Analytical Method: Chlorophyll-a, -b, -c as per ASTM D3731-87 for Prove

Materials and reagents:
Cat.No. 173016  Spectroquant® VIS Spectrophotometer Prove 100 or
Cat.No. 173017  Spectroquant® UV/VIS Spectrophotometer Prove 300 or
Cat.No. 173018  Spectroquant® UV/VIS Spectrophotometer Prove 600
Cat.No. 114946  Rectangular cells 10 mm and/or
Cat.No. 114947  Rectangular cells 20 mm and/or
Cat.No. 114944  Rectangular cells 50 mm
Cat.No. 116754  Water for analysis or distilled water
Cat.No. 100014  Acetone for analysis
Cat.No. 106329  Sodium hydrogen carbonate for analysis
Magnesium carbonate
Filtration apparatus, e.g. water-jet pump with filter flask
Filtration attachment with glass frit or porcelain suction filter
Extraction vessel, light-protected
Glass-fiber filter (pore size 0.45 µm; diameter 47 mm)
Disposable filter (pore size 0.45 µm; solvent-resistant) + 10-ml syringe (solvent-resistant) or
centrifuge (1000 g) and centrifuge tubes with screw caps or stoppers (15 ml, solvent-resistant)


Spectrophotometric measurement of an extract from the filter residue of an aqueous sample 


Measuring range:
Method No. 2507
0 – 50 000 mg/m3 chlorophyll-a, -b, and -c   in the 10-mm rectangular cell
0 – 25 000 mg/m3 chlorophyll-a, -b, and -c   in the 20-mm rectangular cell
0 – 10 000 mg/m3 chlorophyll-a, -b, and -c   in the 50-mm rectangular cell 


Working instructions:
Magnesium carbonate suspension
Suspend 1 g of finely triturated magnesium carbonate in 100 ml of water for analysis and mix. Shake to resuspend before use.


Sodium hydrogen carbonat solution
Dissolve 0.84 g of sodium hydrogen carbonate in 10 ml of water for analysis.

Extraction solution (acetone 90 vol%)
Add 900 ml of acetone and 0.25 ml of sodium hydrogen carbonate solution to 100 ml of water for analysis and mix.


Sample preparation:
Stabilize the chlorophyll by adding 1 ml of magnesium carbonate suspension per liter of sample.
Depending on the trophic status and the anticipated algae concentration, homogenize 0.05 – 2 l of stabilized sample and filter over a glass-fiber filter or centrifuge at 1000 g for 20 minutes. Make a note of the sample volume used.
After filtration tear the glass-fiber filter into pieces and place the pieces in a light-protected extraction vessel; if the sample has been centrifuged, place the pellet obtained by centrifugation in a light-protected extraction vessel.
Add approx. 4 – 5 ml of the extraction solution to the extraction vessel and homogenize the contents (approx. 3 min at 500 rpm). Rinse any fibers still adhering to the homogenizer rod or the sides of the vessel into the extraction vessel with a small portion of the extraction solution, make up the contents to exactly 10 ml with extraction solution, and make a note of "10 ml" (extract volume).
Leave the mixture for extraction to stand in the dark at 4 °C for 0.25 – 24 hours.
Subsequently resuspend the mixture by swirling and filter over a solvent-resistant filter under exclusion of light, or centrifuge at 1000 g for 20 minutes.
Measure the clear filtrate or the clear centrifugation supernatant immediately.


Zero adjustment:
It is recommended to perform a zero adjustment for this method each new working day.
It is recommended to use the same cell for zero adjustment and for sample measurement.
Select method 2507 "Chlorophyll-a,b,c" from method list.
Prior to the zero adjustment enter and confirm the method parameters "Volume sample" and "Volume extract".
In case of an expired zero adjustment the system automatically prompts a zero adjustment, otherwise press "Settings" and select “ZERO ADJUSTMENT” from selection list.
Fill a cell with distilled water (or water for analysis) and insert the cell into the cell shaft. The zero adjustment is executed automatically.
Confirm the implementation of zero adjustment with "OK".


Measurement procedure:
Select method 2507 "Chlorophyll-a,b,c" from method list.
Enter the sample volume in liter and confirm with "OK".
Tip on entry field „Volume extract”, enter the extract volume in milliliter and confirm with "OK".
Press "START" to reach the measurement mode.
Perform a zero adjustment (procedure see "Zero adjustment").
Fill a cell with measurement sample and insert the cell into the cell shaft. The measurement is executed automatically.
Confirm measurement with "OK".
The content of chlorophyll-a in mg/m3, chlorophyll-b in mg/m3 and chlorophyll-c in mg/m3 appears on the display.
Press "START" to start the measurement procedure for the next sample.


ASTM D3731-87 describes a measurement against an extraction solution. When the method was zeroed using the stated reagents, no difference in absorbance was found compared with zeroing the method using water for analysis. It is hence possible to use water for analysis to zero the method.
Alternatively the method can be zeroed with the extraction solution.


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