I think you do not need to run the HPLC with their max UV for A and B. For A you can select UV maximal, but for B please select its UV minimal (if A and B have different UV spectra),  I think you can change the wavelength during the analysis by programming  

Depending on the detector, you can might change the detector sensitity during the measurement (probably, this can be programmed in the method file that you use). This works e.g. for UV and for fluorescence detection in our HPLC machines.

Best, Christoph

Dear Christoph

I tried this but unfortunately the two components have the same UV maxima!

Each detector still has an internal amplification (gain factor, or sensitivity factor), which can be entered in the method file. If you programm your analysis that up the first peak the detector is rather insensiive and then is switched to sensitive (or vice versa) this will shift the areas. Similarly, you could try to measure the high amount compound at an unpreferred wavelength (different from the optimum), which should decrease its area as well.

I would back Christoph answer, but despite being uncomfortable to see, I wouldn´t  bother with this difference which of course reflects just the different response factor of the two analytes. Using the same scale for both is still the best way to get a proper feeling for the response of the 2 analytes.

Try to switch wavelenght during the analysis.

Some detectors and data processing systems have an option to show chromatograms in log scale. If you have this option it will be better than changing analysis conditions, because you can straightforwardly recover the original data.

I think you do not need to run the HPLC with their max UV for A and B. For A you can select UV maximal, but for B please select its UV minimal (if A and B have different UV spectra),  I think you can change the wavelength during the analysis by programming  

Nidal:

Outside of appearance, why do you want to change conditions? Don't the peak areas reflect the relative amounts of compounds? If this is an established method, wasn't the difference seen initially?  If not, is "A" an impurity or degradation product?  Of course, you can always switch detectors (e.g. RI, PAD, etc),as this may change relative peak areas, but this may not be allowed if it is a USP or established procedure. Do you know the identity of A and B?

Dear all, Thank you very much.... for more details ....I'm in the stage of method development, both analytes are API's and thus are identified, I know that the big difference between responses is due to the big difference between concentrations in my drug sample, (the concentration of A is less about 30 times than that of B), but unfortunately the two analytes have the same UV maxima! till now method can be accepted for me as it is and I can go ahead, but I am still in the stage of method development as I mentioned and I am asking if it is technically possible to see both peaks in a convergent sizes or not.

Hi Nidal,

if both components do have the same UV absorption (did you measured their spectrum?) and their concentration varies that much, it is not possible to match their integration area (which is somehow related to their contration and absorption at that certain wavelenght). Other than that, why would you do that if they in fact have a concentration difference? Thats what is usually wanted to show with the analytics. If it is just "uncomfortable" for the "chromatogram appearance", this should not be the first consideration.

You could monitor the UV spectrum during the run (works with most UV detectors), so that you have a spectrum between e.g. 200-800 nm for every second of your run which makes it easier to find absorption maxima for your compounds.

Dear Nidal,

Both paragraphs from Marcus Binner above are quite correct.

Please attempt the suggestion (200-800nm) in his second paragraph.  If you still see the "big difference" then this is what you need to accept because the concentrations are different as you mentioned earlier.

I think Gunawan has answered your question precisely.

I agree with Gunavan and Marcus, and as Brandon says if still there is "big difference", no way you have to accept and considerer different concentration 

I think it is useful to distinguish two aspects of the problem: the first is to obtain a quantitative analysis, the second is the uncomfortable chromatogram appearance. Concerning the first one, I would do a check of the absorption maxima of the two substances, then you can change the wavelength during the analysis by programming, as Gunawan suggests (or run two different analysis if the HPLC you are working with does not allow this function). Concerning the second aspect, as other researchers have already said, I think that the "aesthetic" of the chromatogram can not be improved. May be you can present two different runs at different concentrations for each one of the components.

Regards

Better to follow with Mr. Gunawan

Hi Nidal,

First of all you should check the response factor of both A and B in certain wavelength and check the UV maxima. If maxima are different then follow Mr Gunawan or you can choose a wavelength where both the peak having almost same response. 

Please check before injecting the hplc system both A and B maximum wavelength by using  UV spectrophotometer   (entire  UV range 200 -400 nm) based on the maximum obserbance of both A and B (if both  showing maxima at different wave length) you can go for dual mode HPLC system by selecting dual wavelength then we can  get maximum area in both A & B or another way to increase A concentration or decrease B concentration then you can get similar areas.

While this seems to be primarily a question of convenience, it is certainly interesting and has clearly initiated a lot of discussion and several suggestions.

@Nidal, I am very curious about what you finally decided to do about this issue, if anything at all (and why). I am sure everyone would be interested in an update from you.