Apr 06, 2023

Public workspaceEstimate phospholipids from microalgae V.2

DOI
dx.doi.org/10.17504/protocols.io.q26g74r19gwz/v2
  • 1Dalhousie University
Ying-Yu Hu
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DOI: dx.doi.org/10.17504/protocols.io.q26g74r19gwz/v2
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2023. Estimate phospholipids from microalgae. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74r19gwz/v2Version created by Ying-Yu Hu
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 16, 2023
Last Modified: April 06, 2023
Protocol Integer ID: 77080
Keywords: phospholipids, high temperature dry combustion
Funders Acknowledgement:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
Here we describe a protocol to estimate phospholipids from microalgae.

After extracting and measuring the total lipids from microalgae, the remaining lipid extract is dried using a nitrogen flow, followed by drying with magnesium sulfate at 90°C. However, it has been observed that traditional dry combustion at 500°C only decomposes approximately 50% of phospholipids (Hu et al., 2022). To achieve complete conversion of phospholipids to pyrophosphate, a temperature of around 800°C is required, but such high temperatures cannot be used with glassware. As the acid digestion method involves using only 500 µL of 0.2 M HCl, which must be placed in tightly capped glass vials to prevent concentration changes due to evaporation, combustion must be carried out using glassware instead of crucibles. It should be noted that the recovery rate of phospholipids is around 80% when combusted at 650°C, but this recovery rate is consistent, making the use of glass vials applicable. Therefore, we recommend using 650°C to combust phospholipids and using 80% to correct the final results.

The resulting ash is digested using 0.5 mL of 0.2 M HCl for 30 minutes at 90°C. After digestion, the resulting orthophosphate is detected by mixing the sample with a combination of molybdate and ascorbic acid to produce molybdenum blue, as described in Chen's work (1956).

CITATION
P.S. Chen, T.Y. Toribara and Huber Warner. Microdetermination of Phosphorus. Anal. Chem..
LINK
https://doi.org/10.1021/ac60119a033

CITATION
Ying-Yu Hu, Andrew J. Irwin, Zoe V. Finkel (2022). Improving quantification of particulate phosphorus. Limnology and Oceanography: Methods.
LINK
https://doi.org/10.1002/lom3.10517






Protocol materials
ReagentMagnesium sulfate anhydrousFisher ScientificCatalog #M65500
Step 4
Reagent12 N Hydrochloric acid
Step 9
Reagent18M sulfuric acid
Step 22
ReagentAmmonium molybdateMerck MilliporeSigma (Sigma-Aldrich)Catalog #09878-100G
Step 20
ReagentAscorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G
Step 21
ReagentPotassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
Step 2
Prepare phospholipids sample
Prepare phospholipids sample
Dry remaining organic phase extract of total lipids at Temperature37 °C under a stream of N2 gas (<2 psi)
Phosphate primary standard
Phosphate primary standard
2h
2h
KH2PO4 primary standard stock solution (≈ 1 mM)ReagentPotassium dihydrogen orthophosphateACP ChemicalsCatalog #P-4550
Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
Place the beaker into an oven, dry KH2PO4 at Temperature110 °C for at least Duration02:00:00
2h
Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
Dissolve aroundAmount0.136 g dried KH2PO4 in Amount1 L MilliQ water.
  • Use 1 L volumetric flask
  • Take notes of the actual weight of KH2PO4 for final concentration of standard stock solution
Transfer standard stock solution into a 1 L bottle and store in the fridge.
Note
This stock solution lasts quite a long time, unless there is evidence for growth of algae or other extraneous biotic material.


High temperature dry combustion
High temperature dry combustion
9h
9h
Use diamond pen to engrave the sample vials with numbers. Log number and sample code.
Concentration0.17 M MgSO4 reagent:

Dissolve Amount1.023 g MgSO4 in 50 mL MilliQ water
ReagentMagnesium sulfate anhydrousVWR InternationalCatalog #M65500
Add Amount200 µL Concentration0.17 M MgSO­4 to the dry extract.
Note
Sing-use pipet tip to avoid cross-contamination.

Cover the uncapped vials with foil and place in the oven at Temperature90 °C until samples are completely dry.
Equipment
Forced air oven
NAME
VWR
BRAND
89511-410
SKU

Note
Remove samples out of the oven as soon as they are dried. If muffle furnace is not available, keep samples in vacuum desiccator.


Combust dried samples at Temperature650 °C for Duration09:00:00
Equipment
Muffle furnace
NAME
F30428C
TYPE
Thermo
BRAND
10-505-13
SKU

Note
Only place glass vials in the muffle furnace. Temperature650 °C turns foil into ash.


9h
Allow samples to gradually cool down in the muffle furnace.
Digestion
Digestion
Concentration0.2 M HCl reagent:

In a reagent bottle, dissolve one part of Concentration12 N HCl in 59 parts of MilliQ water
Reagent12 N Hydrochloric acidVWR International

Note
Volume of HCl_0.2M_mL = (0.5_mL) X (#Sample + #Blank)


Preheat oven to Temperature90 °C
Add Amount0.5 mL Concentration0.2 M HCl to each vial.
Tightly cap the vial and vortex.
Place vials in the oven for Duration00:30:00
30m
Cool samples down to TemperatureRoom temperature
Preparing standard working solutions
Preparing standard working solutions
2h
2h
Preheat shaker/incubator to Temperature37 °C
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU



Standard working solutions and reagents can be prepared during sample digestion.


Standard working solution
StandardPrimary (uL)MilliQ (uL)
S101000
S25995
S310990
S420980
S550950
S6100900
S7150850
S8200800

Transfer Amount500 µL of each standard working solution to 2 mL microtube.
Preparing working reagents
Preparing working reagents
All reagents are freshly prepared before colorimetric measurement.
Concentration2.5 % ammonium molybdate reagent:

Weigh Amount0.25 g ammonium molybdate in a Falcon tube and top to Amount10 g with MilliQ water.
Cap and shake until totally dissolved.
ReagentAmmonium molybdateVWR InternationalCatalog #09878-100G
Concentration10 % ascorbic acid reagent (avoid light exposure):

Weigh Amount1 g ascorbic acid in a Falcon tube and top to Amount10 g with MilliQ water;
Cap and shake until all dissolved.

ReagentAscorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5960-100G

Concentration6 N (3 M) sulfuric acid reagent:

Carefully add 1 part Concentration18 M concentrated sulfuric acid into 5 part MilliQ water Reagent18M sulfuric acidVWR International
Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
Mix the reagents into Falcon tube:
ReagentParts as in volume
MilliQ2
6N sulphuric acid1
2.5% ammonium molybdate1
10% ascorbic acid1

Colorimetric measurement
Colorimetric measurement
3h
3h
Add Amount500 µL reagent to each standard, sample (in the vial) and blank, starting from blanks, including blank for standards and blank for samples.
Equipment
Finntip Stepper Tips
NAME
5 mL
TYPE
Thermo Scientific
BRAND
9404200
SKU

Note
Before dispensing the reagent, wipe or dab the liquid drop on the outside of the tip, avoid wiping the open tip.


Vortex.
Incubate at Temperature37 °C for Duration03:00:00 while shaking at 150 rpm
3h
Load microplate with 250 ul reactant from each tube, duplicate.
Example of loading the microplate
Read plate in microplate reader
AB
Shake duration00:00:05
Shaking typeContinuous
Shaking forceHigh
Shaking speed [rpm]600
Wavelength [nm]820
Use transmittanceNo
Pathlength correctionNo
Measurement Time [ms]100

Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

Calculation
Calculation
3h
3h
Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.
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.
Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
Molar Mass of KH2PO4: 136.086 g/mol
Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
(Pmeasured)_umol/sample = (orthophosphate)_uM X (V_HCl)_mL X (0.001)
(Pcorrected)_umol/sample = (Pmeasured) / 0.8
Where, 0.8 is the average recovery of phospholipids after a high temperature dry combustion at Temperature650 °C .
(Phospholipids)_ug/sample = (Pcorrected)X30.97/(0.01X4.3)
Citations
P.S. Chen, T.Y. Toribara and Huber Warner. Microdetermination of Phosphorus
https://doi.org/10.1021/ac60119a033
Ying-Yu Hu, Andrew J. Irwin, Zoe V. Finkel. Improving quantification of particulate phosphorus
https://doi.org/10.1002/lom3.10517