How to design a purity test using HPLC

[winnielee]

I was asked to modify an in-house reverse-phase HPLC method, which determines the concentration of an analyte, so that the new method can check the purity of that analyte in an unknown substance. I've come up with the following test procedures:

(1) Inject a solvent blank, which is used to dissolve the standard in (2).

(2) Inject a standard with known concentration.

(3) Inject a sample (i.e. dissolve the unknown substance in the same solvent as in (2).

(4) Compare the chromatograms obtained in (1), (2) and (3).

(5) Result intepretation: Peaks appear in chromatograms (1) and (3) will be disregarded as they are believed to be generated by the solvent or mobile phase. Refer the analyte peak to chromatogram (2). Peaks only appear in sample chromatogram other than analyte peak and will be treated as impurity peaks.

(6) Purity calculation: Determine peak area percentage of peak analyte in sample chromatogram.

Can any one of you experts give me some advice on the above method design?

[tom jupille]

The problem with that is that it ignores impurities/adulterants which do not show up in your chromatogram (as a simplistic example, the product could be cut with 50% salt, and you would never see it with that procedure). A better approach would be to prepare a standard at a known concentration (let's say, 1 mg/mL) and measure the area of the (hopefully!) single analyte peak. Then prepare your sample at the same concentration and measure the area of the analyte peak. The ratio will give you the purity (e.g., if the analyte peak in your sample has half the area of the standard, then the sample contains half as much).

An even better (if more time consuming) approach is to run your standard at several different concentrations and generate a calibration plot of peak area vs concentration. Hopefully it will be linear, in which case the slope can be used as a response factor. Run your sample and divide the analyte peak area by the response factor to get the concentration of the analyte.

[winnielee]

Thanks!

Could there be any possibility that the peak area of sample is greater than that of standard, which gives a >100% purity?

[tom jupille]

Any measurement has a certain margin for error, which you can determine by running replicates of your standard and your sample. If the results are significantly higher, that suggests "pilot error" in preparation or the possibility of an unresolved impurity which has a higher response than your analyte.

[winnielee]

Your advice is very helpful. In fact, your suggested procedure is simpler than my original proposed one. We will try it and see how it works.

Thank you for your very kind help.

[monsef]

When comparing chromatograms, if you decide to go with a peak % purity, make sure of specificity. I.e. you could have impurity peaks hiding underneath the solvent peaks. Inject at same concentration as sample and compare area counts and peak profiles. Usually a double angle is take on these types of methods.

1) The concentration % purity as suggested above
2) Chromatography profile or new peaks seen

[prepcolumn]

I was asked to modify an in-house reverse-phase HPLC method, which determines the concentration of an analyte, so that the new method can check the purity of that analyte in an unknown substance. I've come up with the following test procedures:

(1) Inject a solvent blank, which is used to dissolve the standard in (2).

(2) Inject a standard with known concentration.

(3) Inject a sample (i.e. dissolve the unknown substance in the same solvent as in (2).

(4) Compare the chromatograms obtained in (1), (2) and (3).

(5) Result intepretation: Peaks appear in chromatograms (1) and (3) will be disregarded as they are believed to be generated by the solvent or mobile phase. Refer the analyte peak to chromatogram (2). Peaks only appear in sample chromatogram other than analyte peak and will be treated as impurity peaks.

(6) Purity calculation: Determine peak area percentage of peak analyte in sample chromatogram.

Can any one of you experts give me some advice on the above method design?

Is your sample containing just your analyte (eg: raw material) or is it in a matrix (eg: raw material +placebo)? If you have the second one you have to compare the effect of other materials in ypur matrix to your chromatogram..