No such thing as a standardless analysis in chromatography. You can ratio to internal standards but without knowing the precise response relationship between internal standard and compound you will not achieve meaningful quantification. UV appears to be as you state but the reality is different than the theory. If you want quantitative results you must derive a calibration curve.

Are you talking about using TIC instead of extracted-ion current profiles for quantification? Yes, you can use TIC. Do you still need to run standards? Yes, you still need standards for quantitative analysis using MS. One issue you run into using TIC is that your peaks may not be well-resolved and thus you may end up with interfering compounds that do not show up using EICP quantification. That is the reason that most published methods using EICP - it is much more selective than using TIC.

Mark Krause thank you for your answer! And what about the ratio of mixture component? For example there are 3 peaks in TIC chromatogram, with integrals ratio 8/5/1, so, does it mean that I have 3 components in mixture with such ratio?

The intensity of the peaks you see in your TIC depends not only on the amount of material present but also on the respective ionisation efficiencies of each of the components and the effectiveness of your extraction and purification before the sample is presented to the MS.

Not really! You need pure internal standards to confirm the retention time and the detector response. You can try SIM (selected ion mode). However, the ratio you cited doesn't tell us much about the molecular structure and it's quantitation.

Bruce Neagle Charles H Hocart Mark Krause

To sum up: the signal intencity (and peak area) in UV chromatogram linearly depends on analyzed compound concentration (Beer–Lambert law). So, ratio in mixture of compounds that have chromophors with similar exctinction coefficient could be directly calculated from areas of peaks on chromatogram (example - mixture of peptides, detection wavelenght 210 nm, amide bound).

In the contrast, the signal intencity (and peak area) in TIC chromatogram depends on ability of analyzed compound to ionize and its tendency to fragment. The ratio of compounds in mixture could be determined in case if compounds have quite close structure (similar ability to ionize) or if internal standarts of all of analyzed compounds were measured.

Am I right?

Not really! While the molecule's concentration versus wavelength is linear, the slope can vary with each detector. The detector response can vary significantly! While the spectrum may be similar and you can make an assumption about an amide bond, you don't know if it internal or terminal. Similarly both Vitamin A Alcohol and Vitamin A Acetate have a phytol chain and thus similar spectrums but different activities.

No such thing as a standardless analysis in chromatography. You can ratio to internal standards but without knowing the precise response relationship between internal standard and compound you will not achieve meaningful quantification. UV appears to be as you state but the reality is different than the theory. If you want quantitative results you must derive a calibration curve.

Mark Krause Bruce Neagle

Thank you very much!

Can you recomend me some textbook(s) or review article(s) about practical aspects of HPLC especially with MS detector? Seems that my analytical chemistry knowledges need an actualization.

www.chromatographyonline.com

Hard to beat the advice and articles on LC*GC. Books take too long to get through the publication process. You'll find up-to-date information here.

No, its not rational to use TIC for integration without pure standards to see response levels. SIM/MRM would be the right approach in my understanding.

There is no such thing as standardless analysis in chromatography. You may have to simulate your sample with dirt + known standard sample. Thank you Mark Krause.

Every peaks of your sample chromotagram will carry one or many compounds. Overlapping of the compounds in the same peak is a common case in TIC chromatogram. U can differntiate the compounds by mass spectra but cannot calculate the area of different compounds in the same peak.