TITLE: Measure chlorophyll-a and pheophytin-a by Turner Designs
AUTHORS: Yingyu Hu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we describe a protocol for measuring chlorophyll-a and pheophytin-a from microalgae by using Turner Designs (10-AU)</div></div>

[STEPS]
?. [Prepare Chlorophyll-a standard]
Primary stock: ≅10 mg/L
?. [Prepare reagent]
Saturated magnesium carbonate solution
?. [Extract chlorophyll sample and blank]
Extract samples on the filter
?. [Daily calibrate]
Allow Turner to warm-up for at least 15 minutes.
?. [Prepare reagent]
90% buffered acetone
?. [Prepare reagent]
Acetone/DMSO extraction solvent
?. [Prepare reagent]
0.1 N HCl
?. [Measure chlorophyll-a and pheophytin-a of samples]
Use extraction of blank filter to zero the instrument on the sensitivity setting that will be used for sample analysis. Or simply log the reading of blank filter extraction and subtract the reading from reading of sample extraction.
?. [Calculation]
Chlorophyll-a (ug/L)=r*(Rb-Ra)*DF/(r-1)
?. [Prepare reagent]
Add 10 grams magnesium carbonate to 1000 mL of MilliQ water. 
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?. [Prepare reagent]
Settle the solution for a minimum of 24 hours.
?. [Prepare reagent]
100 mL clear “powder free” magnesium carbonate solution
?. [Prepare reagent]
900 mL HPLC grade acetone
?. [Prepare reagent]
Mix in an amber reagent bottle.
?. [Prepare reagent]
Mix three parts 90% acetone and two parts of HPLC grade DMSO (Shoaf and Lium 1975)
?. [Prepare reagent]
Dissolve one part of  HCl in 119 parts of MilliQ water
?. [Prepare Chlorophyll-a standard]
Dissolve 1 mg Chlorophyll-a in a 100 mL glass volumetic flask by 90% acetone, top to 100 mL.
?. [Prepare Chlorophyll-a standard]
Aliquote 5 mL to Amber vial with glass serological pipet (5 mL)
?. [Prepare Chlorophyll-a standard]
Store at  to
-80 °C
-50 °C
Flashpoint of acetone is
?. [Prepare Chlorophyll-a standard]
Second calibration standard: (≅400 ug/L)
?. [Prepare Chlorophyll-a standard]
Warm up primary stock to room temperature and transfer 4 mL primary stock to  a 100 mL glass volumetric flask
Adjust the volume of primary stock according to the actual concentration of second calibration standard
?. [Prepare Chlorophyll-a standard]
Top with 90% acetone to 100 mL
?. [Prepare Chlorophyll-a standard]
Working calibration standard (≅16 ug/L)
Maximum concentration of detection range is 15~20 ug/L
?. [Prepare Chlorophyll-a standard]
Fill cuvette and place it in udrop plate, measure absorbance at 664 nm
?. [Prepare Chlorophyll-a standard]
Chlorophyll-a_mg/L = 11.42 X Absorbance at 664 nm, where 11.42 is the extinction coefficient of chlorophyll-a at 664 nm.
Detection limit of spectrophotometrical measurement is about 350 ug/L
?. [Prepare Chlorophyll-a standard]
Label Amber vials with the actual concentration of chlorophyll-a stock solution
?. [Prepare Chlorophyll-a standard]
Aliquote 5 mL to amber vials, label cryobox with actual concentration and store at  to  .
-80 °C
-50 °C
?. [Prepare Chlorophyll-a standard]
Warm up second calibration standard and transfer 2 mL solution to  a 50 mL glass volumetric flask
Adjust the volume of second calibration standard according to the actual concentration of second calibration standard
?. [Prepare Chlorophyll-a standard]
Top with 90% acetone to 50 mL
?. [Extract chlorophyll sample and blank]
Label 20 mL scintillation vials and place them in scintillation vial rack.
?. [Extract chlorophyll sample and blank]
Add 5 mL Acetone/DMSO extraction solvent
?. [Extract chlorophyll sample and blank]
Cap the vial, and vortex.
?. [Extract chlorophyll sample and blank]
Transfer sample and blank filter into scintillation vial
?. [Extract chlorophyll sample and blank]
Cover the scintillation vials with another scintillation rack.
?. [Extract chlorophyll sample and blank]
Let samples be extracted for  at
0 Room temperature
?. [Extract chlorophyll sample and blank]
Vortex samples and allow samples to be extracted for another
?. [Extract chlorophyll sample and blank]
Extract liquid samples
?. [Prepare Chlorophyll-a standard]
Measure absorbance at 664 nm.
?. [Prepare Chlorophyll-a standard]
Chlorophyll-a_mg/L = 11.42 X Absorbance at 664 nm, where 11.42 is the extinction coefficient of chlorophyll-a at 664 nm.
Detection limit of spectrophotometrical measurement is about 350 ug/L
?. [Extract chlorophyll sample and blank]
Freeze liquid sample and blank
?. [Extract chlorophyll sample and blank]
Add extraction solvent into frozen sample
(V_sample) : (V_solvent)= 1:25 ~ 1:50
?. [Extract chlorophyll sample and blank]
Cap the vial, vortex and place sample in the dark at room temperature for
?. [Extract chlorophyll sample and blank]
Vortex and place sample in the dark for another
?. [Calibrate Turner]
Standards
0 Room temperature
?. [Daily calibrate]
Place the solid secondary standard in Turner, wait 15 seconds for the reading to stabilize.
?. [Daily calibrate]
Record the reading on the solid standard calibration record sheet.
?. [Daily calibrate]
Reading should be less than 10% of the previously determined post calibration value.Otherwise, calibrate Turner by liquid standard before measuring samples (Go to the last step: Calibrate Turner)
?. [Calibrate Turner]
Primary Chlorophyll a standards
?. [Calibrate Turner]
Come as a set of one low (15 ~ 20 ug/L) and one high (140 ~ 160 ug/L) concentration in foil wrapped ampules. Use low concentration for the calibration only.
Actual concentration varies by LOT, and is listed on a certificate
?. [Calibrate Turner]
Break the ampule with breaker and pour directly into the test tube
?. [Calibrate Turner]
Blank: 90% buffered acetone
0 Room temperature
?. [Calibrate Turner]
Press  to reach Main Menu screen.
?. [Calibrate Turner]
Press  to reach the Calibration Menu screen. Set the concentration range control to AUTO.
?. [Calibrate Turner]
From screen 2.0, press  to bring up screen 2.4, then  to bring up screen 2.43 (set conc. range control), and press  to toggle.
?. [Calibrate Turner]
Press  to screen 2.0, press  to access screen 2.2 (standard solution concentration). Enter the actual concentration of the primary liquid calibration standard (e.g. the value from the certificate).
?. [Calibrate Turner]
Press  to return to screen 2.0.
?. [Calibrate Turner]
Press  to access screen 2.1 and confirm that option #2 on screen 2.1 reads YES.
?. [Calibrate Turner]
Press  to return to HOME screen.
?. [Calibrate Turner]
While on the HOME screen, fill a clean 13-mm culture tube with 90% acetone (the blank). Put the tube in the sample chamber and replace the light cap.
Because temperature affects fluorescence, do not allow the blank to remain in the instrument any longer than necessary for a stable reading.
?. [Calibrate Turner]
Press  , , , and  to access 2.11 After the Blank % reading is stable (“TC” on screen 2.11 cycles from 1 to 8 seconds) and assuming the Blank % is less than 200%, press .
?. [Calibrate Turner]
When “FINISHED” appears, press  all the way to HOME.
?. [Calibrate Turner]
Remove the blank. Set tube aside.
?. [Calibrate Turner]
While on the HOME screen, place the tube with the LOW primary chlorophyll a standard in the sample chamber and replace the light cap.
Because temperature affects fluorescence, do not allow the blank to remain in the instrument any longer than necessary for a stable reading.
?. [Calibrate Turner]
Pressing , , and  to access 2.3
?. [Calibrate Turner]
Using UP and DOWN arrows to adjust Span% until the FS reading is the closest to actual value of primary standard.
?. [Calibrate Turner]
Wait until reading is stable (“TC” on screen cycles from 1 to 8 sec.), then press .
?. [Calibrate Turner]
When “FINISHED” appears, press  all the way to HOME screen.
?. [Calibrate Turner]
Read the primary liquid standard on the home screen by pressing , to confirm that the calibration was properly set and record the calibrant and reading on the calibration log sheet.
?. [Calibrate Turner]
Place the solid secondary standard in the instrument, read the LOW value. Record the reading on the solid standard calibration record sheet as the reference of daily check.
?. [Measure chlorophyll-a and pheophytin-a of standard]
Use PP pipet tip to transfer 5 mL 90% acetone to disposable glass tube, wipe the outside of the tube dry with kimwipe and place in the instrument. Replace the light cap.
?. [Measure chlorophyll-a and pheophytin-a of standard]
Wait about 15 seconds for the reading to stabilize, and zero the instrument on the sensitivity setting.
?. [Measure chlorophyll-a and pheophytin-a of standard]
Transfer 5 mL working calibration standard (prepared in  ) to a disposable glass tube, measure the fluorescence. Log the reading as "standard before acidification (Rb)".
?. [Measure chlorophyll-a and pheophytin-a of standard]
Add 150 ul 0.1N HCl to working calibration standard.
?. [Measure chlorophyll-a and pheophytin-a of standard]
Carefully mix solution by vortexing for 10 seconds and measure the fluorescence again. Log the reading as  "standard after acidification (Ra)".
?. [Measure chlorophyll-a and pheophytin-a of standard]
Calculate the ratio, r, as follows:r=Rb/Ra
?. [Measure chlorophyll-a and pheophytin-a of samples]
Transfer sample extract to disposable glass tube.
?. [Measure chlorophyll-a and pheophytin-a of samples]
If the display reads OVER, dilute the sample and reread. Log dilution factor (DF) and reading (Rb).
Always bring total volume of diluted to 5 mL, so that the volume of 0.1 N HCl doesn't need to be changed.
?. [Measure chlorophyll-a and pheophytin-a of samples]
Add 150 ul 0.1N HCl. Carefully mix solution by vortexing for 10 seconds and measure the fluorescence again. Log the reading (Ra).
?. [Calculation]
pheophytin-a (ug/L)=r*(rRa-Rb)*DF/(r-1)