UA Biophysics:Protocols:Carotenoid Extration

Carotenoid Extraction, old protocol

• Pigments are extracted from pellet of planktonic
  
• 50mg of the cell pellets are dissolved in 1ml of methanol vortexed for 30 seconds
  
• Centrifuge at 10,000rpm for 10 min
  
• The supernatant containing the pigments is shaken with 1.7M NaCl and ethyl acetate (3:1 v/v) and allow to stand for 10 min to allow a sufficient phase separation
  
• The ethyl acetate layer is then carefully drawn from the top, dried with anhydrous Na₂SO₄, and decant into a clean glass tube
  
• The solvent containing the pigment is remove with N₂, and the residue dissolved in equal volumes of benzene and acetone
  
• This solution was then cooled to -20°C to precipitate the phospholipids, dried with N₂ again, and dissolved in benzene for absorption measurement
  
• The content of carotenoids is quantified by absorption from 400 to 550nm
  

Carotenoid Extraction, New protocol

• Pigments are extracted from pellet of planktonic
• 50mg of the cell pellets are dissolved in 1ml of methanol vortexed for 5 minutes with glass beads
• Centrifuge at 8500 rpm for 10 min
• The supernatant containing the pigments is shaken with 1.7M NaCl and ethyl acetate (3:1 v/v) (first adding ethyl acetate) and the sample is centrifuge at 8500 rpm for 5 min
• The upper (ethyl acetate) phase is is then carefully drawn from the top and the lower phase is reextracte by addition of 1.0 mL of ethyl acetate (perform in duplicate)
• Both organic phases is combine, dried with anhydrous Na2SO4, and decant into a clean glass tube
• The organic solvent containing the pigment is remove with N2 gaseous, and the residue dissolved in equal volumes of benzene and acetone
• This solution was then cooled to -20°C to precipitate the phospholipids, dried with N2 again, and dissolved in benzene for absorption measurement
• The content of carotenoids is quantified by absorption from 400 to 550nm

Bibliografy

• Eggers, L.F., Schwudke, D. (2016). Liquid Extraction: Folch. In: Wenk, M. (eds) Encyclopedia of Lipidomics. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-7864-1_89-1

Carotenoid Extration_ESP

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