments bind to proteins on the thylakoid membrane to form pigment-protein complexes, among which chlorophyll is also known as chloroplast pigments (Chlorophyll).
Detection Principle
Chlorophyll is insoluble in water but soluble in organic solvents. Chlorophyll is extracted using an organic solvent. According to the Lambert-Beer law, the absorbance (A) of a colored solution is proportional to its solute concentration (C) and path length (L), i.e., A = αCL, where α is the proportionality constant. When the solution concentration is expressed in percentage concentration and the path length is 1 cm, α is the absorption coefficient of the substance. In this kit, chlorophyll a and chlorophyll b have maximum absorption peaks at 665 nm and 649 nm, respectively. The contents of chlorophyll a, chlorophyll b, and total chlorophyll can be calculated using empirical formulas. This kit is primarily used for the extraction of chlorophyll from plant tissues and the quantitative detection of chlorophyll a, chlorophyll b, and total chlorophyll content.
Applicable Sample: Plants
Reagents, consumables and Equipments not provided
Procedure
1. Chloroplast Extraction
1.1 Take fresh spinach leaves or other plant leaves, wash, dry, remove the midrib, weigh 0.1 g of the cut sample, place it in a mortar or homogenizer, add a small amount of Extraction Powder (about 50 mg) and 1 mL of Chlorophyll Assay Buffer, and grind or homogenize into a liquid state.
1.2 Transfer the ground or homogenized liquid to a 10 mL centrifuge tube. Rinse the mortar or homogenizer several times with a small amount of Chlorophyll Assay Buffer. Finally, pour everything, including the residue, into the 10 mL centrifuge tube. Add Chlorophyll Assay Buffer to a total volume of 10 mL, mix well, and incubate protected from light for 5 minutes to 2 hours.
Note: Alternatively, the tissue can be cut into pieces, Chlorophyll Assay Buffer added, and incubated protected from light for 12–36 hours, shaking several times during this period to ensure full contact between the tissue and the extraction reagent. When the tissue turns nearly white, it indicates that pigment extraction is complete. If a significant amount of tissue color remains, continue the extraction until the tissue residue color is close to white.
2. Filter through filter paper or three layers of gauze, and collect the filtrate. Alternatively, centrifuge at 4,000 rpm for 5 minutes, and collect the supernatant for testing.
3. Measurement
Turn on and preheat the spectrophotometer for at least 30 minutes. Set the wavelengths to 665 nm and 649 nm. Transfer the chlorophyll extract into a cuvette with a 1 cm light path. Zero the instrument with Chlorophyll Assay Buffer. Measure the absorbance of the extract at 665 nm and 649 nm (A<sub>665</sub> and A<sub>649</sub>) using the spectrophotometer.
4. Calculation of Results
Chlorophyll a content (mg/g) = C<sub>a</sub> × V × N / (W × 1000)
Chlorophyll b content (mg/g) = C<sub>b</sub> × V × N / (W × 1000)
Total chlorophyll content (mg/g) = C<sub>T</sub> × V × N / (W × 1000)
Parameter Description:
Ca = 13.95 × A665 – 6.88 × A649 (mg/L)
Cb = 24.96 × A649 – 7.32 × A665 (mg/L)
CT = 6.63 × A665 + 18.08 × A649 (mg/L)
V: Volume of chlorophyll extract (mL) = 10 (mL)
N: Dilution factor
W: Fresh or dry weight of the sample (g)
1000: Unit conversion factor between mL and L
Notes
To avoid photodegradation of chlorophyll, perform operations under minimal light and keep grinding or homogenization time as short as possible.
If the sample absorbance is greater than 1, dilute appropriately before measurement and multiply by the dilution factor in the calculation.
Chlorophyll Assay Buffer is volatile; always tighten the cap when not in use.
The chlorophyll extract must not appear turbid; if turbidity occurs, filter again.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.
This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
| C1508198 | Component | 50T | Storage |
| C1508198A | Chlorophyll Assay Buffer | 530 mL | RT. |
| C1508198B | 提取粉剂 | 3 g | RT. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0232335 |
分析证书 | C1508198 |
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