Quantitation with no standard

[mardexis]

Hypothetically, if I know the molar absorptivity of a component in my chromatogram then how reliably can I quantitate based on the peak area? If at all?

For example let us say that I inject 10 uL via partial fill mode injection, ie, really 10 uL. Flow rate is 500 uL/min. Molar Absorptivity is 77300 AU/M/L. I measure a peak area of 1000 mAU.S

Can I calculate the concentration? Can I do it reliably and reproducibly? Are there folks out there who do this type of quantitation regularly? ie, use peak area and molar absorptivity for quantitation. Downsides?

Thanks

[HW Mueller]

You must have seen the discussion on injection accuracy. Such problems have to be elucidated with a standard. So, theoretically, it is possible, in practice: don´t trust it. I have used absorption data to calculate expected area to check whether I am in "the ball park" with recovery, etc. That can be very useful, but it is a semiquantitative affair.

[lmh]

If you don't fancy getting bogged down in arithmetic, and you want a ball-park calibration, another option is to run a proper standard curve with some other standard whose extinction coefficient you also know. Obviously the more closely related the second standard, and the more similar it's wavelength maximum etc., the better. Then correct your half-calibrated results using the ratio of the two extinction coefficiencents. It's still far from ideal though.

[tom jupille]

In principle, it should work. Think of a chromatogram as a plot of absorbance (y-axis) as a function of volume (x-axis). The area under a peak would have units of AU-mL (AU times milliliters). If the absorptivity has units AU/(millimole/milliliter), the dividing peak area by absorptivity should leave you with units of millimoles ("demonstration is left for the student as an exercise." ).

In practice, it's more complex, because peak areas are usually reported in units of voltage x time and the cell path length has to be accounted for. I concur with HW: OK for an estimate, but I wouldn't want to bet on it being exact.

[ksharp]

In case you're not already convinced that this practice works in theory but not that well in the lab, I can tell you that I tried it to impress some co-workers and it was ball-park at best.

I ran some samples against a calibration curve, and then took those samples' peak areas to correlate with extinction coefficients. My results were within 15%. Sorta close, but not good enough.

[oscarBAL]

there is a correlation betwen the slope of calibration curve and extition coefficient but in the most of the cases did not work (I tried a coulple of times). I faced the problem of not get an STD a couple of times so I tried to "develop them" by my self; how?
Elemental analysis (you have to have a lot of sample and high concentration ) I tried to take my molecule and analyzed by Titration, TN, TP, TCl or any other. My advntege it was that the most of the time I got raw materials.

For example if have a molecule like C12H15N3O2S and Total Sulfur and total Nitrogen or nonaqueosu titration correlate with the formula (kids don't try to do this in your ouwn labs) and my chromatographic peak was very clean then I used to stimate "purity" with a good degree of confidance; well this was "true" only if you do not have S or N coming from other compunds wich is hard to say and you do not have guarantee about degradation.

[Bruce Hamilton]

One more problem is that many of the reported extinction coefficients are not for the composition of the mobile phase at the time your sample is eluting, and can some values can vary with solvent, and you also need to consider the spectral bandwidth of the respective instruments.

I'd say the technique is useful, and should be quantitative, provided all potential issues are understood and addressed, but I'd only consider using it as a last resort, and with molecules with large absorbance and peaks well above 230 nm.

After all, the external standard technique is similar to your proposal, but performed on the same instrument.

For a few decades, before HPLCs were common, pharmacopoeia used a reported UV extinction coefficient for the assay calculation of active ingredients, even though some impurities could have similar values.