Dear Wajid!

The light scattering curve is wavelength dependent. Depending on its extent, it can run up steeply towards the blue. Thus it can cause the artificial increase of the 645 nm E-value artificially.

A more comlex problem is if your spectrophotometer is out of calibration. If it is shiftewd towards the red, you read the 645 nm E-value close to the 663 nm maximum and the 663 nm E-value on the decreasing slope (on the red-side) of the chlorophyll-a absorption band. This is a double failor: the E-645 will be higher and the E663 will be smaller than their normal E-values.

So, I would suggest you to calibrate your spectrophotometer and anyway to record a spectrum. The shape of the spectrum shows both problems, i.e. the baseline distortion and the spectral shift.

If you don't have a recording spectrophotometer, measure the E-values around 663 nm in series until you find the maximum E-value. Accept it as E-663. Then set the monochormator to 18 nm distance towards to blue and record the E-value there. This will be hopefully the E645 nm value.

Even in this case it can happen that you cannot be absolutely precise since we don't know if the shift of the monochromator is monotonous.

In the Material and Method section of your work, you should carefully describe this problem and don't forget mentioning the type of the spectrophotometer you are using.

Good luck,

Bela Boddi

I suggest you to check your calculations and used formula again.

According to Arnon' s formula,

Chl a (µg g-1) = (12.7 x A663 – 2.69 x A645) x V/W

Chl b (µg g-1)  = (22.9 A645) – 4.68 (A663) x V/W

Total (µg g-1)  = (20.2 A645) + 8.02 (A663) x V/W

Where,

                A663 – OD at 663 nm

                A645 – OD at 645 nm

                V       - Total volume of homogenate (ml)

                W – Weight of tissue (g)

Dear Ratheesh, I have done through this formula but same issue if you can check plz  suppose OD at 645nm= 0.64 and OD at 663nm = 0.70, V=5 ml W=0.5 g 

is there any issues with OD???

i ll be grateful 

The values are out of literature range, being chla higher than b from 1.5 to 3 ratios. You should then try to check phaeophytin made by chl oxidation and acidification by adding MgCO3 and sodium ascorbate. Also the spectrophotometer should be calibrated in wavelength and bandpass  with neutral density filters and holmium oxide liquid solution.

Chlorophyll b is a protector molecule  of chl a that absorb high quantities  of energy, so if you submitt the plants to high temperature is a cause that chl b be higher tahn chl a

Respected Margarita, do you have any literature kindly share with me

Regards  

Dear Wajid,

Experimental error can be behind this high absorbance value at 645 nm. Before running into different theories, please check the following:

1) Calibrate the wavelength accuracy of your spectrophotometer (you may have characteristic filters or a stain with known absorption maximum).

2) Record a spectrum between 550 and 800 nm to be sure not a great baseline distortion (caused by great light scattering) is the reason of the high value at 645 nm.

If so, centrifuge xour samples at 0 degree C with 10 000 g to get rid of the scattering material.

It may happen that the aceton/water mixture (the water content of leaves increased the water proportion) at cold temperatures created an opaque colloid what causes strong baseline distortion, too.

Anayway, via recording the spectrum, you can make clear this baseline story

3) Add small amount of NH4OH to the 80% acetone when extracting the pigments to prevent acidic breakdown of chlorophylls.

Good luck!

Bela Boddi

Respected Bela Boddi, thank you so much for your valuable comments, you have excellently elaborated this issue but i have to ask you something more if we supposed all these reasons are responsible for higher absorbance at 645 nm then at the same time these reasons are also prevailing for absorbance at 663 nm. so the standard difference between 645 and 663 should not be same?  or these conditions only adversely effect value at 645 nm?

Regards 

Dear Wajid!

The light scattering curve is wavelength dependent. Depending on its extent, it can run up steeply towards the blue. Thus it can cause the artificial increase of the 645 nm E-value artificially.

A more comlex problem is if your spectrophotometer is out of calibration. If it is shiftewd towards the red, you read the 645 nm E-value close to the 663 nm maximum and the 663 nm E-value on the decreasing slope (on the red-side) of the chlorophyll-a absorption band. This is a double failor: the E-645 will be higher and the E663 will be smaller than their normal E-values.

So, I would suggest you to calibrate your spectrophotometer and anyway to record a spectrum. The shape of the spectrum shows both problems, i.e. the baseline distortion and the spectral shift.

If you don't have a recording spectrophotometer, measure the E-values around 663 nm in series until you find the maximum E-value. Accept it as E-663. Then set the monochormator to 18 nm distance towards to blue and record the E-value there. This will be hopefully the E645 nm value.

Even in this case it can happen that you cannot be absolutely precise since we don't know if the shift of the monochromator is monotonous.

In the Material and Method section of your work, you should carefully describe this problem and don't forget mentioning the type of the spectrophotometer you are using.

Good luck,

Bela Boddi

really impressive.. thanks for your precious comment. i ll do my best to incorporate your suggestions. Regards