Mar 27, 2024

Public workspaceMeasure chlorophyll-a and pheophytin-a by Turner Designs V.2

This protocol is a draft, published without a DOI.
Measure chlorophyll-a and pheophytin-a by Turner Designs
  • 1Dalhousie University
Ying-Yu Hu
Open access
Protocol Citation: Ying-Yu Hu 2024. Measure chlorophyll-a and pheophytin-a by Turner Designs. protocols.io https://protocols.io/view/measure-chlorophyll-a-and-pheophytin-a-by-turner-d-cxjgxkjwVersion 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: July 21, 2023
Last Modified: March 27, 2024
Protocol Integer ID: 85320
Funders Acknowledgement:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
Here we describe a protocol for measuring chlorophyll-a and pheophytin-a from microalgae by using Turner Designs (10-AU)
Guidelines
  1. The entire procedure should be carried out as much as possible in subdued light (Green) to prevent photodecomposition.
  2. All glassware should be clean and acid free to prevent chlorophyll-a degradation.
  3. Waste disposal:
Follow all laboratory waste disposal guidelines regarding the disposal of acetone, DMSO solutions.
Protocol materials
ReagentChlorophyll-a from spinachMerck MilliporeSigma (Sigma-Aldrich)Catalog #C5753-1MG
Step 5.1
ReagentChlorophyll-a standardCatalog # E-541-0-850
Step 26.1
ReagentMagnesium carbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7179-500G
Step 1.1
ReagentAcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #270725-4X2L
Step 2.2
ReagentDMSO (CAS grade)
Step 3.1
Reagent12 N Hydrochloric acid
Step 4.1
Prepare reagent
Prepare reagent
Saturated magnesium carbonate solution
Add 10 grams magnesium carbonate to 1000 mL of MilliQ water.
ReagentMagnesium carbonateVWR InternationalCatalog #M7179-500G


Settle the solution for a minimum of 24 hours.
90% buffered acetone
100 mL clear “powder free” magnesium carbonate solution
900 mL HPLC grade acetone
ReagentAcetoneVWR InternationalCatalog #270725-4X2L

Mix in an amber reagent bottle.
Acetone/DMSO extraction solvent
Mix three parts 90% acetone and two parts of HPLC grade DMSO (Shoaf and Lium 1975)
ReagentDMSO (CAS grade)VWR International

CITATION
Shoaf WT, Lium BW. Improved extraction of chl and b from algae using dimethyl sulfoxide. Limnology and Oceanography.
LINK
https://doi.org/10.4319/lo.1976.21.6.0926

0.1 N HCl
Dissolve one part of Concentration12 Mass Percent HCl in 119 parts of MilliQ water
Reagent12 N Hydrochloric acidVWR International

Prepare Chlorophyll-a standard
Prepare Chlorophyll-a standard
Primary stock: ≅10 mg/L
Dissolve 1 mg Chlorophyll-a in a 100 mL glass volumetric flask by 90% acetone, top to 100 mL.
ReagentChlorophyll-a from spinachVWR InternationalCatalog #C5753-1MG

Fill cuvette and place it in udrop plate, measure absorbance at 664 nm
Equipment
Spectrophotometer Cells
NAME
12.5W x 12.5L x 48H mm (pathlength 10 mm)
TYPE
VWR® Spectrosil
BRAND
414004-078
SKU

Equipment
µDrop™ Plates
NAME
Thermo Scientific
BRAND
N12391
SKU

Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU



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

Note
Detection limit of spectrophotometrical measurement is about 350 ug/L


image.png

Label Amber vials with the actual concentration of chlorophyll-a stock solution
Equipment
Storage Vials and Closures
NAME
12 mL amber
TYPE
Thermo Scientific
BRAND
B7800-12A
SKU
VWR 66030-686
SPECIFICATIONS

Aliquote 5 mL to Amber vial with glass serological pipet (5 mL)
Store at Temperature-80 °C to Temperature-50 °C
Note
Flashpoint of acetone is Temperature-20 °C


Second calibration standard: (≅400 ug/L)

Warm up primary stock to room temperature and transfer 4 mL primary stock to a 100 mL glass volumetric flask
Note
Adjust the volume of primary stock according to the actual concentration of second calibration standard

Top with 90% acetone to 100 mL
Measure absorbance at 664 nm.
Chlorophyll-a_mg/L = 11.42 X Absorbance at 664 nm, where 11.42 is the extinction coefficient of chlorophyll-a at 664 nm.

Note
Detection limit of spectrophotometrical measurement is about 350 ug/L

Aliquote 5 mL to amber vials, label cryobox with actual concentration and store at Temperature-80 °C to Temperature-50 °C .
Working calibration standard (≅16 ug/L)
Note
Maximum concentration of detection range is 15~20 ug/L

Warm up second calibration standard and transfer 2 mL solution to a 50 mL glass volumetric flask
Note
Adjust the volume of second calibration standard according to the actual concentration of second calibration standard

Top with 90% acetone to 50 mL
Extract chlorophyll sample and blank
Extract chlorophyll sample and blank
Extract samples on the filter
Label 20 mL scintillation vials and place them in scintillation vial rack.
Equipment
Vial rack
NAME
Wheaton
BRAND
Z252425
SKU


Transfer sample and blank filter into scintillation vial
Add 5 mL Acetone/DMSO extraction solvent

Cap the vial, and vortex.
Cover the scintillation vials with another scintillation rack.
Let samples be extracted for Duration00:20:00 at TemperatureRoom temperature
20m
Vortex samples and allow samples to be extracted for anotherDuration00:10:00

10m
Extract liquid samples
Freeze liquid sample and blank
Add extraction solvent into frozen sample
Note
(V_sample) : (V_solvent)= 1:25 ~ 1:50

Cap the vial, vortex and place sample in the dark at room temperature for Duration00:20:00

20m
Vortex and place sample in the dark for another Duration00:10:00

10m
Daily calibrate
Daily calibrate
Allow Turner to warm-up for at least 15 minutes.
Place the solid secondary standard in Turner, wait 15 seconds for the reading to stabilize.
Record the reading on the solid standard calibration record sheet.
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)

Measure chlorophyll-a and pheophytin-a of standard
Measure chlorophyll-a and pheophytin-a of standard
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.
Equipment
Culture Tubes,
NAME
Disposable, Borosilicate Glass
TYPE
VWR
BRAND
47729-572
SKU

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

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

If the display reads OVER, dilute the sample and reread. Log dilution factor (DF) and reading (Rb).
Note
Always bring total volume of diluted to 5 mL, so that the volume of 0.1 N HCl doesn't need to be changed.

Add 150 ul 0.1N HCl. Carefully mix solution by vortexing for 10 seconds and measure the fluorescence again. Log the reading (Ra).
Calculation
Calculation


Calibrate Turner
Calibrate Turner
Standards TemperatureRoom temperature
Primary Chlorophyll a standardsReagentChlorophyll-a standardVWR InternationalCatalog # E-541-0-850
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.
Note
Actual concentration varies by LOT, and is listed on a certificate

Break the ampule with breaker and pour directly into the test tube
Blank: 90% buffered acetone TemperatureRoom temperature
Press <ENT> to reach Main Menu screen.
Press <2> to reach the Calibration Menu screen. Set the concentration range control to AUTO.
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.
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).


Press <Esc> to return to screen 2.0.
Press <1> to access screen 2.1 and confirm that option #2 on screen 2.1 reads YES.
Press <Esc> to return to HOME screen.
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.
Note
Because temperature affects fluorescence, do not allow the blank to remain in the instrument any longer than necessary for a stable reading.

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>.
When “FINISHED” appears, press <ESC> all the way to HOME.
Remove the blank. Set tube aside.
While on the HOME screen, place the tube with the LOW primary chlorophyll a standard in the sample chamber and replace the light cap.
Note
Because temperature affects fluorescence, do not allow the blank to remain in the instrument any longer than necessary for a stable reading.

Pressing <ENT>, <2>, and <3> to access 2.3
Using UP and DOWN arrows to adjust Span% until the FS reading is the closest to actual value of primary standard.
Wait until reading is stable (“TC” on screen cycles from 1 to 8 sec.), then press <*>.
When “FINISHED” appears, press <ESC> all the way to HOME screen.
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.
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.
Equipment
10AU Solid secondary standards
NAME
Turner Designs Digital Fluorometer
TYPE
Turner designs
BRAND
10-AU-904
SKU

Citations
Step 3.1
Shoaf WT, Lium BW. Improved extraction of chl and b from algae using dimethyl sulfoxide
https://doi.org/10.4319/lo.1976.21.6.0926