Hello, Is there an effective and reproducible protocol for converting chlorophyll to water soluble chlorophyllin, particularly one that maximizes yield and preserves chlorophyllin stability? I have been testing few protocols I've found in papers where details were very limited, and none has worked so far. Information on optimal saponification parameters—such as pH, temperature, and reaction time—as well as solutions volumes and post-reaction purification methods that ensure minimal degradation would be highly valuable.
Thank you in advance.
Best regards.
Converting chlorophyll to copper chlorophyllin involves saponification to remove the phytol chain, converting chlorophyll into chlorophyllide, followed by chelation with copper to increase stability and make it water-soluble. Here’s a reproducible protocol with detailed steps and parameters for maximizing yield and stability:
### Materials Needed:
- Chlorophyll source (e.g., spinach extract)
- Ethanol
- 1% sodium hydroxide (NaOH) solution
- Copper sulfate (CuSO₄) solution (1%)
- Distilled water
- Hydrochloric acid (HCl), concentrated
- Ammonium sulfate (optional for precipitation)
- pH meter
- Ice bath
- Rotary evaporator or low-temperature drying equipment
### Protocol:
#### 1. **Chlorophyll Extraction** (if starting from plant material):
- Blend spinach or another chlorophyll-rich plant in ethanol.
- Filter the extract to remove solids.
- Concentrate the extract if necessary by rotary evaporation.
#### 2. **Saponification to Remove Phytol Chain:**
- **Preparation:** Mix the chlorophyll extract with an equal volume of 1% NaOH in ethanol.
- **Reaction Parameters:** Place the mixture in a water bath at 60°C for 30–45 minutes to achieve de-esterification. Stir constantly and avoid temperatures above 60°C to prevent chlorophyll degradation.
- **pH Monitoring:** Keep the pH at 9–10 for optimal saponification.
#### 3. **Neutralization:**
- Cool the mixture on ice, and adjust the pH to neutral (7.0) by adding a few drops of concentrated HCl slowly, checking the pH after each addition.
#### 4. **Copper Chelation to Form Copper Chlorophyllin:**
- Add 10–20 mL of 1% copper sulfate (CuSO₄) solution to the neutralized chlorophyllide mixture. This introduces copper ions, which replace the magnesium in the chlorophyll, forming copper chlorophyllin.
- Stir the solution at room temperature for 30 minutes. You should see a color change to a bright green, indicating chlorophyllin formation.
#### 5. **Purification and Precipitation:**
- **Precipitation (Optional):** Add saturated ammonium sulfate slowly to the solution until you reach approximately 50% saturation, which can help precipitate copper chlorophyllin. Let it sit for 30 minutes.
- Centrifuge the mixture at 5000 x g for 15 minutes to pellet the chlorophyllin.
- Discard the supernatant and resuspend the pellet in distilled water.
#### 6. **Final Washing and Drying:**
- Dialyze the resuspended pellet against distilled water for 24 hours to remove any remaining salts.
- Dry the purified copper chlorophyllin in a rotary evaporator or by freeze-drying, if available.
#### **Storage and Stability Considerations:**
- Store the copper chlorophyllin in a dark, cool environment to prevent degradation.
- Aqueous copper chlorophyllin is stable at a slightly acidic to neutral pH (around 6.5–7) but can degrade if exposed to strong light, extreme pH, or high temperatures.
This method aims to maximize yield and maintain the stability of chlorophyllin by carefully controlling pH, temperature, and reaction time. The final product should be a water-soluble, stable copper chlorophyllin suitable for various applications.
Raghad Mouhamad Hello, thank you so much for your answer. Could you please share the reference or paper from which you got the protocol from?
Raghad Mouhamad - Thank you for sharing the protocol. Is this published in any journal? If so, could you please send me a copy to [email protected]
I greatfully appreciate your response - Thank you.
Schoefs, B. (2002). "Chlorophyll and Carotenoid Analysis in Food Products: Properties of the Pigments and Methods of Analysis." Trends in Food Science & Technology, 13(11), 361-371.
Lanfer-Marquez, U. M., Barros, R. M. C., & Sinnecker, P. (2005). "Physicochemical and Stability Aspects of Chlorophylls and Derivatives." Food Research International, 38(8-9), 885-891.
Raghad Mouhamad - Thank you very much for posting the articles. I was slso looking for the reference for the step by step protocol you had posted. IWondering if it is your own work, not published yet? If you happen to have the oriignal reference, could you please send it me? [email protected] .Thanks.
- Badges
- Science method