# How to calculate the percentage purity using the HPLC?

Any standard I need to use for the calculations? if yes, than which standard, and do I need to run it along with my sample?

How to perform this analysis?

I have run HPLC for my column purified D-gulucose gallate. In HPLC two peaks were analysed for alpha and beta isomers, I'm interested in calculating the percentage purity of my compound.

Any standard I need to use for the calculations? if yes, than which standard, and do I need to run it along with my sample?

How to perform this analysis?

Any standard I need to use for the calculations? if yes, than which standard, and do I need to run it along with my sample?

How to perform this analysis?

- Without Standard, I think that you can not get a 100% black or white answer. With your analysis in hands, the best you can do is to integrate the 2 peaks. An NMR could be of great help. With HPLC, I found that one great trap is what you see can be far from what you get! What sort of detection are you using?

Currious to read some comments. - Here is a discussion from some time ago, it might help

https://www.researchgate.net/post/Can_anyone_explain_when_to_use_an_external_standard_and_when_to_use_an_internal_standard_method_in_HPLC?exp_tc=tprc - The only way to get an accurate purity is to compare your sample to a standard of well known concentration and composition analyzed in the same sequence of runs. However, many compounds do not have well characterized standards available so that is difficult. You can craft your own standard(s) if you have the proper instruments available (MS, HPLC, NMR, GC, and LOD/KF). You should not trust the percent purity of compounds as declared by chemical distributors like Sigma-Aldrich and others. They typically only use one method of analysis and most of the time it is a simple area percent and not a true purity value. You may be able to find standards for your material, but it sounds like probably not. If you cannot buy or make a standard then your answer will be an approximation based on area percent by HPLC. Hopefully you also have the capability to perform a residual solvents analysis, a mass spectral analysis to look for other components that might not be visible to your UV detector and a loss on drying (LOD) at a minimum if you want to try to make your own standard or at least baseline the process.
- Assuming that you do not have a suitable internal standard you could perform a standard addition experiment.

Required is the D-gulucose as reference >99% pure, of which you might want to add a known amount to your product. By subtracting the standard addition area of the neat area you will get a one point calibration which in turn you can use to determine the purity of the neat product.

Creating a standad addition calibration curve of at least 5 points is from an analytical point of view the best, but is rather time consuming. - For purity assay based on peak area analysis three approaches are recommended:

1. Normalization procedure - just calculate percentage of peak area in relation to total area of peaks under interests or in relation to main peak (in case of small impurities to be determined). On the background there is an assumption that (A) extinction coefficient at given wavelength is the same for main peak and all impurities and (B) detector signal is linear in a conc. range between main compound level and impurity level (eg. 0,1%) that is not always the case.

2. Purity assay in relation to main compound content - run your test solution to integrate all impurity peaks; next run test solution dilluted eg. 100x which corresponds to 1% of test solution (1% reference) ; calculate the content of impurities refering to the reference; the dillution of your test solution should be adjusted to expected level of impurities. On the background there is an assumption that extinction coefficient at given wavelength is the same for main peak and all impurities. The advantage of the approach is that U can treat the dilluted test solution as X% reference regardless of its initial concentration and/or purity and U don't need any other standard. For final result it should be stated that the value is expressed in relation to main compound content. The approach is very often applied in Pharmacopoeias.

3. Purity assay in relation to external standard of the impurity - well known procedure with use of qualified material of the impurity requiring weighting, dilluting to the level of the impurity, and calculating against the signal obtained. The method is the most accurate in terms of chemistry if individuals determined, but can be limited with impurity standard availability and/or price and uncertainty of its purity evaluation.

None of above described methods is preferred comparing to others. You can choose anyone but taking into account all assumptions, advantages and disadvantages. - NMR could help
- For quantitative analysis we need a standard compound, but can we found the D-glucose gallate compound as a reference. Later on, you will found HPLC quantitative analysis method in some chromatography journals
- Prepare a known concentration of your sample. Run HPLC, calculate peak area and concentration from the calibration curve. Purity = (concentration calculated/prepared concentration)* 100%
- First prepapre the calibration curve using standard solutions of the analyte in a particular mobile phase. Then take extracted or purified sample's HPLC chromatogram for its peak at the retention time. Then find out the concentration of the analyte in the extracted sample using linear equation. The formula of % purity can be applied.

% Purity = Amount of analyte found / Amount of analyte taken x 100 - You have to take commercially available standard of D-gulucose gallate. Prepare around 5 standard solutions starting from lower ppm level to higher and get the chromatogram for each solution. Note down the peak height and construct the calibration graph by plotting peak height versus concentration of D-gulucose gallate and then check the linear dynamic range. Now separately you have to prepapre the analyte of D-gulucose gallate and up load in the column separately and get the chromatogram. Now the % purity can be calculated on the basis of Amount taken /amount foound and then multiplied by 100.
- Thank you so much to all teachers on answering and guiding me,

from all answers i got the method of calculation and make it try...

Different methods i learned to calculate the purity,

i appreciate you all..thanks again - Besides of what some people have posted you might need to determine the peak purity of your sample. That is for an isocratic run and assuming that you have a multiwavelength detector and your peaks are well resolved you have to determine if the absorptivity ratios at different times within your peak at different wavelengths are similar. If they are then you should proceed to determine purity as stated by other people here.
- Make calibration curve by standard with at least five points (concentrations) without internal standard (not necessary in this case) from the linear equation you can calculate the real peak areas for three controls of calibration curve and then inject the sample with the same three concentrations of controls and then calculate the purity for each control and then take the average

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