I always use the following method:

A half-gram of fresh sugar beet leaves cut into small pieces and grounded in absolute acetone. The homogenate centrifuged at 3000 rpm for 15 minutes, then supernatant complete to 15 ml with acetone. The color intensity was measured at wavelength of 661.6, 644.8, and 470 nm for chlorophyll a and b as well as carotenoids respectively using Milton Roy spectronic 1202. The following equations were applied for calculating the pigments according to Boyer, 1993.

Chlorophyll “a” = (11.24 X E661.6) – (2.04 X E 644.8)

Chlorophyll “b” = (20.13 X E644.8) – (4.19 X E 661.2)

Chlorophyll a+b =7.05 X E661.6 +18.09 X E644.8

Carotenoids = (1000 E470 –1.90 a - 63.14 b)/214

Where E = absorbance wave length.

Hi

here is an easy protocol. It is in german but very easy to translate:

50 mg of plant material need to be homogenised in 500 µl 1 M Tris/HCl pH 8.

take then 20 ul of homogenate and mix it with 980 ul of methanol. centrifuge for 5 min at 6000 rpm. then go to the spectrophotometer and measure. in the protocol you can find the formula to calculate the amount of chlorophyll a and b. 

alternatively check this out:

http://www.specmeters.com/nutrient-management/chlorophyll-meters/chlorophyll/spad502p/

cheers

Thanks

The use of portable chlorophyll meter will be useful too.

http://www.optisci.com/ccm200.htm

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Thanks

Here is an article with information about chlorophyll measurements and clamp on chlorophyll meters.

http://onlinelibrary.wiley.com/doi/10.1111/pce.12324/abstract

There have been several recommendations to use portable chlorophyll meter systems. Most of them measure the transmittance at two wavelenghts (in the red and near infrared) to determine the total amount of chl in the leaf. Howevere, these instruments do not take into account the saturation of red light absorption by Chl despite an increase in Chl concentration. Above a certain level higher concentrations of chl result in the same amount of red light being transmitted, and the effect is visible in the red-edge region, with the red-edge being displaced to the longer wavelengths. Meaning that for high concentrations these instruments do not provide a reliable reading, since the only noticeable effect occurs in a spectral region (red-edge) that is not being monitored.

Now a days we are using portable chlorophyll index meter (CCM 200 chlorophyll content meter) for field level estimation of chlorophyll content of leaf. The chl index meter is good for analysing relative level of total chlorophyll only. However,  for the accurate estimation of chl a and chl b and carotenoids etc the wet methods with organic solvents are  good.

Dr. K. Annamalainathan

Rubber Res. Inst. of India

Kottayam, India

I have used the paper recomended above (Porra 2002) but I am currently using the protocol described by Sims and Gamons:

Sims, D. A. and J. A. Gamon (2002). "Relationships between leaf pigment content and spectral reflectance across a wide range of species, leaf structures and developmental stages." Remote Sensing of Environment 81(2-3): 337-354.

For direct and precise determinations you can follow the next protocol that is pretty well explained and choose the solvent (acetone or methanol)

http://prometheuswiki.publish.csiro.au/tiki-index.php?page=Chlorophyll+extraction+and+determination

For an estimation of chlorophyll content you can use the SPAD-502 as mentioned above. But if you do not have the SPAD (price is about 2000USD), better to use the spectophotometer as descrived above.

Good luck!

Thanks every one .

You can use method Barnes et al. (1992) in DMSO. If you want I send you all recipe about it.

Best regards,

Kasia 

I always use the following method:

A half-gram of fresh sugar beet leaves cut into small pieces and grounded in absolute acetone. The homogenate centrifuged at 3000 rpm for 15 minutes, then supernatant complete to 15 ml with acetone. The color intensity was measured at wavelength of 661.6, 644.8, and 470 nm for chlorophyll a and b as well as carotenoids respectively using Milton Roy spectronic 1202. The following equations were applied for calculating the pigments according to Boyer, 1993.

Chlorophyll “a” = (11.24 X E661.6) – (2.04 X E 644.8)

Chlorophyll “b” = (20.13 X E644.8) – (4.19 X E 661.2)

Chlorophyll a+b =7.05 X E661.6 +18.09 X E644.8

Carotenoids = (1000 E470 –1.90 a - 63.14 b)/214

Where E = absorbance wave length.

Hello Katarzyna Mozdzen, thank you for your answer to Nissar Reshi (thank you for question). I am also very interested in refe that you sent to Nisssar. Please could you share it with me?

This is publication: Barnes J.D., Balaguer L., Manrique E., Elvira, S., Davison, A.E. 1992. A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants. Environmental Experimental Botany 32: 85-100.

Best regards,

Kate

Tank you V much Katarzyna Możdżeń 

you can use the method of Arnon 1949 : chlorophyll estimation