Check the leaf chlorophyll content according to Barnes et al., 1992, method.

From a single plant/plant-let, pool up the different leaves collected at different nodes and cut them in to 1-2 cm.

In determining the chlorophyll concentration, you may have two options in sampling preparation. You may work on leaf area basis or leaf mass basis.

we check chlorophyll content according to arnon s method the procedure is as follow

Estimation of total chlorophyll and carotenoid contents were done according to the method of Arnon (1949). Fresh leaf samples were washed with water and blotted between sheets of filter papers. To estimate pigment contents, 80% acetone (v/v) was used as the extracting medium.

One hundred mg fresh leaf sample was weighed using electronic balance and crushed in 80% acetone using mortar and pestle. The homogenate was centrifuged at 5000 rpm for 10 min at 4oC and the supernatant was

collected. The residue was re-extracted with 80% acetone and centrifuged. The process was repeated till the pellet became colourless. The final volume of the pooled supernatant was noted. The absorbance was read at 663, 646,

750 and 470 nm against the solvent blank (80% acetone). Total chlorophyll (Chl a+b) and carotenoids present in the extract was calculated as microgram chlorophyll and carotenoids per gram fresh weight .

also see the attachment

It is upto you. Generally leaves of different ages will have different amounts of chlorophyll . You can take the leaves from the centre portion of the plant. Otherwise collect equal number of leaves from the different levels of plant, say, top, middle and bottom, and chop them into pieces and mix thoroughly. From this mixture, take the sample as per your requirement.

If you have chlorophyll meter, just take it to the field and take observation on the plant itself. By using chlorphyll meter, you can take a great number of observations from a single plant at diferent levels.

Follow the same procedure for all the treatments/varieties/genotypes.

While writing the paper mention your sampling method clearly including time of sampling and plant portion from which you collected the leaves.

chlorophyll content varies from age to age. What we need is a representative sample from single plant, for which leaves can be taken from central portion and from all the directions. You can mix the discs obtained from them and proceed for chlorophyll estimation. As per my experience, 80% acetone gives consistent results and arguably worldwide accepted method.

Hi, I know this is an an old post. But I had a question. I was going to use the 80% acetone method. My field site is a good few hours from the laboratory so I was wondering how to store these samples until the analysis?

I do not have access to liquid nitrogen.

Any help is appreciated

Anna Gardner

Hai,

If the plant species is having succulent leaves it tends to lose moisture rapidly. If it is an arid plant species it can withstand moisture loss for few hours.

Can't you arrange dry ice? Otherwise, chlorophyll measurement can be made on dry weight basis.

Otherwise, instant extraction can be made, which is difficult. You have to carry a sensitive balance and acetone along with you to the sampling site. The initial sample weight is necessary to determine chlorophyll content/mg of leaf sample.

Another option is chlorophyll meter, which solves your problem.

Koundinya A. V. V. Hi, Thank you for your reply and useful information. It is much appreciated. The species I am studying are oak trees (Quercus robur). I will see if dry ice is a possibility. Thank you for this advice. Best wishes.

SPAD meter

Santoshkumar B Pujer Hello, Thank you for your reply. I have used a SPAD meter for the last few growing seasons. I am now taking chlorophyll measurements in order to calibrate those SPAD readings into actual chlorophyll content.

Anna Gardner How you calibrate SPAD readings?

Dr- Zain Mushtaq Hi, I take SPAD readings of the leaves and then measure the actual chlorophyll content. If you get a good range of leaf colour (from pale leaves to dark leaves) you can get values of chlorophyll content across a large range of SPAD. Then you can correspond any further SPAD measurements to that chlorophyll measurement. This is useful if you need to measure many leaves across a long time frame because you only need to take portion for chlorophyll measurement. I hope that answers your question.

Hi,

I'm working on a project on which biomass calculation is necessary, so I had to dry the leaves at a certain temperature to obtain its crushed powder. Now I must check for its total chlorophyll content. Is it possible to apply the same 80% acetone followed by the spectrum absorbance method? If so, how much amount of the powdered sample must be taken?

Any help is appreciated

Anna Gardner Thank you for the useful information. We often use SPAD meter so this will help us alot.

If fresh leaves of sample is not available, only leaves sample stored in refrigerator is available then how to determine the chlorophylls contents. As any impact of storage on chlorophyll content. How to solve this problem

Anna Gardner Hi Anna. I am an undergraduate about to start a project where I need to measure chlorophyll. How did you end up storing/transporting the samples? I do not have access to liquid nitrogen and idk.

James Nagaoka Hi James. That's ok, I also didn't have access to liquid nitrogen. The important thing is that the samples need to stay cold and dark during transport. As I was in the field, I had to use a cooler box with freezer packs and I also wrapped the leaves in foil (for darkness). Then, as soon as I could, I transferred them into the freezer (a few hours was fine). You can use either a -30C freezer or a -80C freezer. The colder the better. I kept my samples in the freezer for a couple of days before I could analyse them and this was fine.

On the day that you do analyse the samples, make sure you only take out the sample you are analyzing (i.e. one sample at a time). Keep the other samples in the freezer. At this point, it would be useful to have some liquid nitrogen to help you grind up the leaf for chlorophyll extraction. I would also recommend keeping an insulated (polystyrene) box full of dry ice - you can keep all your equipment cold this way. For example, the mortar and pestle can be kept there and you can grind your sample inside the box to keep it cold.

I'd also recommend taking a few extra samples (3-4) so that you can have a few practise leaves to try out before using 'real' samples. This will help you get faster and more efficient.

Good luck with your experiments! Please message me if you need any more information :)

hey Anna Gardner can you please explain the ratios/measurements of the solvents and the sample?

Mohd. Fadhil Hisham Rapahi Hi, I also struggled with this initially. It is not a fixed amount of solvent to sample. The ratio will depend on the leaves you are analysing. For example, if you are using a very dark leaf you will need more than if you are using a pale leaf. The amount of solvent may also change from sample to sample. Generally, after a couple of practise leaves, you will get a feel for the amount of solvent you need. It is very important to record the amount of solvent you use for each sample.

I have explained my method below but just for demonstrating purposes. My experiment may not be the same as yours (I used very mature oak leaves), but this is what I did. I used a set amount of 100mg of leaf. I added 1ml of solvent at a time whilst I crushed the leaf using a mortar and pestle. Once the liquid was green (usually 3ml), I would pipette the liquid into a centrifuge tube and spin. The remaining leaf fragments in the mortar usually needed another 2 or 3ml of solvent and again pipette into a centrifuge tube. The aim is to make sure all the green pigment is extracted. You may need to repeat with another 1ml or 2ml to get the white pellet and green liquid goal. As you can see the amount of solvent really does depend on the leaf which is why it is important to remember how much you have used. I understand this may be hard to follow so I can send you the method I used if it would be helpful to you.

Kind regards,

Anna

Hi, Anna Gardner.

I'm trying to determine the chlorophyll content of fresh leaf of camellia sinensis. I will use methanol or 95% ethanol for extraction (wellburn 1984). Could you please, suggest me about drying temperature and time to grind fresh leaf for extracting chlorophyll?

Muhammad Abid Hasan Chowdhury Hi, I am sorry that I cannot help with this question. I have not used a method that dries the leaf first as I usually use fresh leaf material. I hope somebody else in the chat can help. Apologies. Kind regards, Anna

Anna Gardner, it's ok. Thank you.