Chromatography Forum

Posted: **Mon Jun 26, 2006 9:37 am**

One more thing: If the regulators accept that DAD gives "assurance that no other impurity peak is co-eluting" than the word assurance has been corrupted officially, as assurance should mean the same thing as certainty to the best of human endeavor.

Posted: **Mon Jun 26, 2006 10:42 am**

I have assumed (maybe wrongly) that four compounds were extracted from the herb and prepared as pure isolates whose identities were confirmed spectroscopically (so far so good).

The purity of the four isolates was determined by running them on HPLC and measuring UV absorbance at three wavelengths. I assume that the purpose of this was to use the isolates as calibration standards for determination of the concentration of the compounds in herb material.

The referee is asking for details on how three UV wavelengths were used to calculate purity. I would also be interested to know this because I think that it is impossible, as do others posting on this thread.

Even the absorptions at all wavelengths across the peak (which is what "peak purity" does) cannot be used to calculate the purity of the substance that was injected to the HPLC.

The only way that UV (or any other) peak area alone can be used to determine substance purity is by comparison against a certified reference standard.

Peter

Posted: **Mon Jun 26, 2006 11:59 am**

Hi:
Recently I submitted a papper about the analysis of active compounds in medicinal herb by HPLC method.
Because the compouds could not be purchased, so we prepared them by ourselives. All these compounds were identified by direct comparison of their spectral data (UV, IR, NMR and MS) with those reported in the literature. The purity of the standards was checked by HPLC/DAD at three different wavelengths (210, 252, 300 nm), and the results suggested their purity to be above 98%.
But the reviewer gave us some advices as below:
"it indicated the purity of four standards (prepared by authors) was checked by HPLC/DAD and got a result of >98% purity. To the best of our known, similarity test of spectra at various places in a peak can be employed to calculate the peak purity, but it is imprecise, and there are many other methods of purity test on HPLC/DAD reported. So, the author should give some explanation on the calculation method of peak purity test, and some literatures involved expected to be cited."

Could you give some literatures or some good method for how to identified the purity of srandard substances

Hi,

I have gone thru your query and the comments posted. I guess the reviewer is either concerned about the PEAK PURITY of active ingredient or he is directing towards HPLC purity of the compound. PEAK PURITY can be determined from the software whereas for HPLC purity of the compound espeacially Reference std. you need to characterize the reference std and run it on HPLC to determine the purity of the substance on area% basis.

Regards,

Yogesh

Posted: **Mon Jun 26, 2006 2:01 pm**

Thanks a lot everyone:
An other question :How and what can I reply to the reviewer?

A friend said that the reviewer wants me to find publications published by him and cite them. But I do not know which one is the reviewer.
I do my best to find the literatures and want to solve this problem, but I fail.
I tested the reference standard with IR, NMR, UV and MS, all test results showed that the reference standard is pure. Especially, the grams of H-NMR and C-NMR are very clearn.I realy want somebody give a clear explanation to direct me what experiment should I do.

Posted: **Mon Jun 26, 2006 3:45 pm**

Reading your original statement again it sounds like the referee doesnÂ´t doubt that your standards are pure, but rather wants to know why you did this three wavelength thing and how you arrived at the 98% from that. He apparently thinks that DAD software can do a better job than this, thus he wants a ref. to see whether someone else has an argument for your method (or else he might be affraid that an official method is behind your calc. method so doesnÂ´t dare to tell you to use a different calculation). From that I deduce that he is not interested in knowing what the possibillity is that your peaks are composed of additional unknown substances. To rule out such possibilities you would need different techniques in addition to your HPLC.

Chromatography Forum

How to identified the purity of standard substance?

Re: How to identified the purity of standard substance?