I came across some journals without a clear description on how the researchers determine the purity of their isolated compounds. Usually, isolation was done via HPLC coupled with a fraction collector followed by identification and confirmation via mass spectrometry and, all of a sudden, the percentage of purity will be mentioned (95%-98%). I am curious to know:
1. Do I need to use a standard of the same isolated compound to determine the purity based on DAD absorbence? E.g. I infuse 100ppm of standard A and the area under the graph (AUG) of DAD gives me a certain value. Next, I infuse 100ppm of the isolated compound A and determine its AUG value. I compare both AUGs and check for the purity. Is this the correct way of determining the purity?
2. Is there any other way to check the purity of an isolated compound without comparing the available standard, like using NMR etc.?
Hello Mr chandra devan
purity of the compound can be determined by HPLC but for that u need standard for sure. without standard comparing you cannot determine the % purity of the purified/isolated compound.
in that case u can first draw the standard curve of the standards by injecting different concentrations with the same method. then plot a graph in excel(preferable) of concentration v/s area under curve and you should get a straight line .calculate the standard curve .now put the value of the area under curve of your sample and then u will get the % purity.
2) there are other methods also for the calculation of % purity i.e by spectrophotometer and a more simple but old method of Titration.
NMR only tells you the structure of the compound(if its a single isolated compound) but it will not tell the % purity .
all the best
:)
Yes you have to use standard of the same isolated compound to determine the purity based on DAD absorbance as said by Dr Ashrey for exact comparison.
Exactly .........without standard a comparison is not possible.
You can do all the analyses with the isolated compound like FTIR, NMR, MASS and elemental analysis to know the exact nature of functional groups, the arrangement of protons and carbons, the molecular mass and fragmentation pattern and the percentage of the constituting elements respectively. But by this you come to know the structure of the compound and become clear about the nature and chemical formula of the compound. Once you have all the data in hand, there are two options:
1. Compare it with a standard to find its relative purity as compared to the standard. This can be done by HPLC as suggested.
2. You can use the already available data about that compound regarding the analyses you performed and compare it with your analytical results and find out how much your isolate is close to the available standards. This is an alternative method in absence of a standard but can be applied effectively to judge the purity of the isolate.
Gud Luck and best regards
as previously indicated getting purity informations as rather complicated.
therefore you should think about the reason you need the purity information. Is it because you need 100% pure compound ? or is it because a paper ask you the information for submitting.
You can also use HPLC with an ELSD detector. The ELSD detector gives signal closely related to the amount of compound injected in your HPLC (Adnani, N.; Michel, C. R.; Bugni, T. S. Universal Quantification of Structurally Diverse Natural Products Using an Evaporative Light Scattering Detector. J. Nat. Prod., 2012, 75, 802-6) it can help estimate quite accurately the purity of your compounds. However, sensitivity is not so good (except for brand new apparatus) you might not see very minor impurities present in your purified compound.
Another solution is quantification by NMR (Bharti, S. K.; Roy, R. Quantitative 1H NMR spectroscopy. TrAC Trends in Analytical Chemistry, 2012, 35, 5-26 / Dreier, L.; Wider, G. Concentration measurements by PULCON using X-filtered or 2D NMR spectra. Magn. Reson. Chem., 2006, 44(S1), S206-S212) which can help also to estimate purity of compounds without standards. This last solution is quite elegant. However, as for ELSD detection NMR id not highly sensitive... You might not see very minor impurities present in your purified compound. But it should not introduce error in your measurements.
Greetings Mr. Ashray Gupta, Mr. Robin Joshi and Dr. Alok Nahata,
Thanks for the very informative answers. As what I thought earlier, it is going to be hard to determine the purity of an Isolate without using a standard as a reference. You guys provided me with a assuring answer :-)
it was our esteem pleasure providing you the information,
if any problems u can mail Mr Robin Joshi as he is the expert in this line.
all the best for your experiments.
Greetings Mr. Samuel,
I am trying to isolate a compound from a herbal source and would like to know its purity. The purity will help me to generate a better data for publication in future :-) as I need to compare different techniques used in isolating the compound. Since I don't have a NMR, determination of purity of that particular compound via NMR will be hard.
However I am interested to know how ELSD detector can be used to determine the purity of a compound,Mr. Samuel. Do we need to pre-run a standard compound before running the isolated compound? Does ELSD calculate the purity relative to the standard? Or it just count the amount of isolated compound from the total injection volume in a run? Thanks as well for the reference paper
the ELSD signal is directly correlated to the amount of compound. but it changes slightly according to the molecule.
One easy solution could be to use as a standard a very similar compound which is commercially available. Then do a calibration curve and use this calibration curve.
just check in the publication if it is better tu use moles or mg for the calibration curve and the modification of the molecule.
anyway, you can also roughly used % of overall peaks area to estimate purity.
Basically ELSD is used for non-chromophoric types of compounds or for sugar analysis etc. But if you are using any detector you have to go with some commercial detector.
Thanks so much Mr. Samuel and Mr. Robin :-) I just learnt something new about ELSD. Thanks guys.
The isolated compounds always have no standard as reference. If the compound is stable before its melting point, you may use differential scanning calorimetry (DSC) in absolute terms to measure the total impurity. However, DSC usually need 3-5 mg samples which you might not affordable. The quantitative NMR could be another option.
Purity estimations using NMR can be performed even in the absence of standard of the same compound.
Check this link: http://mestrelab.com/blog/purity-calculation/
I am surprised no one mentioned classical elemental analysis. It cant stand alone, but if you have other analysis (clean PDA, ELSD, RID chromatograms) Good looking NMR spectrum. Then I would consider my sample clean (95%+)
To determine the purity of an isolated compound simplest way use thin layer chromatography(TLC).
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