From this peak table how we know our sample purity? Am analyzing Synthetic peptides and proteins. After HPLC we are doing MALDI,ESI-MS etc but from HPLC what what info we can able to find, please help me Experts to give clear info for my understanding....
Hi
In HPLC we do separation and quantification of the components of a mixture of compounds regarding certain UV_VIS light absorption and /or Fluorescence activity of the compound of interest, and we must focus on the compound of interest before we start our experiment and that's why we need pure compound to develop the HPLC method and optimize it before running any samples containing that compound.
The results of the sample mixture will refer to how much compounds you have in that mixture (no.of peaks you got in the chromatogram) and this is the qualitative part of the results i.e the more peaks you got the less pure sample you have, the peaks that appear earlier means they are more polar than you compound in most instances where your mobile phase contain more organic phase, and using the time of retention of the peak you will be able to know which one is corresponding to your compound by comparing to the pure compound run under same conditions. While the area of each peak will refer to the concentration of that compound in the sample according to the standard curve you already have made, and the area percent give you an idea about how much the compound of interest constitute of the total sample in test.
While the height of the peak will have little to do with the concentration relationship but it has a great role to understand the separation efficiency of your method the highest and sharpest peak means it has been separated very well from others and it also refer to the high selectivity of your method to separate and quantitate the compound of interest and the height percent will accordingly refer to which compound has been separated efficiently in this method, the wider peaks and low height are normally not counted as well separated as it may involve more that on compound in it.
if you can collect your compound after its separation by HPLC it would be very efficient method for the purification as well.
Hope these information will be helpful for you.
Regards
JUMAH
Dear Selva, You have combined a number of questions into one!
1. Peak area and Peak height in sample - If you also have run the pure reference compound by HPLC under the same condition, peak area and peak height can be used for quantitative estimating of the compound in your sample.
2. Peak area % and Peak height %: indicate the % of the peak component among the total 'detectable' peaks (constituents) in the sample chromatogram (taken as 100%). This is a relative estimate. For example, if you use a single wavelength (UV) detection, all the detected compounds in that wavelength show up as peaks in the chromatogram. It does not mean that all the compounds in the sample matrix are detected. There may be compounds which do not absorb in that single wavelength and may not be seen in the chromatogram. Hence, the peak area % and peak height % are relative to the total 'detectable' constituents. This will not give accurate quantitative estimation of a given peak as in 1.
3. Regarding your 'question' on sample purity - are you referring to sample purity or peak purity? If you have got only one peak for example, you can check the peak purity by over-laying the UV spectrum (if you use a PDA, say from 190 nm to 400 nm) at different points of the peak. If you are using an MS detector, you can overlay the mass spectrum at different points of the peak. This will give you some indication of the peak purity.
You need to clarify what you mean by 'sample purity'
Good luck
Dear Selva Chemist,
1.Peak area shows how much of your sample concentration, that means utilized for UV Detection and also your particular sample so you just conduct reference experiment and compare this for your understanding
2.Peak Height Shows your target concentration that means how much your target in your sample.
3. If You get sharp single peak and 100%peak area means your sample is >99%pure
Hope You Clear...
Regards Sridharani...
HPLC. purification and separation technique, from that Peak area % you will get your target peak % that means your target in total sample.,
Peak height % shows your compound purity concentration.,
For Separation and purification purpose you use HPLC for other info you go for other analysis..
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