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Analytical Method: Chlorophyll-a and phaeophytin-a as per APHA 10200-H

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. 100014  Acetone for analysis
Cat.No. 109060  Hydrochloric acid 0.1 mol/l Titripur® 
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 1 µm; solvent-resistant) + 10-ml syringe (solvent-resistant) or centrifuge (1000 x 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.
10-mm rectangular cell (method No. 2504)
20-mm rectangular cell (method No. 2505)
50-mm rectangular cell (method No. 2506)

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.
Extraction solution (acetone 90 vol%):
Add 900 ml of acetone to 100 ml of the magnesium carbonate suspension and mix.
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 or centrifuge at 1000 x 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. 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 at least 2 hours.
Subsequently resuspend the mixture by swirling and filter over a round paper filter under exclusion of light, or centrifuge at 500 x g for 20 minutes.
Measure the clear filtrate or the clear centrifugation supernatant (= non-acidified sample) immediately.
For the differentiation of the chlorophyll-a content and the measurement of the phaeophytin-a content acidify 5 ml of the extract with 150 µl of hydrochloric acid 0.1 mol/l and measure after 90 seconds.
Measurement procedure:
Select the method (2504, 2505, or 2506).
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 volumetric 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 mg/m3 appears in the display.
The phaeophytin-a content in mg/m3 appears in the display by pressing the START/ENTER key. 

APHA 10200-H 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. To do this press the BLANK/ZERO key and insert the rectangular cell containing the extraction solution into the cell compartment.


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