Lipids in microalgae: Quantitation by acid-dichromate method in microtiter plate
Ying-Yu Hu, Zoe V. Finkel
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.
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.
Steps
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
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 1mg GTP, take note of the actual weight.
Dissolve GTP by 1mL chloroform in amber vial, gently vortex.
Prepare working standards:
5 ug/vial:
In two 12 ml amber vials, add 5µL GTP primary standard to each vial. Cap the vial to avoid contamination.
10 ug/vial:
In two 12 ml amber vials, add 10µL GTP primary standard to each vial. Cap the vial to avoid contamination.
20 ug/vial:
In two 12 ml amber vials, add 20µL GTP primary standard to each vial. Cap the vial to avoid contamination.
40 ug/vial:
In two 12 ml amber vials, add 40µL GTP primary standard to each vial. Cap the vial to avoid contamination.
80 ug/vial
In two 12 ml amber vials, add 80µL GTP primary standard to each vial. Cap the vial to avoid contamination.
100 ug/vial
In two 12 ml amber vials, add 100µL 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
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 . 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
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 0.5mL of acid-dichromate reagent to each vial (standards, +Blank and –Blank, samples and sample blanks). Cap and vortex right after.
Keep reaction vials in boiling water for 0h 15m 0s.
Cool vials to Room temperature in the fumehood
Prepare 0.2g/ml sodium sulphite solution
Weigh 0.2g sodium sulphite in a 2 ml microtube.
Add 1mL MilliQ water into the tube.
Vortex
Add 1.125mL MilliQ (1 mL + 125 uL by pipet) to each vial. Cap immediately and vortex.
Cool vials to room temperature.
Add 25µL 0.2g/ml sodium sulphite solution to the “+Blank” vial. Vortex.
Colorimetric analysis
Vortex each vial and load 250µL reactant into microtiter plate.
Read absorbance at 348 nm
Calculation
Subtract absorbance of "+Blank" from the absorbance of standards.
Plot the resulted absorbance versus mass of GTP (ug).
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.
