Public Lab Research note


Absorbance of grass in blue light

by vanjo63 | February 21, 2016 18:45 | 233 views | 10 comments | #12717 | 233 views | 10 comments | #12717 21 Feb 18:45

Read more: publiclab.org/n/12717


Hello everybody, I am a student in biochemistry at the University of Liège in Belgium, and I have a huge scientific curiosity so I decided to build my own spectrometer, with the help of Spectral Workbench informations of course, in order to analyse the spectrum of many many samples. I realised many tests, and I decided to focus my "work" on concrete things.

I think that is important to clarify that is my first research note and that I speak French, so I am sorry for the possible mistakes of English language.


I will present you the spectrum of grass extract. To do that, I picked some grass in my garden, I add water and press the grass to extract ... chloroplasts I presume :D This is my "stock solution".

Here is a picture of my spectrometer : Spectro_André_V2.0.jpg

So, I analysed my stock solution in a dilution 8X. I used water for the blank and I used a LED RGB but i lighted only with a blue light. I collected results and analysed them. Here is a graph with the spectrum of the blank, the sample and the difference of both (it is absorbance) :

Extrait_d'herbe.jpg

We observe 2 maximums of absorbance, at wavelength corresponding of maximum of absorbance of chlorophyll A (~430 nm) and chlorophyll B (~445 nm).


The spectrum is not very accurate and the resolution is weak, so it is a good idea to improve my spectrometer to increase the resolution and accuracy of my future spectrum. I realised the same experiment with a red light, but results are not enough usable.


I think that my results are significant, and that stimulate myself to continue and perform a lot of spectrum of many samples.


Here is the links of my data : Blank : https://spectralworkbench.org/spectrums/71896 Grass diluated 8X : https://spectralworkbench.org/spectrums/71896


10 Comments

That's a good idea to use only one color of light. It can make it easier to interpret the result. Your result is very consistent with the two peaks of chlorophyll a. I would have also expected to see some strong absorbance between 450 and 500 nm attributable to chlorophyll b. Do you know why this peak was absent in your results?

What does your spectrometer use as a sensor? It would be good to learn more about your design.

Thanks,
Chris

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In fact, chlorophyll a has 2 peaks : the first around 430 nm and the second around 660 nm ; while chlorophyll b has also 2 peaks : the first around 445 nm and the second around 640 nm. In my spectrum, we see the first peak of chlorophyll a (~430nm) and the first peak of chlorophyll b (~445nm). To see the second peak of chlorphyll a and b, I should be analyse the sample with a red lamp.

My spectrometer is very basic : the light cross the sample, it strike on a fragment of DVD (that decompose the light in its constituents) and a webcam (attached against the DVD fragment) capture the spectrum :

Spectro_André_V2.0_capture_chamber.jpg

:)

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This is very cool, thanks for posting! I also wanted to point out that comparisons of this type are possible in Spectral Workbench 2:

And so are subtractions; note that since your spectrum has high baseline noise, the subtraction actually removes this:

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Oh tank you for this information, I didn't know that was possible to do that via the application Spectral Workbench 2 :)

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This is very very cool! I'm looking forward to more of your work.

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I guess you are right about the two peaks. I was thinking that you had captured the lesser peak of Chlorophyll a shown here at about 410 nm:

chlorophyll_a_b_spectrum_3.jpg

Thanks, Chris

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Maybe you should try to do your extraction with some alcohol. Chlorophylls are, after all, lipophilic and you have to get them out of the chloroplast membranes. Desinfectant alcohol is probably the safest, though if you work in a ventilated kitchen (under a hood) or outside you could use methanol of acetone from your local DIY store. It may clean up your baseline and enhance the vies of the chl b shoulder in your spectrum.

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I have wondered about how important it is to really extract the plant pigments for this type of absorbance observation. If you had an intact transparent leaf and put it between the light source and the spectrometer entrance slit, you might get a result very similar to vanjo63's result. The colors not absorbed by the leaf will pass through it.

I have tried extracting plant pigments with grain alcohol (ethanol), and made a nice colored liquid which absorbed just as theory would predict (see https://publiclab.org/notes/cfastie/10-10-2013/lycopene). But I left the vials sitting around and after a few days all the color sank to the bottom of the vials suggesting that I did not have an extract but just a suspension of tiny pieces of plant tissue. I don't know if a true extract would provide better absorbance information.

Chris

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I am going to try the experiment with an extraction of grass pigments by ethanol even if I don't think there is a big difference. That will allow to have a comparison of two different extraction methods :)

In plant cells, there is also an other pigment that chlorophyll, the carotenoids. And this pigment absorb in the range of 400-500 nm, so we must to consider that in this spectrum.

I think that try on only chlorophyll is a good comparison to make, but the problem is to have only chlorophyll ... So, create an "amateur" chromatography to separate the different pigments is an ambitious idea whose I am going to cogitate :)

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@cfastie: well, I recall that we made whole leaf spectra in the lab (me = biologist, Ph D of UAntwerp). Guess with a decent light source you can do that with your equipment too ? So no, it is not necessary... I just wondered if it could enhance the signal (you have a noisy background). Just a question - if you shook that extract, could you still measure a spectrum ? or was it just degraded chlorophyll ? Also - please don't take this as criticism!! You're doing great work (I love it, and hope to join the fun as soon as I have some time).

@vanjo63: in the student lab I taught, we separated the two (even chl a and b and different carotenoids) on a column of potato starch. Elution with petroleum ether and toluene, but maybe something else (cheaper or less toxic) works also.

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