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

[DESCRIPTION]
Here we describe a protocol for measuring chlorophyll-a and pheophytin-a from microalgae by using Turner Designs (10-AU)

[GUIDELINES]
The entire procedure should be carried out as much as possible in subdued light (Green) to prevent photodecomposition.
All glassware should be clean and acid free to prevent  chlorophyll-a degradation.
Waste disposal:
        Follow all laboratory waste disposal guidelines regarding the disposal of acetone, DMSO solutions.

[STEPS]
SECTION: Prepare Chlorophyll-a standard
5. Primary stock: ≅10 mg/L
SECTION: Prepare reagent
1. Saturated magnesium carbonate solution
SECTION: Extract chlorophyll sample and blank
8. Extract samples on the filter
SECTION: Daily calibrate
10. Allow Turner to warm-up for at least 15 minutes.
SECTION: Prepare reagent
2. 90% buffered acetone
SECTION: Prepare reagent
3. Acetone/DMSO extraction solvent
SECTION: Prepare reagent
4. 0.1 N HCl
SECTION: Measure chlorophyll-a and pheophytin-a of samples
20. 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.
SECTION: Calculation
24.   

SECTION: Prepare reagent
1.1. Add 10 grams magnesium carbonate to 1000 mL of MilliQ water. 
 


SECTION: Prepare reagent
1.2. Settle the solution for a minimum of 24 hours. 
SECTION: Prepare reagent
2.1. 100 mL clear “powder free” magnesium carbonate solution 
SECTION: Prepare reagent
2.2. 900 mL HPLC grade acetone
 

SECTION: Prepare reagent
2.3. Mix in an amber reagent bottle.
SECTION: Prepare reagent
3.1. Mix three parts 90% acetone and two parts of HPLC grade DMSO (Shoaf and Lium 1975)
 

 

SECTION: Prepare reagent
4.1. Dissolve one part of 12 N HCl in 119 parts of MilliQ water 
 

SECTION: Prepare Chlorophyll-a standard
5.1. Dissolve 1 mg Chlorophyll-a in a 100 mL glass volumetric flask by 90% acetone, top to 100 mL.
 

SECTION: Prepare Chlorophyll-a standard
5.5. Aliquote 5 mL to Amber vial with glass serological pipet (5 mL)
SECTION: Prepare Chlorophyll-a standard
5.6. Store at -80 °C to -50 °C 
 


SECTION: Prepare Chlorophyll-a standard
6. Second calibration standard: (≅400 ug/L)

SECTION: Prepare Chlorophyll-a standard
6.1. Warm up primary stock to room temperature and transfer 4 mL primary stock to  a 100 mL glass volumetric flask
 

SECTION: Prepare Chlorophyll-a standard
6.2. Top with 90% acetone to 100 mL
SECTION: Prepare Chlorophyll-a standard
7. Working calibration standard (≅16 ug/L)
 

SECTION: Prepare Chlorophyll-a standard
5.2. Fill cuvette and place it in udrop plate, measure absorbance at 664 nm
 

 

 



SECTION: Prepare Chlorophyll-a standard
5.3. Chlorophyll-a_mg/L = 11.42 X Absorbance at 664 nm, where 11.42 is the extinction coefficient of chlorophyll-a at 664 nm.

 


 
SECTION: Prepare Chlorophyll-a standard
5.4. Label Amber vials with the actual concentration of chlorophyll-a stock solution
 

SECTION: Prepare Chlorophyll-a standard
6.5. Aliquote 5 mL to amber vials, label cryobox with actual concentration and store at -80 °C to -50 °C .
SECTION: Prepare Chlorophyll-a standard
7.1. Warm up second calibration standard and transfer 2 mL solution to  a 50 mL glass volumetric flask
 

SECTION: Prepare Chlorophyll-a standard
7.2. Top with 90% acetone to 50 mL
SECTION: Extract chlorophyll sample and blank
8.1. Label 20 mL scintillation vials and place them in scintillation vial rack.
 


SECTION: Extract chlorophyll sample and blank
8.3. Add 5 mL Acetone/DMSO extraction solvent

SECTION: Extract chlorophyll sample and blank
8.4. Cap the vial, and vortex.
SECTION: Extract chlorophyll sample and blank
8.2. Transfer sample and blank filter into scintillation vial
SECTION: Extract chlorophyll sample and blank
8.5. Cover the scintillation vials with another scintillation rack.
SECTION: Extract chlorophyll sample and blank
8.6. Let samples be extracted for 20 min at Room temperature 
SECTION: Extract chlorophyll sample and blank
8.7. Vortex samples and allow samples to be extracted for another10 min

SECTION: Extract chlorophyll sample and blank
9. Extract liquid samples
SECTION: Prepare Chlorophyll-a standard
6.3. Measure absorbance at 664 nm.
SECTION: Prepare Chlorophyll-a standard
6.4. Chlorophyll-a_mg/L = 11.42 X Absorbance at 664 nm, where 11.42 is the extinction coefficient of chlorophyll-a at 664 nm.

 

SECTION: Extract chlorophyll sample and blank
9.1. Freeze liquid sample and blank
SECTION: Extract chlorophyll sample and blank
9.2. Add extraction solvent into frozen sample 
 

SECTION: Extract chlorophyll sample and blank
9.3. Cap the vial, vortex and place sample in the dark at room temperature for 20 min 

SECTION: Extract chlorophyll sample and blank
9.4. Vortex and place sample in the dark for another 10 min 

SECTION: Calibrate Turner
26. Standards Room temperature 
 
SECTION: Daily calibrate
11. Place the solid secondary standard in Turner, wait 15 seconds for the reading to stabilize. 
SECTION: Daily calibrate
12. Record the reading on the solid standard calibration record sheet. 
SECTION: Daily calibrate
13. 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)

SECTION: Calibrate Turner
26.1. Primary Chlorophyll a standards  
SECTION: Calibrate Turner
26.2. 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.
 

SECTION: Calibrate Turner
26.3. Break the ampule with breaker and pour directly into the test tube 
SECTION: Calibrate Turner
27. Blank: 90% buffered acetone Room temperature 
SECTION: Calibrate Turner
28.  Press <ENT> to reach Main Menu screen. 
SECTION: Calibrate Turner
29. Press <2> to reach the Calibration Menu screen. Set the concentration range control to AUTO. 
SECTION: Calibrate Turner
30.  From screen 2.0, press <4> to bring up screen 2.4, then <3> to bring up screen 2.43 (set conc. range control), and press <ENT> to toggle. 
SECTION: Calibrate Turner
31. Press <Esc> to screen 2.0, press <2> 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). 


SECTION: Calibrate Turner
32. Press <Esc> to return to screen 2.0. 
SECTION: Calibrate Turner
33. Press <1> to access screen 2.1 and confirm that option #2 on screen 2.1 reads YES. 
SECTION: Calibrate Turner
34. Press <Esc> to return to HOME screen. 
SECTION: Calibrate Turner
35. 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. 
 

SECTION: Calibrate Turner
36. Press <ENT> , <2>, <1>, and <1> 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 <0>. 
SECTION: Calibrate Turner
37. When “FINISHED” appears, press <ESC> all the way to HOME. 
SECTION: Calibrate Turner
38. Remove the blank. Set tube aside.
SECTION: Calibrate Turner
39. While on the HOME screen, place the tube with the LOW primary chlorophyll a standard in the sample chamber and replace the light cap. 
 

SECTION: Calibrate Turner
40. Pressing <ENT>, <2>, and <3> to access 2.3
SECTION: Calibrate Turner
41. Using UP and DOWN arrows to adjust Span% until the FS reading is the closest to actual value of primary standard. 
SECTION: Calibrate Turner
42. Wait until reading is stable (“TC” on screen cycles from 1 to 8 sec.), then press <*>. 
SECTION: Calibrate Turner
43. When “FINISHED” appears, press <ESC> all the way to HOME screen. 
SECTION: Calibrate Turner
44. 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. 
SECTION: Calibrate Turner
45. 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.
 

SECTION: Measure chlorophyll-a and pheophytin-a of standard
14. Use polypropylene 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.
 

SECTION: Measure chlorophyll-a and pheophytin-a of standard
15. Wait about 15 seconds for the reading to stabilize, and zero the instrument on the sensitivity setting.
SECTION: Measure chlorophyll-a and pheophytin-a of standard
16. Transfer 5 mL working calibration standard (prepared in   ) to a disposable glass tube, measure the fluorescence. Log the reading as "standard before acidification (Rb)".

SECTION: Measure chlorophyll-a and pheophytin-a of standard
17. Add 150 ul 0.1N HCl to working calibration standard.
SECTION: Measure chlorophyll-a and pheophytin-a of standard
18. Carefully mix solution by vortexing for 10 seconds and measure the fluorescence again. Log the reading as  "standard after acidification (Ra)".
SECTION: Measure chlorophyll-a and pheophytin-a of standard
19. Calculate the ratio, r, as follows:
 
SECTION: Measure chlorophyll-a and pheophytin-a of samples
21. Transfer sample extract to disposable glass tube. 

SECTION: Measure chlorophyll-a and pheophytin-a of samples
22. If the display reads OVER, dilute the sample and reread. Log dilution factor (DF) and reading (Rb).
 

SECTION: Measure chlorophyll-a and pheophytin-a of samples
23. Add 150 ul 0.1N HCl. Carefully mix solution by vortexing for 10 seconds and measure the fluorescence again. Log the reading (Ra).
SECTION: Calculation
25.