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Jacqueline
Joey
18
Ngee Ann Poly
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    Preparation of Lab Sample
    Tuesday, July 3, 2012 @ 1:58 AM | comment (0)

    Extraction of Tocopherols from Human Milk
    - Milk specimens were warmed to a temperature of 38 degree Celsius after defrosting (on the actual day of analysis) and sonicated for a short while.
    - The sampling of milk is then performed at 38 degree Celsius with continuous stirring. These steps are done to make sure that the sample is homogenous. 
    - There are 2 methods, and 3 samples will be used for each method. 


    *All milk specimens were extracted using the  method and do not involve δ- tocopherol spike.
    Method 1:

    1.  1mL of milk will be spiked with 10µL of δ- tocopherol standards(around 7-10µg)
    2. Add 1mL of methanol containing pyrogallol (3% w/v)
    3. Add 1mL of aqueous potassium hydroxide (KOH) (10%, w/v)
    4. Vortex sample until it is thoroughly mixed.
    5. Place sample into a water bath for 30 minutes at 70 degree Celsius. This step is called saponification.
    6. After the first 15 minutes of saponification briefly vortex the tubes of samples.
    7. Cool the samples on ice.
    8.  Add 6M of hydrochloric acid (HCl)to acidify the sample to around pH 2. Use a pH probe to detect the pH value of the sample.
    9. Add 4mL of hexane
    10. Vigorously vortex the samples for 20seconds. Keep it on ice for a short while.
    11. Repeat step 10 for another 2 times.
    12. Centrifuge the samples at 1300g for 10 minutes to separate the emulsion.
    13. Carefully transfer the top, organic layer into a clean Pyrex container.
    14. Evaporate the layer under a gentle stream of nitrogen and on a warm plate at 40 degree Celsius.
    15. The fatty residue will be reconstituted in 0.5mL methanol-propan-2-ol (1:1 v/v) and warming up to 30 degree Celsius.    

    Method 2:  

    1. 1 mL of milk was spiked with 10 μL δ-tocopherol standard (7–10 μg)
    2. 1 mL methanol containing pyrogallol (2%, w/v) was added, to disrupt milk globules.
    3.  Keep the tubes on ice for 10 mins
    4. Add 3mL of hexane
    5.  Vigorously vortex the samples for 20seconds. Keep it on ice for a short while.
    6. Repeat step 10 for another 2 times.
    7. Centrifuge the samples at 1300g for 10 minutes to separate the emulsion.
    8. Carefully transfer the top, organic layer into a clean Pyrex container.
    9. Evaporate the layer under a gentle stream of nitrogen and on a warm plate at 40 degree Celsius.
    10. The fatty residue will be reconstituted in 0.5mL methanol-propan-2-ol (1:1 v/v) and warming up to 30 degree Celsius.    

     *Pyrogallol( 1% final concentration) was used to protect vitamin E from possible degradation during the extraction and saponification process.