Science topics: Chlorophyll b
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The question is in another way.
What is the relationship between SPAD chlorophyll value and Chlorophyll a, b and total Chl?
For example, if the lettuce plant has high SAPD content value that means the Chlorophyll must be high?? or any relationship with other chlorophyll components??
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As a general response, SPAD index and chlorophyll are correlated. However the SPAD measurement do not correspond to chlorophyll content rather it gives an indication of an increase or reduction of chlorophyll.
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Dear researchers,
I am currently helping one of my seniors in her experiment. And yesterday, we calculated the amounts of chlorophyll a and b. The results turn out that b is higher than a. We expected a would surely exceed the amount of b. So, I'd like to know in which conditions chlorophyll b can be higher than a. Please kindly answer my question. Thank you very much.
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This can be observed in early developmental periods under stress conditions, especially deficit irrigation, low light intensity and high UV trigger acumulation of Chlorophyll b for gaining more from photosythesis.
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Does anyone know the appropriate equation for chlorophyll a, chlorophyll b,total chlorophyll & carotenoids extracted by DMSO
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Dears,
I have used fresh leaves methods (1 g of the leaf tissues using 80% acetone) using these equations:
[Chl a (mg.g-1) = (13.95OD665−6.88OD649)V/200 W];
[Chl b (mg.g-1) = (24.96OD649− 7.32OD665)V/200W];
[Car. (mg.g-1) = (1000OD470−2.05Chl a−114.80Chl b) V/ (245×200 W)].
Where; Chl a– chlorophyll a, Chl b – chlorophyll b, Car. – carotenoid, V –volume, and W – sample weight.
what about dry leaves methods??
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The procedure and formulas are pretty much the same.
However, unless you freeze-dry the leaves, you'll lose a lot of chlorophyll through the process of drying. Chlorophyll is degraded very easily. Normal drying methods like heat drying or sun drying will make the leaves lose a substantial amount of chlorophyll.
If you don't care about that, though, and just want to, say, see how much chlorophyll is left after drying, then just do like with fresh leaves.
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Hello! I am trying to extract chlorophylls from microalgae, and I would love to ask how can I separate chlorophyll a from chlorophyll b? I want to get each of them separately. Thank you in advance.
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Hello. I've been working on photosynthesis pigment content in in vitro grown trees and was examining the formulas for determining the concentration of chlorophyll a and b and carotenoids based on absorbance, i.e. using a spectrophotometer. I've found various formulas, and even though they all share the same general design: concentration=coefficient1*adsorbance1-coeficeint2*adsorbance2, the coefficients differ among sources. I was curious as to the reason behind this. Is it due to the use of different equipment, i.e. every spectrophotometer is different, or something else? I'm new to this field, thus any help, directions to relevant introductory literature, articles, would be great.
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Philipp Girr Thanks, you made my day.
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I am performing UV/Vis spectroscopy using for pesticide effects on chlorophyll a and b. Chlorophyll a is having a tendency to bring about negative values. The absorptions are performed on three separate solutions from three different extractions of chlorophyll a and chlorophyll b is giving positive results.
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Thank you for your help :)
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I estimate chlorophyll A and chlorophyll B as well as total chlorophyll is present in pointed gourd leaf sample per 100 gram but there were much variation for various sample.
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Perhaps the samples were very variable - for example if some samples had more vein material which has (I expect) less chlorophyll per unit fresh mass than lamina. This would cause the apparent variation. Otherwise increase the number of samples to obtain a mean and apply appropriate statistics to detect differences caused by any treatments applied..
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Hi,
I am cultivating my chlorella sorokiniana as heterotrophic. When the fermentation is ended the algaes are green. However, after some time (and not consistent time for each individual batch) the colour changes into brown. I've done pigments analysis which states that the chlorophyll a and b goes from around 25% for both down to 1% and the lutein goes from around 20% up to 50%. Overall, I wonder why my algaes are turning from green to brown? Also, why does the chlorophyll a and b goes down while the lutein goes up? Is it because of oxidation of chlorophyll a and b?
In regards, thanks.
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please explain in which case the amount of chlorophyll a be less than chlorophyll b
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The ratio Chl a/b is a good indication of acclimation of the plant species to the environment such as nitrogen content and light. Fo further information, I advise you read the following article:
Increases of chlorophyll a/b ratios during acclimation of tropical woody seedlings to nitrogen limitation and high light
Thank you for asking this important qouestion!
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I had measured values of chlorophyll a, b and total by using a spectrophotometer.
After calculation by the equation, the values of chlorophyll b showed the negative.
Can I use these values for the research?
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Dear Kim,
Check the equation used for calculating chlorophyll a, b and total. Maybe the equation is incorrect. May contain an incorrect constant number (factor). Because the quantity of chlorophyll b is less than the quantity of chlorophyll a, but it is a positive value, not a negative. The sum of ch a and ch b is the total. Thank you and good luck.
Best Regards
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I requirement to know the most trusty extractant for chlorophylls a and b.
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Thank you Kathryn De Abreu
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What is the correlation between Chl a/ b ratio, Fv/Fm and ETR? please help me to understand.
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Dear Darshan,
under deep shade you may expect plants to undergo a reorganization of their photosynthetic apparatus at many levels, from the morphological one to the biomolecular one. The extent of this response may depend also on the time during which the plant has undergone low light. However, regarding your question, a general response of plants to shade is a lower Chl a/ b ratio, which is indicative of a higher investment in antenna complexes (or, at least, a lower disinvestment in antenna complexes, if the resources are scarce), where chl B is located. A shade-acclimated plant should also display high values of Fv/Fm, because its potential photochemical efficiency is not affected by those processes that may be generally considered the cause of chronic photoinhibition, which may be induced under prolonged exposure to high photon flux density. Last: obviously, in shaded environments ETR is limited by the low light energy.
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chlorophyll a and chlorophyll b will always positively correlated?
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Chlorophyll a is considered as primary pigment , while chlorophyll b as accessory pigment supplying energy to chlorophyll a.Thetefore , both the pigments are supposed to be positively correlated , since both the pigments compliment each other.But r=1.00 , I doubt such a perfect correlation...
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photosystem I (in Stroma) or photosystem II (in Granna)?
chlorophyll a or chlorophyll b?
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Millton Lima Neto thank you. It's so perfect.
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How does the growth conditions affect chlorophyll cycles? (both improved conditions and stress conditions)
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Please check these files
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Helo researchers.
I estimated chlorophyll content under heavy metal stress plant.
I have read in many papers that chlorophyll "a" value always double from chlorophyll "b".but if their is opposite situation so what does it means.I mean chlorophyll "a" is lower almost half of chlorophyll "b".
Like chlorophyll "a" value is 6.71mg/g where as chlorophyll "b" 13.14 mg/g.
so i need your scientific explanation and if any research paper support it please share.
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I am doing study on Chlorophyll. The majority of the plant has higher Chl a than Chl b.
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Hi Kishor:
Generally the content of chlorophyll a is higher than chlorophyll b, and this is possibly because chlorophyll a is found in all the complexes of the reaction center, as well as in the antennas, while chlorophyll b is found only in complexes of peripheral antennas, and a higher content of the chlo a respect to chlo b, will do higher the quantum yield in photosystems. That is why some researchers use the Chla/Chlb relationship as an expression of photosynthetic efficiency.
regards
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Dear everyone,
I have a question regarding calculations of chlorophyll extraction.
after different steps of the protocol and measurements of the photometer, I have these formulas to measure chlorophyll a,b and a+b:
Chlorophyll a (μg/ml) = ((12.7 *E663) - (2.69 *E645))*dilution factor
Chlorophyll b (μg/ml) = ((22.9 *E645) - (4.86 *E663))*dilution factor
Total chlorophyll (μg/ml) = ((20.2*E645) + (7.52 *E663))*dilution factor
*E=extinction value
Shouldn't there be another calculation afterwards with the material weight that I took (0.01 g) and the acetone weight used in different steps?
Thank you
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Dear Setareh,
Calculation of chlorophylls and carotenoid content in plants, definitely related to solvent you used for the assay. 
Hartmut Lichtenthaler, distinguished professor at Karlsruhe Institute of Technology, has the best papers about photosynthetic pigments assay. I am sure you can find your answer about the calculation of chlorophylls and carotenoid in them. I attached them to you for your reference.
Best regards, Homayoon. 
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Dear colleague,
Can you tell me if my calculations of chlorophyll and carotenoid content are correct, I used Arnon's equations for 80% acetone.
Chlorophyll a (mg/g) = [12.7 (A663) - 2.69 (A645)]*V/1000*W
Chlorophyll b (mg/g) = [22.9 A645) - 4.68 (A663)] *V/1000*W
Total chlorophyll (mg/g) = [20.2 (A645) + 8.02 (A663)] *V/1000*W
Carotenoid (mg/g) [A480+ (0.114 A663)-(0.638 A645)] *V/1000*W
With : V: final volume of chlorophyll extracted in 80% acetone; W: fresh weight of the algal powder used.
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Dear Colleague,
Calculation of chlorophylls and carotenoid content in plants, definitely related to solvent you used for the assay. 
Hartmut Lichtenthaler, distinguished professor at Karlsruhe Institute of Technology, has the best papers about photosynthetic pigments assay. I am sure you can find your answer about the calculation of chlorophylls and carotenoid in them. I attached them to you for your reference.
Best regards, Homayoon. 
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I am working in Hyperspectral Remote Sensing. I should correlate chlorophyll a , chlorophyll b with each wavelength of hyperspectral. The blue and red band are exactly correlate with chlorophyll a but the green bad does not correlate with chlorophyll b. Help me.
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Following websites and article may be helpful in this regard:
Hyperspectral remote sensing of plant pigments - Researchgate article
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Chlorophyll a ~ chlorophyll b ~ carotenoids
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Chlorophyll a absorbs energy from wavelengths of blue-violet and orange-red light at 675 nm. It reflects green light which gives chlorophyll its green appearance. While , chlorophyll b absorbs energy from wavelengths of green light at 640 nm. It is the accessory pigment that collects energy and passes it on to chlorophyll a. It also regulates the size of antenna and is more absorbable than chlorophyll a . Chlorophyll b complements chlorophyll a. Its addition to chlorophyll a increases the absorption spectrum by increasing the range of wavelengths and broadening the spectrum of light that is absorbed. When there is little light available, plants produce more chlorophyll b than chlorophyll a to increase its photosynthetic ability. This is necessary because chlorophyll a molecules capture a limited wavelength so accessory pigments like chlorophyll b are needed to aid in the capture of a wider range of light. Therefore , chlorophyll a and chlorophyll b are inter-related and positively related as well.
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Is it possible that the content of chlorophyll "b" is greater than the content of chlorophyll "a"? Abiotic stress can lead to this? Anyone know any literature?
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I think this is quite unusual.
How did you determine your chlorophyll contents? Spectrophotometric? The method by Lichtenthaler ( ) or by Lichtenthaler and Buschmann ( ) are quite reliable, but there are also HPLC methods separating both chlorophylls very well.
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The Chlorophyll b pigment is found in Euglena gracilis algae which I want to study using fluorescence microscopy.
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Fluorescence emission wavelengths are characteristically longer than corresponding absorption peaks, namely 648 cf. 642 nm for Chl b.
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I have grown vicia plants and wanted to know the impact of osmotic stress on the ratio of chl a to chl b
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under osmotic stress, both Chla and Chlb tend to to decrease while, the chlorophyll a/b ratio tend to increase due to greater reduction in Chlb compared to Chla. Any situation plant possesses more Chla than Chlb. Maintenance of more Chla than Chlb is vital for survival. So, during the process of Chl degradation, Chlb may be converted into Chla, thus resulting in the increased content of Chl a. hope the attached articles help in further clarification.
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We made a measurement on chlorophyll content for plants under sun and shade. We found that the shaded plants have less chlorophyll b.
This is in contrast to the theory where in shades, the LHCII are expanding in contrast to the reaction cores. Hence the chlorophyll b will be higher than chlorophyll a.
Please help. Thank you.
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Sunlit leaves have higher photosynthesis rate because these leaves are exposed to both direct and diffuse light. Shaded leaves, in contrast, are only exposed to diffuse light. Shaded leaves are not exposed to direct light because they are behind (or "in the shadow of") the sunlit leaves. Chloroplasts of shaded leaves contain more thykaloid stacks made up of wider grana than the chloroplasts of sunlit plants. Plants in shade have a lower chlorophyll a/b ratio. The result is a wider catchment array to snag what light does reach the leaf and pass it down to the reaction center, but with a lower over all reaction rate since they also have fewer reaction centers in the chloroplasts
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I have chlorophyll b standard purchased from company, however there no available information on its concentration. The standard is in liquid form. 
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A good reference for chlorophyll an and b extinction coefficients in various solvents and at different wavelengths is Porra et al (1989) Biochim Biophys Acta 975, 384-396.
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Dear Colleagues 
I am working on a spathe of an epiphyte plant, we saw no chlorophyll b in our samples. Do you know is it possible to have chl a while no chl b being detected?
Thanks
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Chlorophyll a is universal while chlorophyll b, c, d and e are optional. For instance in brown algae chlorophyll c is present in place of chlorophyll b. Similarly in red algae chlorophyll d is present in place of chlorophyll b. Hence chlorophyll a can be detected in absence of chlorophyll b.
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We put our Chlamy cells under several stress conditions and we would like to have an idea on how the chlorophyll content is affected.
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Just a little note, If you do the extraction in acetone, keep your extract in the dark as much as possible and let it extract in the cold (-20°C) overnight. 
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I determined the chlorophyll a and b content (mg g-1 F. wt.) with the method as described by Arnon (1949) by using the following formulae.
Chlorophyll a (mg/100 mL) = 0.999 A663- 0.0989A645
Chlorophyll b (mg/100 mL) = -0.328 A663 + 1.77 A645
But the value for chlorophyll b is greater than for chlorophyll a, I tried a few other equations but same trend is being observed. 
I cut the fresh leaves into 0.5 cm segments and extract over night with 80% acetone at 10C.
I am writing here absorbance  for 645nm: 0.64 and 663nm 0.70, kindly help me to resolve this issue. 
Regards 
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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
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Dear Colleagues, I am preparing a paper about Chlorophyll a and Chlorophyll b in the rosa species. Can anybody help me with the in the Different chlorophyll formulas ?
in the Different chlorophyll formulas The amount of Chlorophyll a and chlorophyll b in the rosa species are different. How should I understand this difference?
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Maybe the following papers would be interesting to you:
Porra, R., et al. (1989). "Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy." Biochimica et Biophysica Acta (BBA) - Bioenergetics 975(3): 384-394.
Lichtenthaler, H. K. and A. R. Wellburn (1985). "Determination of total carotenoids and chlorophylls a and b of leaf in different solvents." Biochemical Society Transactions 11: 591-592.
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why chlorophyll a is always heigher than chlorophyll b in plant cultivated  under salt stress ?
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Chl a is the site of photochemical reaction. It absorbs light which is used in the reaction. Chlb supports Chla by supplying more light energy to Chla. Under shade when Chla fails to absorb more light, Chlb take leading part in supplying more light to Chla just to continue more photochemical reaction. Chlb is more adaptive to stress condition. Any situation plant possesses more Chla than Chlb. Maintenance of more Chla than Chlb is vital for survival . 
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Can any one tell me,
        What is the the average chlorophyll and protein content of paddy leaves under normal condition?
1. Protein 
2. Chlorophyll a , Chlorophyll b and Total chlorophyll (mg/g or mg/ml)
Under normal condition, What is the average protein & chlorophyll content (chl a, chl b & chl t in terms of mg/g or mg/ml ) of paddy leaves after 45 & 60 days of transplanting ?
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Best method to calculate chlorophyll  contents is use of  SPAD-502.
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Arnon's equations for 80% acetone is common in literature.
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Arnon (1949) gave the equations for extraction with 80% acetone and absorbances read at 645nm and 663 nm as
Chlorophyll a (μg/ml) = 12.7 (A663) - 2.69 (A645)
Chlorophyll b (μg/ml) = 22.9 (A645) - 4.68 (A663)
Total chlorophyll (μg/ml) = 20.2 (A645) + 8.02 (A663)
Lichtenthaler & Welburn (1983) gave the equations for extraction with 80% acetone read at 646 nm and 663 nm as
Chlorophyll a (μg/ml) = 12.21 (A663) - 2.81 (A646)
Chlorophyll b (μg/ml) = 20.13 (A646) - 5.03 (A663)
Lichtenthaler (1987) as cited by Royse (2012) gave the equations for 100% acetone read at 661.6 nm and 664.5 nm as
Chlorophyll a (μg/ml) = 11.24(A661.6) - 2.04 (A664.8)
Chlorophyll b (μg/ml) = 20.13 (A664.8) - 5.03 (A661.6)
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Can any one tell me the what is the average content of
  1. proline
  2. chlorophyll a
  3. chlorophyll b
  4. total chlorophyll
content in bougainvillea and nerium plants under normal conditions as well as stress conditions.........
  • If u have any references also please attach it......
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Hello,
Sorry, but the question is actually impossible to answer. There will be ranges in all species and under different conditions and the question is far to general.  Why not measure them yourself? The methods are not too difficult with simple chemistry.
Regards
David Lawlor
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It is required apply some factor to the obtained data? The equations to calculate chl a,  chl b and carotenoids could be the same as used for the determination of photosynthetic pigments in spectrophotometer?
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The paper mentioned by Sunil is really worth reading.
Now for the question posted by Marcia; a microplate reader can be used to calculate chlorophyll content, - equations may be modified as per the paper cited.
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So does anyone know among various chlorophyll traits (Chlorophyll A, Chlorophyll B, Total Chlorophyll and Chlorophyll A/B ratio), is there any one best trait that can be used for measuring drought or heat 
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Drought tolerance can mean many things and many plant processes can be affected by drought, which can also mean many things. So it is not possible to use one measure to describe a complex situation. The question is too simplified. I have dealt with the problem in different publications listed in Research Gate and suggest  reading  them and the literature given there.  Chlorophyll fluorescence is a valuable technique but only under particular conditions, such as rapid and relatively severe dehydration but may not be useful under mild drought which affects growth processes and yield without altering photosynthetic metabolism greatly and so does not alter chlorophyll fluorescence. Chlorophyll content /concentrations in leaves are not a useful indicator of early drought effects unless the water deficit is rapid and large. If a specific list of references is required I can supply later. 
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I want to know the most reliable extractant for chlorophylls a and b.
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Does the nature of the material (e.g algal cells, plant leaves) affect the type of extract to use? Because I used 90% ethanol to extract chlorophyll from some leaf samples and got a consistent reading.
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I want to use it in ATK Quantum wise.
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...and with OpenBabel http://openbabel.org/wiki/Main_Page you can convert between various chemistry formats.