TITLE: Lipids in microalgae: Quantitation by acid-dichromate method in microtiter plate
AUTHORS: Yingyu Hu, Zoe V Finkel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for quantitating total lipids in microalgae. </div><div class = "text-block">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.  </div><div class = "text-block">In addition, comparing with the absorbance at 440 nm, the absorbance at 348 nm yields five-time higher sensitivity in lipids quantitation.</div><div class = "text-block">Total lipids in the samples should not excess 80 ug. Accurate quantitation can be achieved with as little as 20 ug. A working detection limit is about 5 ug. </div></div>

[STEPS]
?. [Preparation of Standard]
Prepare  glyceryl tripalmitate (GTP) primary standard solution (around 1 mg/ml)
?. [Preparation of Standard]
Prepare working standards:
?. [Preparation of acid-dichromate reagent]
Estimate the total volume of potassium dichromate required:Number of standards and standard blanks: 12Number of samples and sample blanks: NV=0.5x(N+12) ml
?. [Preparation of Standard]
Dry working standards at    under N2 gas stream (
0 Room temperature
?. [Preparation of acid-dichromate reagent]
Transfer concentrated sulphuric acid to a glass vial for temporary storage
?. [Preparation of acid-dichromate reagent]
Weigh a glass vial, and tare the balance
?. [Preparation of acid-dichromate reagent]
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 Step 4. Write down the weight of sulphuric acid.
?. [Reaction of lipids and acid-dichromate reagent]
Allow frozen extract warm up to  .
0 Room temperature
?. [Reaction of lipids and acid-dichromate reagent]
Dry extract at  under a stream of N2 gas (
0 Room temperature
?. [Reaction of lipids and acid-dichromate reagent]
Label two 12 ml vials with “+ Blank” and “- Blank”."-Blank" is 0 ug GTP."+Blank" is the reference of absorbance.
?. [Reaction of lipids and acid-dichromate reagent]
Prepare boiling water bath on hot plate, place a vial rack in the water bath
?. [Reaction of lipids and acid-dichromate reagent]
Add  of acid-dichromate reagent to each vial (standards, +Blank and –Blank, samples and sample blanks). Cap and vortex right after.
0.5 mL
?. [Reaction of lipids and acid-dichromate reagent]
Keep reaction vials in boiling water for .
?. [Reaction of lipids and acid-dichromate reagent]
Prepare  sodium sulphite solutionWeigh   sodium sulphite in a 2 ml microtube.Add  MilliQ water into the tube.Vortex
0.2 g
1 mL
?. [Reaction of lipids and acid-dichromate reagent]
Cool vials to  in the fumehood
0 Room temperature
?. [Reaction of lipids and acid-dichromate reagent]
Add  MilliQ (1 mL + 125 uL by pipet) to each vial. Cap immediately and vortex.
1.125 mL
?. [Reaction of lipids and acid-dichromate reagent]
Cool vials to room temperature.
?. [Reaction of lipids and acid-dichromate reagent]
Add  sodium sulphite solution to the “+Blank” vial. Vortex.
25 µl
?. [Colorimetric analysis]
Vortex each vial and load  reactant into microtiter plate.
250 µl
?. [Colorimetric analysis]
Read absorbance at 348 nm
?. [Calculation]
Plot the resulted absorbance versus mass of GTP (ug).  Use this as standard curve.
?. [Preparation of acid-dichromate reagent]
Weigh dichromate and dissolve it into concentrated sulphuric acid. Cap the vial and vortex gently.
?. [Reaction of lipids and acid-dichromate reagent]
Gently vortex the extract.
?. [Reaction of lipids and acid-dichromate reagent]
Transfer less than 80 ug extracted lipids to 12 ml vial by glass serological pipet. Take notes of the volume and calculate the fraction of extract used in the analysis.
?. [Calculation]
Subtract absorbance of "+Blank" from the absorbance of samples and sample blanks.
?. [Calculation]
Subtract absorbance of "+Blank" from the absorbance of standards.
?. [Calculation]
Calculate the mass of lipids by using the standard curve and the resulted absorbance.
?. [Calculation]
Subtract the lipids mass of the blank filter from the lipids mass of the samples.
?. [Calculation]
Convert the resulted mass to total extracted lipids based on the fraction of extract used in the reaction.
?. [Reaction of lipids and acid-dichromate reagent]
Freeze the rest of the extract in ULT freezer  .
-80 °C
?. [Preparation of Standard]
Place frozen GTP in vacuum desiccator with lose cap until it is warmed to   before making primary standard solution
0 Room temperature
?. [Preparation of Standard]
Weigh around  GTP, take note of the actual weight.
1 mg
?. [Preparation of Standard]
Dissolve GTP by  chloroform in amber vial, gently vortex.
1 mL
?. [Preparation of Standard]
5 ug/vial:In two 12 ml amber vials, add  GTP primary standard to each vial. Cap the vial to avoid contamination.
5 µl
?. [Preparation of Standard]
10 ug/vial:In two 12 ml amber vials, add   GTP primary standard to each vial. Cap the vial to avoid contamination.
10 µl
?. [Preparation of Standard]
20 ug/vial:In two 12 ml amber vials, add   GTP primary standard to each vial. Cap the vial to avoid contamination.
20 µl
?. [Preparation of Standard]
40 ug/vial:In two 12 ml amber vials, add   GTP primary standard to each vial. Cap the vial to avoid contamination.
40 µl
?. [Preparation of Standard]
80 ug/vialIn two 12 ml amber vials, add   GTP primary standard to each vial. Cap the vial to avoid contamination.
80 µl
?. [Preparation of acid-dichromate reagent]
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).