We are trying to isolate some carotenoids from algae with semi-prep HPLC. As literature says, a few mg will be enough. Is this true?
As always, the answer is "it depends". Mainly it depends on the purity of the material, the nature of the carotenoid and, most of all, what information you are trying to gather. You don't mention whether you have a cryoprobe. If you do, then you could get some excellent structural data on 1mg of ultra pure material. If you don't have a cryoprobe or if you material isn't pure then you may struggle with anything less than 10mg.
It also depends on how much time you want to spend. One can get a lot of information with a small amount of pure compound, but at the cost of having to acquire many free induction decays (FID) for a given pulse sequence. This may need several hours of acquisition time for a small amount of compound. I'd suggest it depends on the number of molecules. Depending on how one defines a "carotenoid", a compound with a mass of 200 Da (Daltons) has twice as many molecules in a milligram as a compound at 500 Da. I've noticed that some classify some of the degradation products of carotenoids, such as damascenones, to also be carotenoids.
Erdoğan
For small molecules (less than 1000 g/mol), typical 1H NMR spectra require 5-25 mg of material. Typical 13C spectra require 50-100 mg of material. This amount of material will allow you to obtain a 1H spectrum in a few minutes or a 13C spectrum in 20-60 minutes. When the amount of material is doubled, the resultant signal will be doubled. Bear in mind that a very concentrated sample will produce a quick 13C spectrum, but may result in a broadened 1H lineshape. Overly concentrated samples can also be difficult to shim. See
--- https://www.cif.iastate.edu/nmr/nmr-tutorials/sample-preparation
Good luck
When you have access to a dual-frequency micro probe - such as the Bruker 1.7mm TXI MicroProbe - a few mg of material is sufficient. With a micro probe the mass sensitivity can be 4 times greater compared to a standard 5mm probe. This advantage decreases compared to a 5mm cryo probe.
Since the 1.7mm sample volume is only 5 microliters the carotenoids must be soluble in this solvent volume. Also, sample preparation is more tedious than normal without the optional Gilson 215 Liquid handler.
John Hollerton
Thank you for your informative reponse. We expect that it is a highly pure carotenoid. I will collect as much as material and send to NMR analysis. Unfortunately, they do not have cryo probe. I hope we get good results. Thanks. All the best.
Dear William C Hutton, Adel Amer , Jack Silver
Thank you very much for your help and answers. I will keep them in mind. We expect that the carotenoid we have collected is highly pure. But, for sure we will see via NMR analysis.
All the best
Dear Ayşegül,
The amount estimates discussed above are all correct, but a important point to mention is the following: the amount you need also depends very much on the NMR time your NMR co-worker is willing/able to invest!
As a rule of thumb half of the material requires 4 times more NMR time..there is NO general limit of detection (LOD) for NMR. If we really need to know the structure of a small amount of a compound we use up to one weekend of NMR tîme in our lab.
I hope this helps
Alfred
Minimum amount of carotenoid for 600 MHz H and C NMR analysis in characterization required is about 15-20 miligram.
- Badges
- Science method