25mg tissue in 10 to 20 ml DMSO keep it in dark for over night and tack OD at different nm like 480nM , 665nM and 649nM . than using some formula we gate chl a , Chl b total Chl and carorinoids. mg/ml.

Some accuracy is eventually lost with small amounts of tissues, but we have done the analysis with approx. 50 mg samples. There are many variations of chemical (spectrophotometrical) chlorophyll analysis, but basically you need to disrupt your tissues (or use small discs instead), extract them with a known amount of solvent proprotional to the amount of plant material (most often ethyl alcochol or aceton), and read OD spectrophotometrically at several wavelegths. Then by use of formulae (there are several) calculate the amount of chlorophyll a, chlorophyll b, and carotenoids.

simply with a spectrophotometer,in which you determine the amount of chlorophyll a, chlorophyll b, and total carotenoid content.

or

you may try for your little meterial

The material was processed in the fresh state immediately

after collection. After fine chopping, portions

weighing 0.5 g were measured off on an analytical balance.

The measured-off material was then homogenized

in a homogenizer with the addition of 10 ml of 80 % acetone.

A primary acetone extract containing all chloroplast

pigments was obtained in this way. The extract was then

centrifuged at 2500 rpm for 5 min. Since the concentration

of pigments was in most cases too great for reading

to be performed on a spectrophotometer, the obtained

extract was diluted by adding 9 ml of 80% acetone per ml

of extract. The extract produced in this way was subjected

to reading on a spectrophotometer. Chlorophyll content

was calculated according to We l l b u rn (1994)

(source-Arch. Biol. Sci., Belgrade, 57 (4), 283-290, 2005.)

Dear Caio, see the book: WITHAM, F.H.; BLAYDES, D.F.; DEVLIN, R.M. Experiments in plant physiology. New York: D. Van Nostrand, 1971. p. 55-58.

The procedure is very simple and reliable.

a leaf of Arabidopsis is enough

SPAD values itself is not a problem, as you can calibrate SPAD readings against spectrophotometrically measured chlorophyll concentration. The problem arises when small, curly, thick etc. leaves need to be measured. Or when treatment leads to morphological changes in leaves (pubescence, vax layer, thickness etc.).

I have also used the method described above by Tamal Mondal. After measuring absorbance at 663 and 645 nm, you can easily calculate the content of Chl a and Chl by using Arnon's equations. You should be fine with 80 mg of fresh material.

Dear Caio you can use Chlorophyll meter, SPAD-502 (Minolta), however there is lab protocoles for measuring chlorophyll a and b but it needs more time.

you may try the chlorophyll content estimation by adopting the procedure and extraction or Chlorophyll was done with DM50 (Dimethyl sulphoxide) methods. The leaf samples weighing 0.375 g were added in test tubes containing 10 ml DM50 solution and kept in BOD incubator for 2 hours at 60°C (the time in BOD incubator depends on succulency of leaf i may more more or less) for extraction of chlorophyll. The supernatant was used for estimation of pigments. The optical density of the aliquot was measured on spectrophotometer at the wavelength of 663 nm for chlorophyll a, 645 nm for chlorophyll b and 652 nm for total chlorophyll with red filter.

You can use calibrated SPAD meter to measure leaf chlorophyll content. Here first you should calibrate the meter and then you can take measurements of representative samples and finally you can average the measurements. It is so easiest than other manual extracting methods.

Hi,

this is the method I applied in my work (maybe the problem is not the amount of material but the amount of replicates you can analyse to have a good accurancy):

Leaves (50 mg fresh weight for each sample)

were crushed by adding liquid nitrogen, ground in 10 ml

N,N-dimethylformammide (DMF) on an orbital shaker for

2 h and then centrifuged at 4,000 g for 20 min. Absorbance

of the supernatant was measured at 663.8 nm, 646.8 nm and

480 nm with a spectrophotometer. Ca, Cb and Cx + c

concentrations per unit leaf fresh weight (μgg−1) were

calculated using extinction coefficients and simultaneous

equations for DMF extraction derived by Porra (2002).

And here a good reference: Porra RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophyll a and b. Photosynth Res 73:149–156.

I hope it's of help

The easy way to determine chlorophyll content in plants is just by taking 0.2 gm from the fresh leaves, immerse in 5 ml Dimethyl formamide in a test tube covered with aluminium foil. The tube should kept overnight in a referigrator. next morning chlorophyll A, B and the Carotenoids can be determine using Spectrophotometer. 664.5 nm for Chl. A, 647.4 nm for Chl. B and 452,5 nm, for Carotenoids

Very simple. You can extract chlorophyll with small amount of leaf without maceration. Go through the method of Hiscox and Israelstam 1979 using DMSO

Another simple method for a small quantity of sample is maceration of the tissue in 4ml acetone and 6 ml hexane using ultra turex at 14000 rpm for 2 min and measure absorbance reading at 663,645, 505 and 453 nm in a spectrophotometer. then use formula as described by Nataga et . al 1992 to calculate Chl a and b.

What if the weight of the sample is very less, say about 50mg. Will the volume of the solvents differ...???

Hi Farhan,

Please take advice of Batoul and Nafees for small amount of sample. Acetone: Hexane extraction is suitable for at least sample wt of 0.5g.

what temperature is the orbital shaker set to?

There is method for estimation of photosynthetic pigments without macerating leaves. The chopped leaves are to be placed in DMSO for a fixed period of time at a particular temperature, after that the pigments are estimated spectrophotometrically. The timing for incubation and temperature are to be standardized for the sample in question. 

dear my colleague

it is very simple

you can get about 0.05-0.1 gram from fresh materials and soaked it for minimum 24 hrs in refregerator with methanol after adding a traces amount from sodium carbonate. after that you can read the abosorption at 470, 653 and 666 nm on spectrophotopmeter (Lichtenthaler and Wellburn, 1985)

25mg tissue in 10 to 20 ml DMSO keep it in dark for over night and tack OD at different nm like 480nM , 665nM and 649nM . than using some formula we gate chl a , Chl b total Chl and carorinoids. mg/ml.

you can use 80% acetone and keep your sample in the dark for 48 h then measure it on the common three wavelength

Using 80% acetone will on a ground portion of the leaf (0.02-0.05 g) and measure absorbance at different wavelength ( 480, 649 and 665 nm ) the the various chlorophyll concentrations can be calculated using the Arnon's (1949) equation .

Ransford Okley Tetteh gave the best suggestion (Arnon 1949) it is a reliable method, but takes a week time. Caio Guilherme Pereira you can go for it if you have time, dilutions matter.

it takes just from 0.1g till 0.05 g

25mg tissue in 10 to 20 ml DMSO keep it in dark for over night and tack OD at different nm like 480nM , 665nM and 649nM . than using some formula we gate chl a , Chl b total Chl and carorinoids. mg/ml.

You may also follow this .

http://www.saps.org.uk/saps-associates/browse-q-and-a/375-how-can-i-quantify-the-chlorophyll-in-leaves

Kindly go through the following PDF attachments.

Just use Aceton 80% for 48 h

You can use 0.1 gm as sample