Analytical Method: Chlorophyll-a and phaeophytin-a as per DIN 38412 / ISO 10260

Materials and Reagents:

Cat.No. 100706 Spectroquant® Spectrophotometer Pharo 100 or
Cat.No. 100707 Spectroquant® UV/VIS Spectrophotometer Pharo 300
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
Homogenizer
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)

Method:

Spectrophotometric measurement of an extract from the filter residue of an aqueous sample.
10-mm rectangular cell (method No. 2509)
20-mm rectangular cell (method No. 2510)
50-mm rectangular cell (method No. 2511)

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. 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. 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.
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.

Measurement Procedure:

Select the method (2509, 2510, or 2511).
To key in the sample volume press START/ENTER, key in the noted sample volume [l], and acknowledge by pressing START/ENTER .
To key in the extract volume press START/ENTER , key in the volume of the measurement flask used [ml], and acknowledge by pressing START/ENTER .
Fill the non-acidified measurement sample into a rectangular cell, press START/ENTER , and insert the rectangular cell into the cell compartment to automatically start the measurement.
Fill the acidified measurement sample into a rectangular cell, press START/ENTER , and insert the rectangular cell into the cell compartment to automatically start the measurement.
The chlorophyll-a content in µg/l appears in the display.
The phaeophytin-a content in µg/l appears in the display by pressing the START/ENTER key.

Note:

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. To do this press the BLANK/ZERO key and insert the rectangular cell containing the extraction solution into the cell compartment.