HPLC chromatogram

[mandytan]

In a HPLC chromatogram for a standard, the peak area is 20000.
2000 times dilution of the standard gave a peak area of 8.

The peak area of this diluted standard is used for impurities limit test (i.e. the area of any impurity must be less than the peak area in the diluted standard, which in this case it would be 8. )

Theoretically,a 2000 times dilution of the standard should give an area count of 20.

Is this huge difference in area count (8 versus theoretical 20) due to the large dilution factor?

[bisnettrj2]

Pardon my math, but isn't 20000/2000 = 10? So a peak area of 8 = pretty darn close to expected, especially when a peak area of "8" has to be an extraordinarily tiny peak?

[mandytan]

Oops, I'm sorry for the typo error. The peak area of the Standard chromatogram is 40 000, so the diluted standard should have a theoretical area of 20.

[unmgvar]

have you done a linearity test?
either the compound chromophore is not linear
or the detector.
or the column.
you also have to take into account the precision of the sampler
and the precision of your dilution steps

what is the peak height of the standard
and what is you dilution procedure?

[tom jupille]

And what is the repeatbility of that low level peak (mean and std deviation of 5 injections). You may be below the limit of quantitation