Jun 07, 2022
Version 2
Lipids in microalgae: Quantitation by acid-dichromate method in microtiter plate V.2
- Ying-Yu Hu1,
- Zoe V. Finkel1
- 1Dalhousie University
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2022. Lipids in microalgae: Quantitation by acid-dichromate method in microtiter plate. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw9dpzgmk/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: June 01, 2022
Last Modified: June 07, 2022
Protocol Integer ID: 63699
Keywords: acid-dichromate, lipids, microalgae, microtiter plate
Abstract
This is a protocol for quantitating total lipids in microalgae.
The acid-dichromate method is widely used to perform colorimetric analysis of extracted lipids. Here we present a protocol using 96-well microtiter plate for safe and efficient sample handling with high throughput. Only 500 ul of 0.15% acid-dichromate is required for each sample, which greatly reduces the amount of corrosive and toxic reagent.
In addition, comparing with the absorbance at 440 nm, the absorbance at 348 nm yields five-time higher sensitivity in lipids quantitation.
Total lipids in the samples should not excess 100 ug. Accurate quantitation can be achieved with as little as 20 ug. A working detection limit is about 5 ug.
CITATION
Guidelines
Lipids of microalgae is extracted by Folch solvent. The extract is dissolved in chloroform and stored at -80 degree ULT freezer.
Protocol
NAME
Lipids in microalgae: The Extraction by modified Folch solvent
CREATED BY
Ying-Yu Hu
Estimate the mass of extracted lipids as 10~30% of total cell mass.
The quantitative range of this method is 0~80 ug.
Materials
MATERIALS
Potassium dichromateFisher ScientificCatalog #P188-100
Concentrated sulphuric acid
Glyceryl tripalmitate
Sodium sulphite
Chloroform (HPLC grade)Sigma AldrichCatalog #439142-4L
Equipment
Storage Vials and Closures
NAME
12 mL amber
TYPE
Thermo Scientific
BRAND
B7800-12A
SKU
VWR 66030-686
SPECIFICATIONS
Equipment
VWR® Vials, Borosilicate Glass, with Phenolic Screw Cap
NAME
22.18 mL
TYPE
VWR
BRAND
66012-044
SKU
LINK
24-400 cap: VWR 89076-764
SPECIFICATIONS
Equipment
Boil-Proof Microcentrifuge Tubes
NAME
Microtube
TYPE
Axygen Scientific
BRAND
MCT-200-C
SKU
Equipment
VWR® Volumetric Pipets, Reusable, Color Coded, Class A
NAME
0.5 mL and 5 mL
TYPE
VWR
BRAND
10546-004 and 10546-014
SKU
Equipment
Safetypette
NAME
Jencons
BRAND
75856-442
SKU
Equipment
Gastight® 1700 Series Syringes
NAME
1710N
TYPE
Hamilton
BRAND
81000
SKU
Equipment
Microbalance
NAME
Cubis series
TYPE
Sartorius
BRAND
MSE6.6S-000-DM
SKU
Equipment
Reacti-Vap Evaporator
NAME
Thermo Scientific
BRAND
TS-18825
SKU
Equipment
VWR ANALOG VORTEX MIXER
NAME
VWR
BRAND
10153-838
SKU
With tube insert
SPECIFICATIONS
Equipment
VWR® Advanced Hot Plates
NAME
VWR
BRAND
97042-658
SKU
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU
Equipment
96-Well Microplates, Polystyrene, Clear,
NAME
Greiner Bio-One
BRAND
655101
SKU
Safety warnings
Safety information
Any items contaminated by potassium dichromate should be disposed as hazardous waste.
Before start
Pre-combust 12 ml amber vials (one for each sample/sample blank/standard/standard blank) and glass vials (for MilliQ water, concentrated sulphuric acid, acid-dichromate reagent).
Rinse vial caps with 95% ethanol and dry.
Rinse serological pipet (0.5 and 5 mL) with chloroform. The pipets are used to measure and transfer extract, concentrated sulphuric acid and acid-dichromate reagent.
Rinse syringe with chloroform and dry.
Note
Note
If the biomass is unknown, process one of the replicates first, and then decide the dilution factor and the amount that can be used for phospholipids.
If the result from Step 1 shows the lipids collected is lower than low-limit-of-detection, consider combine replicates as one sample.
Preparation of Standard
Preparation of Standard
Prepare glyceryl tripalmitate (GTP) primary standard solution (around 1 mg/ml)
Place frozen GTP in vacuum desiccator with lose cap until it is warmed to Room temperature before making primary standard solution
Weigh around 1 mg GTP, take note of the actual weight.
Dissolve GTP by 1 mL chloroform in amber vial, gently vortex.
Prepare working standards:
5 ug/vial:
In two 12 ml amber vials, add 5 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
10 ug/vial:
In two 12 ml amber vials, add 10 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
20 ug/vial:
In two 12 ml amber vials, add 20 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
40 ug/vial:
In two 12 ml amber vials, add 40 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
80 ug/vial
In two 12 ml amber vials, add 80 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
100 ug/vial
In two 12 ml amber vials, add 100 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
Dry working standards at Room temperature under N2 gas stream (<2 psi).
Preparation of acid-dichromate reagent
Preparation of acid-dichromate reagent
Estimate the total volume of potassium dichromate required:
Number of standards and standard blanks: 12
Number of samples and sample blanks: N
V=0.5x(N+12) ml
Transfer concentrated sulphuric acid to a glass vial for temporary storage
Weigh a glass vial, and tare the balance
Use 5 ml serological pipet to measure and transfer concentrated sulphuric acid to this vial. The volume of sulphuric acid is several milliliter more than estimated in go to step #6 . Write down the weight of sulphuric acid.
The weight of dichromate required for the 0.15% (w/w) acid-dichromate reagent equals the weight of sulphuric acid multiplied by (0.15/99.85).
Weigh dichromate and dissolve it into concentrated sulphuric acid. Cap the vial and vortex gently.
Reaction of lipids and acid-dichromate reagent
Reaction of lipids and acid-dichromate reagent
Allow frozen extract warm up to Room temperature .
Label two 12 mL vials with “+ Blank” and “- Blank”.
"-Blank" is 0 ug GTP.
"+Blank" is the reference of absorbance.
Prepare boiling water bath on hot plate, place a vial rack in the water bath
Add 0.5 mL of acid-dichromate reagent to each vial (standards, +Blank and –Blank, samples and sample blanks). Cap and vortex right after.
Note
Use 5 mL glass serological pipet, fill to "4 mL", dispense 0.5 mL .
Keep reaction vials in boiling water for 00:15:00 .
Cool vials to Room temperature in the fumehood
Prepare 0.2 g/ml sodium sulphite solution
Weigh 0.2 g sodium sulphite in a 2 ml microtube.
Add 1 mL MilliQ water into the tube.
Vortex
Add 1.125 mL o MilliQ (1 mL + 125 uL by pipet) to each vial. Cap immediately and vortex.
Cool vials to room temperature.
Add 25 µL 0.2 g/ml sodium sulphite solution to the “+Blank” vial. Vortex.
Colorimetric analysis
Colorimetric analysis
Vortex each vial and load 250 µL r reactant into microtiter plate.
Note
Reverse pipetting
Read absorbance at 348 nm
Calculation
Calculation
Subtract absorbance of "+Blank" from the absorbance of standards.
Plot the resulted absorbance versus mass of GTP (ug).
Expected result
Note
The high limit of detection is 100 ug. Higher concentration from 150 ug to 300 ug shows different linear response, which should be avoided.
Subtract absorbance of "+Blank" from the absorbance of samples and sample blanks.
Calculate the mass of lipids by using the standard curve and the resulted absorbance.
Subtract the lipids mass of the blank filter from the lipids mass of the samples.
Convert the resulted mass to total extracted lipids based on the fraction of extract used in the reaction.
Citations
Pand SV, ParvinKhan R, Venkitasubramanian TA.. Microdetermination of lipids and serum total fatty acids.
https://doi.org/10.1016/0003-2697(63)90094-0