I followed this person’s research (Artxy-http://www.art-xy.com/2013/03/isolation-of-chlorophyll-and-beta.html) but altered slightly the method of the solvent preparation and ended up dissolving most of the chlorophyll A and B, but retained a large quantity of B-carotene.

The concentrations of chlorophyll (a)(b) were extremely low, although there peaks showed on the data but as little bleeps. The B-carotene though was clearly extracted in a big way since it is a non-polar compound, so the ethyl acetate solvent was able to do most of the work extracting it.

Below are the 2 cuvettes containing the samples, the first mainly chlorophyll and the second shows that B-carotene settled at the bottom

This next pic is the sample after solvent preparation and filtering into a test tube, again you can clearly see the two distinct separation layers, top layer of course is chlorophyll and the bottom is B-carotene.

Some discussion on all this, I expected to have successful separation because I have done this before several times before I did it officially online, this time though I think I let the sample sit too long in the solution (a full 24 hrs.) I tried different dilutions but the chlorophyll A-B just showed up as minute concentrations and appeared as very small spikes on the data graph. Everything that I have studied on this subject, ie,. spectrometry, and analytical chemistry suggests that concentrations below 0.2 are very suspect.

So for this study, my focus was on the successful extraction of B-carotene and my subsequent data to support that assertion.

As in the study that was done in the reference I stated earlier, my data was spot on in places such as the predicted peaks for B-carotene and also for chlorophyll A-B, I will include this data in a future research post.

Some conclusions, first, is this spectrometer kit version 2.5, with the slight modifications I have made with slit width and DVD grating, I have to say, it is really a fine instrument within it's range. Point two, cuvette holder design, VERY important, I have found that enclosing at least 75 percent of the cuvette re-directs the light back to it and increases the intensity of fluorescence, on my design the side of the cuvette that faces the slit is 12mm square, it makes a difference.

References: https://commons.wikimedia.org/wiki/File:Chlorophyll_ab_spectra2.PNG http://www.chm.bris.ac.uk/motm/carotene/absorp~3.gif https://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy http://www.art-xy.com/2013/03/isolation-of-chlorophyll-and-beta.html