Hi, Everybody,

I would like you all to discuss this issue. When peak purity ie. area percentage by HPLC is lower than the value got from its assay method, what are the possible reasons other than one use impure standard? Thank you very much in advance.

1. May be the assay method is not specific , some unknown impurity might be eluting along with main peak.
2. May be the wavelength selected for purity and the assay method are different and hence the response factors of the impurities.

Hi,

Peak purity is a term that is usually used for the results of tests carried out by some special algorithms (evaluating data from different wavelengths and/or DAD data. Peak purity should prove that no other compounds elutes together with the peak of interest.
Purity of a compound can be examined by summarising al peak areas and calculation the fraction of each peak. I think thats what you mean.
Often methods for purity and assay are optimised for the intended purpose. When you do assay at 250 nm (UV maximum) and purity at 210 nm (more universal) I would be suprised if you would get the same result. What are your detections?
If your chromatographic methods are different (solvent, gradient, column etc) then the assay method might not be as specific as the purity method (Well, it should...Look into the validation documentation.).Are the methods different?
Standards itself usually have Certificates of Analysis that state purity, content, residual solvents and whatever is appropriate. Do you have CoAs?
Please provide more information on the method, detection ... when asking a question, that makes it easier to answer.

purity at 210?I always thought that purity was calculated comparing many spectrum from sample peak(spectrally pure) ?

Yes, usually people talk about peak purity refering to DAD data. The result will tell you if the sample peak is spectrally pure and no co-eluent. But, here I think he is talking about peak area percentage of sample peak vs. total peak area.

It is not clear to met from the original question what is being asked and whether DAD data was being referred to at all. Perhaps what was meant is a comparison of (1) an assay of a component versus an external standard, to (2) the area percent of that component in the chromatogram?

If so, then the answer could be very simple. The response of a detector (including UV detectors) is not the same for different materials. So the area percent in a chromatogram cannot generally be relied upon to give a meaningful number.

UV data cannot be used alone to determine the purity of a peak. If there are 2 perfectly co-eluting peaks, UV alone will not pick up the impurity. If the two peaks have the same spectra, again, UV will can not be used to GUARANTEE that the peak is pure. MS is the only way to guarantee that a peak is pure, UV will not prove that beyond a doubt.

I have to disagree with Anon 09/09/02. Peak purity with MS has no guarante either. The impurity might not ionize, my scan range might be outside my impurity MW, adducts and other artifacts masses can blur the purity picture as well. In low resolution instruments, many compound can have the same nominal mass. The moral of the story is simple. It always takes a variety of techniques (PDA, MS, ELSD, gravametric) to really get a handle on purity questions.

Anon from 10Sep02 is absolutely correct, but back to the original problem. I am making the assumption that the assay value is due to more than just the variability of the standard/sample preps and the HPLC system. The area percent of the sample may look better than it is because there may be impurities that do not show up on LC. The sample may have impurities (salts, water, res. solvents, etc) that the UV will not show. Based on the HPLC area percent alone will not account for these types of impurities and the sample will look more pure than it actually is.