[redtree]

Hello everyone,

I'm in my first year of uni and already struggling with my chemistry classes. I've been hours trying to figure out the answer but I just can't, I'm stuck and can't move forward.
The did a practical the other day with using a spectrophotometer to measure the absorbance of pigments of a leaf and now we need to answer some questions about it. The question goes as follows:
calculate the amount of chlorophyll in each cm2 of the leaf, knowing that you can calculate the dilution factor with the volume of leaf extract you started with and the amount of extract at the end of the process.

I already calculated the amount of chlorophyll in 1 ml of 80% acetone, and I used 5 cm2 of leaf.

I didn't come here to find someone who can do my work for me. I just want to know who to do this, I'm sure its very simple but right now I seem unable to work it out. If anyone can help I'd really appreciate it. Thanks

[Borek]

We won't be able to help not knowing the procedure you have followed.

Generally speaking you need to calculate how much chlorophyll was there in the 2 cm² of the leaf that you used, then multiply it by the ratio of surfaces. Or you can calculate surface concentration of chlorophyll (amount per one square centimeter) and multiply it by the 5 cm². Many ways of skinning that cat.

[redtree]

Thanks for replying, but I'm still confused. I'll try to give more details.

We used 5cm2 of a leaf and added 3ml of 80% acetone up to 4 times before measuring the weight of the sample. the weight of the tube plus the extract was 13.12, but since we were working in pairs we had to add a little bit more of 80% acetone to both our tubes so that the weight of our solutions was each within 0.1 grams. Then we weighted the tubes again and now they were 13.35 g.
We centrifuged the solution and took 3 mL of the supernatant to measure the absorbance at different wavelengths.

Now one of the questions is to calculate the amount of chlorophyll in each mL of the 80% acetone solution I analysed using this equation  20.2 x A645 + 8.02 x A663
I already did that part but now there's the second part of the question,

Calculate the amount of chlorophyll in each cm2 of the leaf knowing that you can obtain the dilution factor with the volume of the leaf extract you started with and the amount of extract at the end of the process.

I suppose I can calculate the dilution factor with the two weights of the tubes ( the before and after weights) but I'm not sure. Even if I did I don't know in which equation I have to use that result, the only law we've been using in class in the Beer-lamber law, but there's no dilution factor in that law..

[Borek]

Lambert-Beer law is for determining the concentration in the final solution. Knowing this concentration you should be able to calculate total amount of chlorophyll present in all the solution.

And dilutions is not part of the L-B law as calculation of concentrations during preparation is a separate process from the determination of the concentration with a spectrophotometer. Dilutions and calculations related to the sample preparation are just supporting calculations, that are never part of the main method.

[redtree]

I understand what you're saying but I still don't know what equation or equations I need to use to calculate the amount of chlorophyll in each cm2

[Borek]

Try to calculate total amount of chlorophyll in the solution.

If I understand correctly what you did, you have measured the concentration in the solution directly, without diluting - so all you need to do is to calculate amount of the substance in the whole volume. C=n/V, solve for n.

[RhysLloyd]

I know this is old but I'm in a similar situation, I've calculated the chlorophyll in every ml, how do I calculate the chlorophyll in every cm^2?

[Borek]

I've calculated the chlorophyll in every ml

How many mL in total? How much chlorophyll in total?

how do I calculate the chlorophyll in every cm^2?

How many cm² used?

[RhysLloyd]

I think I've done it now, thanks. If it says you have 9ml but you do a dilution by adding 2ml of fresh acetone to 1ml of your pigment extract, does that mean the total ml is 27ml?

[Borek]

If it says you have 9ml but you do a dilution by adding 2ml of fresh acetone to 1ml of your pigment extract, does that mean the total ml is 27ml?

Yes, these are equivalent ways of calculating the dilution.