A typical formula often used for the concentration of an unknown sample concentration as follows:
Conc in ppm= (standard area/ sample area)* stnadard conc.* Dilution factor
Can anybody derive this formula? I would really appreciate that.
When use term standard that means you have agent same as your agent or is very very likely in structure with yours.
So in this formula you have known num, standard con, standard area(that software calculate for you neither do for sample) and dilution that determine yourself
what is the problem?
I cant understand dive formula
whats your point.
please explain more.
I advice you to make a standard sample first in order to get the calibration curve, then you can use the HPLC ( area under the peak ) to calculate the concentration of you unknown sample, this will be more accurate.
Dear Asif
Your formula appears to be wrong.
Normally you compare a peak area of the analyte from an unknown concentration sample with the peak area of a standard, but as it is difficult to always get the same concentration in the standard and unknown we generally use a calibration curve which is derived from running a set of different concentrations of the standard, which proves the linearity of the response and the fact that you are working within the detectors linear dynamic range. The choice of standard concentrations for the calibration must bracket the expected concentrations of the unknown samples.
This is generally performed on between 3 and 9 different concentrations of the standard plus a blank. A calibration curve is then generated from the data points with the peak area of the standard plotted against the known concentration for that data point. The slope of the linear regression line through the peak area versus standard concentration provides an average response factor (that should have a correlation coefficient of >0.95) that is then applied to the peak areas of the analyte from your unknown samples. This calculation is included as part of most vendors modern control and data collection software and all you must do is identify the standards as such and give their concentrations before they are run.
The concentration used for the standard is in the same unit that will be reported for the concentration of the unknown (PPM in your example).
You need to take any dilution factor used in the preparation of the samples into account hence the dilution factor term in the formula. A 1/10 dilution of the sample prior to analysis would equate to a final concentration in the unknown sample being 10 times higher than the diluted aliquot analysed.
The response factor is the ratio of the peak area over the concentration for the standard therefore your formula should be:
[C]unk = (Peak_Areaunk/Response factorstd)/dilution factor
I would suggest you look up the ICH and FDA guidelines for performing quantitative HPLC assays. They are both available on the web.
Nice job Duncan! In addition Asif, recall that the peak area of the analyte is the equivalent concentration response on the chromatogram which is dependent on standard response factors.
Try to lookout for further guide in "performing quantitative HPLC assay by ICH and FAD" as recommended by Duncan.
I wish you best.
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