Protocol

 

Authors

Sven-Erik Jacobsen, Eva Rosenqvist and Mira Bendevis

Author Affliations

Department of Plant and Environmental Sciences, Copenhagen University, Denmark

Overview

Chlorophyll extraction with DMF is an easy and quick extraction method. The method results in very stable measurements compared to methods using acetone.

Background

NOTE: DMF is a strong solvent and is highly toxic and carcinogenic! Wear gloves at all times, double if possible! All spills or sprays must be wiped up immediately!

Materials/Equipment

• Set of cork borers
• N,N-Dimethylformamide (DMF) Note: DMF is expensive. Calculate how much you will need before beginning this procedure.
• 5 ml. Syringes with 1.2x40mm needles
• 5-8 ml Glass sample containers with plastic snap-lids
• 1.5 ml disposable spectrophotometer cuvettes appropriate for solvents (otherwise the cuvettes turn milky white and the sample is wasted). Suggested item: Plastibrand, UV-Cuvette semi-micro 100 pc. – Cat.No.: 7591 50
• Spectrophotometer (receive instruction from lab. tech prior to use)
• Distilled or mili-Q water for rinsing syringes
• Fumehood – All activities are done in the fumehood, spectrophotometer needs to be placed in the hood as well.
• Scale, sensitive to small weight
• Fridge (4^∘C) with light bulb unscrewed or removed

Procedure

1. Take fresh leaf sample using a cork borer. Measure the diameter of the borer size and calculate the area. Weigh each leaf sample and record. With both known area and weight chlorophyll content can be expressed on both fresh weight and area basis.

NoteA few trial samples have to be taken to know which borer is suitable for the desired range of leaf mass in order to achieve and absorbance ranging from 0.02-1.7. For quinoa a FW mass of 0.04-0.08 g was appropriate, Riethmuller-Haage et al. (2006) used 0.1-0.2 g, it will depend on maturity and type of leaf and 0.1-0,2 g is too much for a mature leaf. This is done to avoid having to dilute the samples later, which would waste DMF!

2. Place each sample in labelled glass container, add 3ml. of DMF into each container and secure snap lid. For accuracy and minimization of contact with the solvent, use a syringe to dispense the DMF into each glass

3. Place the samples in the fridge, in darkness, (in a small tray or box with lid or foil cover) at 4^∘C. The complete extraction takes around 2-4 days, depending on the leaf material. Shake the bottles a little every now and then to ensure complete extraction (Leaf disks appear white). The samples can be kept refrigerated for up to 20 days before the Chlorophyll reading is done, but it is advised to get it done sooner rather than later.

• For thicker leaf specimens like orchids or kalanchoe, slice through the leaves to create thinner disks for easier extraction.

4. Prepare the spectrophotometer according to lab protocol and set it to manual scan. Run the samples first at 647.00 nm and then immediately after that at 664.5 nm. Record theabsorbance readings manually on paper.

• Place the cuvettes according to spectrophotometer guidelines
• Note: Remember to also adjust the number of sample cuvettes in the initial spectrophotometer set-up.

Number 1 sould be a blank DMF. (add a small piece of scotch tape covering the top, not sides at all, of that cuvette to avoid evaporation during the chlorophyll readings.

Although the DMF/Chl extract is relatively stable, keep each set of the extracted samples in dark and at cool temperatures until the spectrophotometer is ready!

5. Transfer the extracted sample (approximately 1 ml) to the cuvette using a syringe through the snap lids of the sample containers. In between each sample transfer, rinse the syringewith water. Tiny bits of plastic from the snap lids can get stuck in the needle opening, remove as necessary with another syringe needle.

Note:It is not necessary to fill the cuvettes completely, which will help avoid and minimize any spills and also leave enough extract for a 2nd analysis if needed.

6. Calculate chlorophyll content according to Inskeep et al (1985).

7. Dispose of empty containers, waste DMF extract, and cuvettes according to regulations. DMF waste should be collected in a clearly labelled glass bottle which can be kept in the fume hood while the measurements are going on and disposed off when the experiment is over. Check with lab techs to find out where to put it. Cuvettes and syringes and glass sample containers can be left to dry out under the fume hood and then disposed of as per standard laboratory procedures.

Notes and troubleshooting tips

Related wiki pages:

Chlorophyll extraction and determination

Chlorophyll determination

Leaf chlorophyll and carotenoid determinations

Suppliers

Plastibrand, UV-Cuvette semi-micro

Literature references

Inskeep, W.P., P.R. Bloom. 1985. Extinction coefficients of Chlorophyll a and b in N,NDimethylformamide and 80% Acetone. Plant Physiology 77: 483-485.

Riethmuller-Haage, I., L. Bastiaans, M.J. Kropff, J. Harbinson, C. Kempenaar. 2006. Can photosynthesis-related parameters be used to establish the activity of aceltolactate synthaseinhibiting herbicides on weeds 54: 974-982

Health, safety & hazardous waste disposal considerations

DMF is a strong solvent and is highly toxic and carcinogenic! Wear gloves at all times, double if possible! All spills or sprays must be wiped up immediately!

Dispose of empty containers, waste DMF extract, and cuvettes according to regulations.

DMF waste should be collected in a clearly labelled glass bottle which can be kept in the fume hood while the measurements are going on and disposed off when the experiment is over. Check with lab techs to find out where to put it.

Cuvettes and syringes and glass sample containers can be left to dry out under the fume hood and then disposed of as per standard laboratory procedures.