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Old Apr 8th 2017, 08:02 PM   #1
nobodytobe123
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Question Analyzing astaxanthin content with spectrophotometer

Hi chem wizards!

I'm not a chemist, only had a few basic chem classes in college. But I happen to have some H. pluvialis powder here that I need to analyze for cheap and I can get a cheap spectrophotometer on ebay, so I've been looking at articles to try to figure out what wavelength to look for.

Strangely, different sources disagree on the wavelength! So I'm confused.

One source says, "The data show that 530 nm is the most suitable wave length for spectrophotometric determination to the astaxanthin in Haematococcus crude extract."

https://link.springer.com/article/10...343-012-1217-5

But another source, https://www.ncbi.nlm.nih.gov/pubmed/22320081 seems to indicate that 432nm is the magic wavelength.

And other sources say other wavelengths!

Please end this confusion, someone! Thank you!
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Old Apr 11th 2017, 01:29 AM   #2
MatthijsM
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You can end this yourself by measuring a full wavelenght scan on the sample. So you just measure every possible wavelenght the machine can measure so you can look at the maximum absorption. Buy some pure astaxanthin and check it out, I'd say that could be a possible solution.

If you need more information just ask away
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Old Apr 11th 2017, 04:24 AM   #3
nobodytobe123
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Thanks MatthijsM, but from what I've read, such a simple idea would not work (otherwise there wouldn't be so much dispute already between the studies). It seems that the other carotenoids in the H. pluvialis create some distortion in the overall curve, so the wavelength for pure astaxanthin is not the wavelength of astaxanthin within H. pluvialis.

If I'm wrong about this, could you please post a link to some info, because this is definitely the first idea I came up with as well...
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Old Apr 11th 2017, 05:20 AM   #4
MatthijsM
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What do you exactly need to know? That might give me more information, so I could be more of an assistance
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Old Apr 11th 2017, 07:57 PM   #5
nobodytobe123
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I need to know how much astaxanthin is in a sample of H. pluvialis algae.

Now thinking about what you said, I'm realizing there is probably a fairly simple way of figuring this out... I can get some known-pure astaxanthin (hopefully), and then dissolve that into a sample of the H. pluvialis, and compare the spectrum of that to the spectrum of the unadulterated H. pluvialis. That should show me where the astaxanthin peak is in context for sure... at least according to my amateur mind.

Altho, I still do not understand why professional analytical chemists publishing papers cannot agree on this wavelength.
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Old Apr 12th 2017, 12:30 AM   #6
MatthijsM
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your idea to add some pure powder is a good idea to start, look for some information about internal standards, might help you with it.
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Old Apr 14th 2017, 12:26 PM   #7
nobodytobe123
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Reply from a chemist friend

Hi again... I asked a chemist friend about this strange disagreement in the literature, and he offered his take on it, which I'll share just in case anyone else is interested in this topic:



"I haven't had a chance to dive into the literature, but I suspect that the wavelength variations are due to the excitation of different transitions. Pi and nonbonding electron pairs of organic chromophores can be promoted to higher energy levels via excitation. Each pair will excite at a unique radiation frequency and hence wavelength.

As far as analysis goes, you can do absorbance measurements for any sufficiently strong excitation wavelength. It is standard practice to first do an absorbance scan of your analyte. This means that you excite your sample over a spectrum of different wavelengths and you observe which wavelengths are absorbed the most. You can select the peak absorbing wavelength in the spectrum and then set the instrument to excite at that wavelength for your analysis.

This does however assume that you've purified your sample first. If you haven't purified the sample, the scan won't be of much help as you've got peak absorbance from all your contaminants in the spectrum as well. Unless you can deconvolute those from the spectrum, it's best to just try exciting at a wavelength suggested in the literature. You could try both and see which one works best. Typically the wavelength that absorbs more strongly will give a better signal to noise ratio. Also concentration should be sufficiently dilute otherwise you'll have signal to noise ratio problems as well (consult the literature for recommended concentrations).

Hope this helps, and best of luck.

Chris"

and then:

"One thing I forgot to mention in my hasty response is that if you decide to go the way of spectrophotometry, in order to get a measurement of concentration for your analyte (astaxanthin), you would first need to make a calibration curve to standardize your absorbance measurement. This means that you would need to obtain a pure sample and prepare several solutions of known concentration. Then you run them through the instrument and measure their absorbance. That way you could presumably take a sample of unknown concentration (your supposed 5% one) and compare its absorbance with that of the calibration curve to infer its actual concentration.

TLC would be a good technique to identify if your sample consists of multiple components, however it would be difficult to deduce concentrations as it is a separation method. You could perhaps use it to purify astaxanthin from your extract (assuming it contains some), and use that to prepare a calibration curve for spectrophotometric analysis as I mentioned above.

Alternatively, potentiometry (electrochemistry) is an inexpensive analytical technique that you could probably do. I reckon that the keto/hydroxy groups in its structure would be susceptible to some redox reactions. You would have to search for literature to see if any groups have measured redox potentials.

I suppose if all else fails, you may consider getting a second opinion from reputable lab, eh? "
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