Jan 28, 2025

Public workspaceLipids in microalgae: Quantitation by acid-dichromate method in microtiter plate V.3

DOI
dx.doi.org/10.17504/protocols.io.e6nvw9dpzgmk/v3
  • 1Dalhousie University
  • Marine Microbial Macroecology Lab
    Tech. support email: ruby.hu@dal.ca
Ying-Yu Hu
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DOI: dx.doi.org/10.17504/protocols.io.e6nvw9dpzgmk/v3
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2025. Lipids in microalgae: Quantitation by acid-dichromate method in microtiter plate. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw9dpzgmk/v3Version 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: March 06, 2024
Last Modified: January 28, 2025
Protocol Integer ID: 96269
Keywords: acid-dichromate, lipids, microalgae, microtiter plate
Funders Acknowledgements:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
This protocol describes a method for quantitating total lipids in microalgae using the acid-dichromate method, a widely used colorimetric analysis technique. We present a procedure utilizing a 96-well microtiter plate for safe and efficient sample handling, enabling high throughput. Only 200 - 500 µl of 0.15% acid-dichromate is required per sample, significantly reducing the amount of corrosive and toxic reagent used.

Furthermore, we demonstrate that measuring absorbance at 348 nm provides five times higher sensitivity in lipid quantitation compared to absorbance at 440 nm.

A preliminary test for lipid-unknown samples is included to minimize uncertainty in the measurements. This test ensures that the method performs reliably within the detection range of 20 to 80 µg of lipids. Without this test, samples with lipid concentrations outside this range (either less than 20 µg or greater than 80 µg) may result in inaccurate or failed measurements. Specifically, samples with concentrations above 80 µg exhibit a linear response with an opposite slope, which could cause lipid concentrations to be underestimated if the calibration curve based on the 20 to 80 µg range is used. Accurate quantification can be achieved with as little as 20 µg, and the working detection limit is approximately 5 µg.
CITATION
Pand SV, ParvinKhan R, Venkitasubramanian TA.. Microdetermination of lipids and serum total fatty acids.. Analytical Biochemistry..
LINK
https://doi.org/10.1016/0003-2697(63)90094-0

Protocol materials
ReagentSodium sulphite
ReagentConcentrated sulphuric acid
ReagentGlyceryl tripalmitate
ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
ReagentPotassium dichromateFisher ScientificCatalog #P188-100
Safety warnings

Safety information
Any items contaminated by potassium dichromate should be disposed as hazardous waste.


Before start
Clear vials are pre-combusted at Temperature500 °C for Duration06:00:00
Reagent bottle for dichromate reagent is rinsed by 95% ethanol and air-dried.
Glass serological pipets are rinsed by 95% ethanol and air-dried.
All caps are rinsed by 95% ethanol and air-dried.
Barrel and needle of the glass syringe is rinsed by chloroform and air-dried.
Preliminary test of sample biomass
Preliminary test of sample biomass
For microalgae samples that have been rarely studied with no available information on lipid content, it is essential to conduct a preliminary test before processing all samples.
Blank
Note
The process of redissolving and subsampling is essential for purification purposes, aimed at removing water-soluble organic substances such as proteins, amino acids, sugars, RNA, and DNA, which could potentially interfere with the dichromate assay.

Add 5 mL chloroform into the dried extractReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L

Transfer 4.5 mL into a new vial.
Dry this 4.5 mL extract solution for assay.
Discard the remaining extract solution into waste tank.
One replicate from each sample
Add 4 mL chloroform into the dried sample extract.
Transfer 2 mL extract into another new vial.
Dry the extract in the new vial for assay.
Store the remaining extract Temperature-80 °C (for phospholipids assay).

Preparation of standard
ReagentGlyceryl tripalmitate Contributed by users

Place frozen glyceryl tripalmitate (GTP) in vacuum desiccator with lose cap until it is warmed to TemperatureRoom temperature before making primary standard solution
Weigh and transfer GTP (around Amount2 mg ) to a clear vial , log the actual weight.

GTP (mg)

Dissolve GTP by chloroform for a concentration of about 1 mg/mL, gently vortex.

chloroform (mL)

The following steps require a 100 uL glass syringe: the syringe barrel and needle is chloroform rinsed and air-dried
Equipment
Gastight® 1700 Series Syringes
NAME
1710N
TYPE
Hamilton
BRAND
81000
SKU

5 ug/vial:
In a 12 ml clear vial, add Amount5 µL GTP primary standard to each vial.
10 ug/vial:
In a 12 ml clear vial, add Amount10 µL GTP primary standard to each vial.
20 ug/vial:
In a 12 ml clear vial, add Amount20 µL GTP primary standard to each vial.
40 ug/vial:
In a 12 ml clear vial, add Amount40 µL GTP primary standard to each vial.
80 ug/vial
In a 12 ml clear vial, add Amount80 µL GTP primary standard to each vial.
100 ug/vial
In a 12 ml clear vial, add Amount100 µL GTP primary standard to each vial.
120 ug/vial
In a 12 ml clear vial, add 2X Amount60 µL GTP primary standard to each vial.
160 ug/vial
In a 12 ml clear vial, add 2X Amount80 µL GTP primary standard to each vial.
200 ug/vial
In a 12 ml clear vial, add 2XAmount100 µL GTP primary standard to each vial.
240 ug/vial
In a 12 ml clear vial, add 3X Amount80 µL GTP primary standard to each vial.
300 ug/vial
In a 12 ml clear vial, add 3XAmount100 µL GTP primary standard to each vial.
Dry working standards at TemperatureRoom temperature under N2 gas stream (<2 psi).

Estimate the total volume of potassium dichromate required:
Number of standards and standard blanks: 12+2
Number of samples and sample blanks: N


Transfer concentrated sulfuric acid from the original package to a 95% ethanol rinsed and air-dried glass reagent bottle for temporary storage.
ReagentConcentrated sulphuric acidVWR International (Avantor)
Weigh a 95% ethanol rinsed and air-dried glass reagent bottle, and tare the balance
Use 5 ml serological pipet to measure and transfer concentrated sulfuric acid to this vial. The volume of sulfuric acid is several milliliter more than estimated in Go togo to step #6 . Write down the weight of sulfuric acid.

Sulfuric acid (mL)Sulfuric acid (g)

The weight of dichromate required for the 0.15% (w/w) acid-dichromate reagent equals the weight of sulfuric acid multiplied by (0.15/99.85).
ReagentPotassium dichromateVWR International (Avantor)Catalog #P188-100
Dichromate (g)

Weigh dichromate and dissolve it into concentrated sulfuric acid. Cap the vial and vortex until complete dissolve.
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 Amount0.5 mL of acid-dichromate reagent to . Cap and vortex right after.

Note
Use 5 mL glass serological pipet, fill to "4 mL", dispense Amount0.5 mL .

Add Amount0.2 mL of acid-dichromate reagent to . Cap and vortex right after.

Note
Use 5 mL glass serological pipet, fill to "4 mL", dispense Amount0.2 mL .

Keep reaction vials in boiling water for Duration00:15:00 .

Cool vials to TemperatureRoom temperature in the fumehood
Prepare Concentration0.2 g/ml sodium sulphite solution
ReagentSodium sulphite VWR International (Avantor)
Weigh Amount0.2 g sodium sulphite in a 2 ml microtube.
Add Amount1 mL MilliQ water into the tube.
Vortex
Add Amount1.125 mL o MilliQ (1 mL + 125 uL by pipet) to . Cap immediately and vortex.

Add Amount450 µL MilliQ to . Cap immediately and vortex.

Cool vials to room temperature.
Add Amount25 µL Concentration0.2 g/ml sodium sulphite solution to the “+Blank” vial. Vortex.
Reverse pipetting, load Amount200 µL r reactant into microtiter plate.

Read absorbance at 348 nm
Subtract absorbance of "+Blank" from the absorbance of standards.
Plot the resulted absorbance versus mass of GTP (ug).
Expected result


Note
Take notes of the color for using the corresponding curve.

Concentration of lipids (left to right): 5 ug, 10 ug, 100 ug, 200 ug and 300 ug

Calculate the mass of lipids by using the standard curve and the absorbance, including sample blanks and samples.


For samples:
For blanks:
Determine the subsampling method for the remaining replicates
Preliminary result of is between 20 to 80 ug
Use the same method to subsample and assay

Preliminary result of is above 80 ug
Use smaller portion from the extract or use 0.5 mL dichromate for an estimated value between 20 to 80 ug
Preliminary result of is lower than 20 ug
Use larger portion from the extract and use 0.2 mL dichromate for an estimated value between 20 to 80 ug
Subsample for the assay
Subsample for the assay
For sample blanks, add 5 mL chloroform and transfer 4.5 mL to a new vial. Discard the remaining extract.
For samples, subsample following the preliminary test.
Dry the extract in the new vial for assay.
Store the remaining extract Temperature-80 °C (for phospholipids assay).
Assay for the samples
Assay for the samples
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 TemperatureRoom temperature before making primary standard solution

Weigh and transfer GTP (around Amount1 mg ) to a clear vial , log the actual weight.

GTP (mg)

Dissolve GTP by chloroform for a concentration of about 1 mg/mL, gently vortex.

chloroform (mL)

Prepare working standards:
5 ug/vial:
In a 12 ml clear vial, add Amount5 µL GTP primary standard to each vial.
10 ug/vial:
In a 12 ml clear vial, add Amount10 µL GTP primary standard to each vial.
20 ug/vial:
In a 12 ml clear vial, add Amount20 µL GTP primary standard to each vial.
40 ug/vial:
In two 12 ml amber vials, add Amount40 µL GTP primary standard to each vial.
80 ug/vial
In a 12 ml clear vial, add Amount80 µL GTP primary standard to each vial.
Dry working standards at TemperatureRoom temperature under N2 gas stream (<2 psi).

Estimate the total volume of potassium dichromate required:
Number of standards and standard blanks: 6
Number of sample blanks and samples using 0.2 mL dichromate: N
Number of samples using 0.5 mL dichromate: M

Transfer concentrated sulfuric acid from the original package to a 95% ethanol rinsed and air-dried glass reagent bottle for temporary storage.
Weigh a 95% ethanol rinsed and air-dried glass reagent bottle, and tare the balance
Use 5 ml serological pipet to measure and transfer concentrated sulfuric acid to this vial. The volume of sulfuric acid is several milliliter more than estimated in Go togo to step #6 . Write down the weight of sulfuric acid.

Sulfuric acid (mL)Sulfuric acid (g)

The weight of dichromate required for the 0.15% (w/w) acid-dichromate reagent equals the weight of sulfuric acid multiplied by (0.15/99.85).
Weigh dichromate and dissolve it into concentrated sulfuric acid. Cap the vial and vortex until complete dissolve.
Dichromate (g)

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 Amount0.5 mL of acid-dichromate reagent to and some of the samples. Cap and vortex right after.

Note
Use 5 mL glass serological pipet, fill to "4 mL", dispense Amount0.5 mL .

Add Amount0.2 mL of acid-dichromate reagent to (1) , (2) some of the .
Cap and vortex right after.

Note
Use 5 mL glass serological pipet, fill to "4 mL", dispense Amount0.2 mL .

Keep reaction vials in boiling water for Duration00:15:00 .

Cool vials to TemperatureRoom temperature in the fumehood
Prepare Concentration0.2 g/ml sodium sulphite solution
Weigh Amount0.2 g sodium sulphite in a 2 ml microtube.
Add Amount1 mL MilliQ water into the tube.
Vortex
Prepare two vial racks, one is for those with 0.5 mL dichromate, the other is for those with 0.2 mL dichromate.
Label the racks to avoid adding MilliQ in wrong volume.
Add Amount1.125 mL o MilliQ (1 mL + 125 uL by pipet) to those reacted with 0.5 mL dichromate.
Cap immediately and vortex.

Add Amount450 µL MilliQ to those reacted with 0.2 mL dichromate.
Cap immediately and vortex.

Cool vials to room temperature.
Prepare sodium sulphite Concentration0.2 g/ml

In a 2 mL microtube, add 0.4 g sodium sulphite and 2 mL MilliQ, vortex.
Add Amount25 µL Concentration0.2 g/ml sodium sulphite solution to the “+Blank” vial. Vortex.
Reverse pipetting, load Amount200 µL r reactant into microtiter plate.

Read absorbance at 348 nm
Subtract absorbance of "+Blank" from the absorbance of standards.
Plot the resulted absorbance versus mass of GTP (ug).
Calculate the mass of lipids by using the standard curve and the absorbance, including sample blanks and samples.

, where is the actual volume of dichromate reagent added.

, where is the volume of chloroform added into the dried extract, is the volume of extract used in the assay.

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