TEST METHOD: Chlorophyll

Revised: 19 Feb 2014 (details on longer path lengths and syringe filtration added)

RISK ASSESSMENT

Assess Safety, Health, Quality and Environmental aspects of each specific step.

OVERVIEW

Chlorophyll may be measured in the laboratory using the spectrophotometer. The data obtained from the following process can be used to determine Chl-a in the presence of pheophytin as well as the determination of Chl-a, b, c by the trichromatic method.

REAGENTS and EQUIPMENT

90% acetone/deionised water Filtration apparatus
0.1M HCl GF/F glass fibre filter pads
  Alfoil
  Black lidded glass reaction cells (Merck)
  10mm, 20mm, 50mm cuvettes and Vega spectrophotometer
  Varipippette and tips
  Fine dropper for stirring cuvettes
  Syringes and syringe filters (disposable one-piece filters or sartorius filter holders)
  25mm GA55 glass fibre filter pads if using filter holders
  Small weighing glasses for transferring extract (wide enough for syringe filter)

 

SPECIFIC JOB STEPS RISK ASSESSMENT RISK CONTROL

In the field:

To collect chlorophyll samples, a quantity of water is filtered in the field, through a GF/F grade glass fibre filter. For low chlorophyll waters, filtering 2 litres or more is appropriate.

Turbid waters or those with an algal bloom will clog up the filter earlier.

 

No vacuum pump available in the field

Filter a smaller amount. Always record the VOLUME that was filtered and forward this to the lab with the filter pads.

Use a large syringe body to apply pressure to the filter apparatus (see image below).

Remove the filter pad. If it is very wet, set it on a piece of blotting paper (eg "roar" paper) clean side down to wick some of the water away. Then fold it in half with the clean side outwards, wrap in alfoil to prevent light from entering, label the package and immediately put on ice or into a portable freezer. Sunlight degrades the chlorophyll Work in the shade, fold and wrap the filter pad immediately it is removed from the filtration apparatus
 

Frozen samples from neutral to alkaline waters can be stored for up to three weeks.

Acid waters break chlorophyll down. Process promptly.

In the lab:

Label black lids of sufficient glass reaction tubes with the sample names. Place 5mL of 90% acetone (10% deionised water) into each glass reaction tube. This quantity of acetone will make sufficient extract to enable reading with a 10mm or 20mm cuvette.

Only 100% acetone is available

Very little colour is visible on the pad (can't really see any)

Use the 100mL volumetric flask to make up 90% acetone. Store well stoppered in the dark.

Use 10mL 90% acetone (10% deionised water) which will make sufficient extract for a 50mm cell

Open a foil wrapped filter pad, roll the pad into a sausage and insert it into a labelled tube containing 5mL (10mL) of acetone. Tightly cap the glass reaction tube. The filter pad sticks up past the acetone Use fine nosed forceps to push the pad under the acetone
Immediately wrap the glass reaction tube with alfoil ensuring no light can penetrate, and place in a rack. Treat each filter pad in the same way.Once the rack is filled, invert the tubes several times to ensure the acetone penetrates well, and place the rack in the refrigerator at 5oC for 24 hours. The acetone/chlorophyll extract may be uneven Invert the tubes again an hour before you plan to read them.
Transfer 3mL of extract into a 10mm or 20mm cuvette (or 6mL into a 50mm cuvette if you used 10mL acetone) The extract may be turbid

If the extract in the extraction tube is not completely clear, or contains sediments that may be disturbed, pour the extract into a shallow weighing glass and use a syringe with a filter to ensure a clear extract is obtained. Ideally use disposable syringe filters, as acetone causes deterioration of the filter holders. If Sartorius filter holders must be used, use ones that are marked "for use with acetone". Use glass fibre GA 55 25mm filter pads.

More on transfer of extract into appropriately sized cuvettes...

The extract may be too weak for reliable results in the 10mm cuvette (3mL samples).

The extract may be too strong for reliable results in the chosen cuvette (all sized samples)

Do a quick test using the 10mm cuvette at 665nm absorbance. The reading should be between 0.1 and 1.

Very dilute samples (reading < 0.1) can use the longer path length (20mm) cuvette.

Strong extracts (reading > 1.0) in long path length cuvettes can use a shorter path length (with appropriate volume). In the case of strong extracts in 10mm cuvettes the extract can be diluted.

In all cases you will need to correct for these changes using the spreadsheet that has been set up for determination of chlorophylls. Calculations are provided based on two sources: Aquatic Biology and Standard methods 19th ed.

Read absorbance directly at 750nm, 665/664nm, 645/647nm and 630nm    
Acidify the sample by using the varipippette to add 100uL of 0.1M HCl directly to the 10mm or 20mm cuvette. Stir with a fine dropper. Let stand 90 seconds. The change in absorbance after acidification is sensitive to volume of extract/acid and time.

Use standard volumes of extract and acid, and a timer.

If using larger amounts of extract in a longer path cuvette, increase the acid proportionately, by adding 200uL of 0.1M HCl to 6mL of extract in a 50mm cuvette.

Read the absorbance of the acidified sample at 750nm and 665nm    
Transfer your readings to the spreadsheet that has been set up for determination of chlorophylls. Calculations are provided based on two sources: Aquatic Biology and Standard methods 19th ed. Which should I use?

The Standard Methods results are reported on lab reports. Where the pheophytin ratio is less than 1.7, the pheophytin-corrected Chl-a result is reported. Where the ratio is 1.7 the trichromatic Chl-a is reported.

The two methods provide very similar results for the trichromatic calculation where there is little or no pheophytin present. Where this breakdown product is present, the Standard Methods calculation seems to provide a more robust determination. We are collecting both sets of results for comparative purposes.

     

 

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