Dear Habib

You can use the portable SPAD-502 chlorophyll meter after correlating the SPAD 

readings with actual chlorophyll content. Firstly, you have to construct a calibration curve for the plant species you are interested.

You have to record the SPAD measurements from leaves of different greenness (I think that 30 leaf samples are quite enough) and then you have to estimate their chlorophyll content with a biochemical method.

Then, the calibration curve of SPAD readings versus leaf chlorophyll content can be used for chlorophyll content estimation using only the respective SPAD readings of the unknown samples of the certain plant species.

According to the certain biochemical method that you will use, you must convert the spectrophotometric measurements to chlorophyll concentration (for example mgr/gr fresh weight, mgr/cm2, mgr/gr dry weight) in order to construct the calibration curve.

In the attached paper you can see one of these methods.

Opti-Sciences chlorophyll content meter CCM300 measures not relative units but chlorophyll concentration using fluorescence ratio. There is no need for any calibration. Another advantage is possibility to measure very small samples nondestructively, e.a. lichens on tree trunk, conifer needles etc.

More information can be found at http://www.optisci.com/ccm300.htm

I am using one myself with great satisfaction

 If you are looking for number of Chl molecules, first estimate the concentration of Chl a and Chl b [or total Chl]. Then you can calculate the number by dividing the molecular weight of Chl 1K (apprx.).

For exact numbers, you can divide the MW of Chl a and Chl b separately.

I think that the Arnon method is better. 

1) The Spadometer is a hand-held device that is widely used for the rapid, accurate and non-destructive measurement of leaf chlorophyll concentrations. It has been employed extensively in both research and agricultural applications, with a range of different plant species. [Reference: Ling Q, Huang W, Jarvis P. "Use of a SPAD-502 meter to measure leaf chlorophyll concentration in Arabidopsis thaliana" Photosynth Res, 2011, 107(2):209-214].

2) The CCM-300 (Chlorophyll Content Meter) uses a proven fluorescence ratio technique for chlorophyll content measurement at an affordable price. With this technology, chlorophyll content can now be measured from germination through maturity.

I concur with Juan. The method by Arnon, 1949 could suffice your needs.

There is another method, very simple, with DMSO (for chlorophyll and carotenoids) and acidified methanol (for compound that absorbed at 305 nm). 

Chlorophyll and carotenoids were extracted from 20 mg (FW) of leaf tissue using 2 ml of dimethysulphoxide (DMSO) at 45 ºC for 12 h in darkness (Chapelle et al.

1992). Chlorophyll and carotenoid concentrations were calculated according to Wellburn (1994) procedure and expressed as mg g-1 DW. We  are using a borer (diameter 3 mm) with 3 "borer" for chlorophyll and 2 "borer" for abs 305.

Porra (Photosynthesis Research 73: 149–156, 2002) is a nice update on the Arnon paper for chl extraction. Table 3 provides extinction coefficients for extraction in acetone, DMF or methanol, both in nmol/ml or ug/ml chl a and b. 

According to Juan and Alistair, I do agree their proposals are really suitable for your meanings. Another option is following Sims and Gamon (2002) protocol. It's also simple and fast. 

Dear Habib,

As you can see in the previous answers, you can have basically two methods. One is non-invasive, when you use an equipment with an external sensor what you attach to the given leaf and measure  relative absorbance or light scattering (probably both) signal what can give you some results with the help of calibration curves.

A more accurate method is invasive when you extract the pigment from a plant material of known mass and measure the absorption spectrum. Using  the given equations (the work of Porra 2002 is excellent) you get concentration values. If you know the volume of the extraction and the mass of the plant material (and the molecularmasses of Chl-a and Chl-b) you can simoly calculate the "number of Chl molecules" over one gramm fesh mass of the plant material.

Thanks every one who guide me. 

First you measure the lab based chlorophyll extraction then relate to molecular wt and volumetric calculation.

Thanks dear Murat

Regards,