Adam peak purity allows one to determine whether or not they have a co-eluting peak. Blank subtraction such as you are doing can be done by making up the standard in the matrix to start with. Better yet do SPE and remove the matrix.

I'm not a chromatography expert, but in order to get an answer that's helpful you should maybe post an example and specify what's your problem with the interpretation. Don't forget to include an internal standard.

Hi Afroza Bulbul

Kindly elaborate your query

Regards

If your device equipped with Chromeleon Data System Software, you can easily get the peak purity from the analysis report.

Sometimes a peak on HPLC may contain two or more compounds, if you use diode array UV-visible detection, try to use the collective spectrum function to see spectrum on every point on the peak to see: are the spectra on all the points the same, then this peak is same compound, if not, the peak may contain two or more compounds (sometimes may be only just buffer, you can tell from the spectrum). Hope this can help you a little!

Or may be you are talking about the purity of a peak in a HPLC trace as a whole peak, then use intergration, can be done by the HPLC program or you can do it manually .

Hi Afroza,

I would agree with Nidal's response. Your system software (Chromeleon CDS?) should be able to determine peak purity as long as you are running full spectrum analysis for your test method. By the way, what is your detection method? UV-Vis, DAD, other?

If you have specific details, you might contact Thermo Fisher technical services directly.

Regards,

Chris

While I don't know the details for accessing peak purity in the software you are using, all peak purity measurements are derived from the same algorithms. The information below will help you understand the limitations of these techniques.

We do peak purity on the instrument by comparison of different parts of the spectrum.

The easiest method is the ratio, which measures the absorbance at two wavelengths, then divides the absorbance at one wavelength by the absorbance at another. If the peak is pure, the ratio is a constant value. This technique has a few limitations:

Another method is looking at the entire spectrum- usually using vector analysis. It is the mathematical equivalent of Dr. Xiang’s suggestion of looking at the UV-vis (or other type of spectrum). The advantage of this method is that it avoids issue #1 above since the entire spectrum is measured. It also seems more sensitive for related compounds (issue #4 above). Some of these techniques were applied to mass spectra. See the references in the link below for more details of the math behind these techniques.

https://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Information%20Rich%20Flash%20Chromatography%20II%20All-Wavelength%20Collection%20and%20Purity%20Measurement%20App%20Note.pdf

You are not supplying any specifics regarding the mode of detection. If you are using a DAD the instrument should come equipped with software to allow a look into your peak purity. As Chris said, contact Thermo with questions regarding the software.

Wow, I have learned a lot from these expert answers. I normally make sure I run a blank in the exact same sample matrix as the sample. If this is possible your answer is simple to obtain. This may not be possible if you are dealing with an endogenous compound in a biological matrix. For example, glutathione, which will not be possible to have a true blank in sample matrix if you are studying blood.

Adam peak purity allows one to determine whether or not they have a co-eluting peak. Blank subtraction such as you are doing can be done by making up the standard in the matrix to start with. Better yet do SPE and remove the matrix.

First, "Peak Purity Determination By HPLC" (specifically by use of a diode array detector) is a null test at best. It can only show possible impurities which may be present, but can NOT show true purity. As used by most HPLC instrument vendors, it is subject to many limitations and assumptions (such as all possible compounds absorb light with similar extinction coefficients, which is of course not realistic).

I teach the use of this feature on several software platforms and perhaps the two most important aspects of using it are: (1) That you have a great deal of experience and training in HPLC analysis and operation of the HPLC system and (2) that the analysis method used has been fully optimized and reviewed that it is in compliance with following good chromatography fundamentals (e.g. Proper K prime's for peaks). This software feature is an advanced feature and can be easily manipulated to show any purity value you want. It requires formal training to use. Good documentation of the method settings and proof that the method is selective for the given compound is required before use (which means that a typical Method Validation procedure is not enough).

For more information on this topic, here is a link to a free, short article I wrote on the topic from my classes. Perhaps it will assist you with your questions.

• "Peak Purity Determination by HPLC Diode Array Chromatography Software: Limitations and Uses"; https://hplctips.blogspot.com/2016/12/peak-purity-determination-by-hplc-diode.html

Each HPLC vendor has their own definition of 'peak purity' and thus the equipment will give a different answer. While 'peak purity' reduces the possibility of a co-eluting peak (probably a molecule or related substance that is structurally and chemically similar to your molecule of interest) it cannot detect a molecule that is invisible in the UV like a 'common salt'.

Do you know the absorption maxima of the the analyte(s) corresponding to the peak(s) you would like to determine the purity for? That might help.

Peak purity analysis (PPA) in chromatography is to see that is chromatogrpahic peak eluted is attributed you analyte peak only and there is no interference from other peaks. You require PDA detector to determine the peak purity. Following is link for to download the chromeleon software tutorial where you can find how to determine peak purity.

http://tools.thermofisher.com/content/sfs/manuals/Man-Chromeleon-670-EN.pdf