unknown volume hplc

Basic questions from students; resources for projects and reports.

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I have a known concentration and peak area is it possible to work out the volume?
Perhaps. It would be;

Concentration (let's say ug/mL) x Injection Volume (in mL) = ug (Mass)

where 1 mL = 1000 uL

Peak area has nothing to do with the calculation. Remember, HPLC is a 'mass' device and not volume. Why do you want to know?
To calculate the actual volume of the peak you will need to know the detector response versus the concentration of the target molecule. It should be a linear curve.
There's more than a bit of ambiguity here:

1. Where/how is your "known concentration" determined? Is it the concentration of the sample that was injected? Is it the concentration at the peak maximum (and if so, how did you determine it)?

2. What units are you using for peak area? Integrator counts? mV?

3. What volume are you trying to determine (the volume injected? the volume of the peak itself?)

4. If you're after the volume of the peak, what fraction of the analyte do you wish to account for? Chromatographic peaks are, to a good first approximation, Gaussian. The "baseline" width of a peak is approximately 4 x sigma, and so includes roughly 95% of the area. Is that what you want, or are you looking at 99%, or . . . ?
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
i have a calibration curve of conc vs peak area
I am trying to determine how much volume of a drink you have to consume to be toxic (500mM)
the peak area units is (iss)
Ok. So you inject a sample of your drink and measure the peak area of the compound of interest. Use your calibration curve to convert that to concentration.

Concentration is mass (or moles) divided by volume. You should be able to take it from there.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
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