TITLE: Estimate phospholipids from microalgae
AUTHORS: Yingyu  Hu

[DESCRIPTION]
Here we describe a protocol to estimate phospholipids from microalgae. 
After the extraction and measurement of total lipids from microalgae, the rest of lipids extract is dried by nitrogen flow and then dried with magnesium sulphate at 90 °C. A 650 °C dry combustion is used to decompose 80% phospholipids.   The ash is digested by 0.5 mL 0.2 M HCl for 30 minutes at 90°C. The resulting orthophosphate is detected by mixing the digested sample with a mixture of molybdate and ascorbic acid to produce molybdenum blue (Chen 1956). 

 




[STEPS]
SECTION: Prepare phospholipids sample
1. Dry remaining organic phase extract of total lipids at 37 °C under a stream of N2 gas (<2 psi)
SECTION: High temperature dry combust
2. Use diamond pen to engrave the sample vials with numbers. Log number and sample code.
SECTION: High temperature dry combust
3.  0.17 M MgSO4 reagent:
Dissolve 1.023 g MgSO4  in 50 mL MilliQ water 
 
SECTION: High temperature dry combust
4. Add 200 µL 0.17 M MgSO­4 to the dry extract.


 

SECTION: High temperature dry combust
5. Cover the uncapped vials with foil and place in the oven  at 90 °C  until samples are completely dry. 
 

 



SECTION: High temperature dry combust
6. Combust dried samples at 650 °C for  540 min
 

 

SECTION: High temperature dry combust
7. Allow samples to gradually cool down in the muffle furnace.
SECTION: Digestion
8. 0.2 M HCl reagent:
In a reagent bottle, dissolve one part of 12 N  HCl in 59 parts of MilliQ water 
 

 


SECTION: Digestion
9. Preheat oven to 90 °C 
SECTION: Digestion
10. Add 0.5 mL 0.2 M HCl to each vial. 

SECTION: Digestion
11. Tightly cap the vial and vortex.
SECTION: Digestion
12. Place vials in the oven for 30 min 
SECTION: Digestion
13. Cool samples down to Room temperature 
SECTION: Preparing standard working solutions
14. Standard working solutions and reagents can be prepared during sample digestion.
SECTION: Preparing standard working solutions
15. KH2PO4 primary standard stock solution (≈ 1 mM)  

SECTION: Preparing standard working solutions
15.1. Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
SECTION: Preparing standard working solutions
15.2. Place the beaker into an oven, dry KH2PO4 at 110 °C for at least 120 min 

SECTION: Preparing standard working solutions
15.3. Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
SECTION: Preparing standard working solutions
15.4. Dissolve around0.136 g dried KH2PO4 in 1 LMilliQ water.
Use 1 L volumetric flask
Take notes of the actual weight of KH2PO4 for final concentration of standard stock solution


SECTION: Preparing standard working solutions
15.5. Transfer standard stock solution into a 1 L bottle and store in the fridge.
 

SECTION: Preparing standard working solutions
16. Standard working solution

 
 
 

 
 
SECTION: Preparing standard working solutions
17. Transfer 500 µL of each standard working solution to 2 mL microtube. 

SECTION: Preparing working reagents
18. 
 

SECTION: Preparing working reagents
19.  6 N (3 M) sulfuric acid reagent:
Carefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water   

SECTION: Preparing working reagents
20.  2.5 % ammonium molybdate reagent:
Weigh 0.25 g ammonium molybdate in a Falcon tube and top to 10 g with MilliQ water.
Cap and shake until totally dissolved.  
 

SECTION: Preparing working reagents
21. 10 % ascorbic acid reagent:
Weigh 1 g ascorbic acid in a Falcon tube and top to 10 g with MilliQ water;
Cap and shake until all dissolved. 

 

 

SECTION: Preparing working reagents
22. Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
SECTION: Preparing working reagents
23. Mix the reagents into Falcon tube:

 
 
 

 
 
SECTION: Colorimetric measurement
24. Preheat incubator/shaker to 37 °C 
 

SECTION: Colorimetric measurement
25. Add 500 µL reagent to each standard, sample (in the vial) and blank, starting from blanks, including blank for standards and blank for samples.
 

 


SECTION: Colorimetric measurement
26. Vortex.
SECTION: Colorimetric measurement
27. Incubate at 37 °C  for 180 min  while shaking at 150 rpm
SECTION: Colorimetric measurement
28. Load microplate with 250 ul reactant from each tube, duplicate.
  

 

SECTION: Colorimetric measurement
29. Read plate in microplate reader

 
 
 
 
 

 

SECTION: Calculation
30. Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.
SECTION: Calculation
31. Subtract the average absorbance at 820 nm of the blank sample (i.e. blank filter) replicates from the absorbance at 820 nm of all other individual samples.
SECTION: Calculation
32. Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
SECTION: Calculation
33. Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
SECTION: Calculation
34. (Pmeasured)_ug/sample = (orthophosphate)_uM X (V_HCl)_mL X (0.001) X (30.97)

(Pcorrected)_ug/sample = (Pmeasured) / 0.8
 
Where, 0.8 is the average recovery of phospholipids after a high temperature dry combustion at 650 °C .

   

SECTION: Calculation
35. (Phospholipids)_ug/sample = (Pcorrected)/(0.01X4.3)