Analytical Method: Chlorophyll-a and phaeophytin-a as per DIN 38412 / ISO 10260 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
Cat.No. 159010  Ethanol 96 % for analysis
Cat.No. 109063  Hydrochloric acid 2 mol/l Titripur®
Filtration apparatus, e.g. water-jet pump with filter flask acc. to DIN 12476
Filtration attachment with glass frit or porcelain suction filter
Extraction vessel, light-protected
Glass-fiber filter made of borosilicate glass, without organic binder (0.2 – 0.4 mm thick)
Round filter acc. to DIN 53135 (permeability very dense) or
centrifuge (5600 g) and centrifuge tubes with screw cap or stopper (30 – 50 ml, solvent-resistant)


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


Measuring range:
Method No. 2509
0 – 50 000 µg/l chlorophyll-a and phaeophytin-a   in the 10-mm rectangular cell
0 – 25 000 µg/l chlorophyll-a and phaeophytin-a   in the 20-mm rectangular cell
0 – 10 000 µg/l chlorophyll-a and phaeophytin-a   in the 50-mm rectangular cell 


Working instructions:
Extraction solution (ethanol w = 90 %)
Mix 500 ml of ethanol 96 % with 33 ml of water for analysis.


Sample preparation:
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. 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.
Under reflux heat approx. 50 ml of the extraction solution to boiling (78 °C).
Transfer approx. 30 ml of the boiling extraction solution to the shredded glass-fiber filter in the extraction vessel, allow to cool to room temperature, and homogenize using a homogenizer. 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.


Leave to stand at room temperature for 6 – 24 h to complete the extraction.
Subsequently filter the extract over a round paper filter under exclusion of light or centrifuge at 5600 g for 10 – 20 minutes. Transfer the clear centrifugation supernantant quantitativily into a 100-ml volumetric flask or collect the clear filtrate in a 100-ml volumetric flask under exclusion of light. Rinse the paper filter with extraction solution and make up the contents of the volumetric flask to the mark with extraction solution. Make a note of "100 ml" (extract volume).
For the differentiation of the chlorophyll-a content and the measurement of the phaeophytin-a content acidify
50 ml of the extract with 0.15 ml of hydrochloric acid 2 mol/l and measure after 3 to 30 minutes.


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 2509 "Chlorophyll-a DIN" 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 2509 "Chlorophyll-a DIN" 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 the non-acidified measurement sample into a rectangular cell and insert the cell into the cell shaft. The measurement is executed automatically.
Fill the acidified measurement sample into a corresponding rectangular cell and insert the cell into the cell shaft. The measurement is executed automatically.
Confirm measurement with "OK".
The content of chlorophyll-a in µg/l and phaeophytin-a in µg/l appears on the display.

Press "START" to start the measurement procedure for the next sample.


The DIN 38412 standard describes a measurement against an extraction solution and acidified 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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