Dear Robert Mosley

I worked on this device in the USA

It's a great device.

You can change the wavelength after completing the scan

Even when the device is turned off

Just use the program and change the wavelength until you get the wavelength that shows the peaks appropriately

Greetings.

see this picture Robert Mosley William Letter Dhifaf Zeki Zahraa Kamil Kadhim Lawi Maysaa Adil Hadi

You should begin your initial method development by running each of the two standards separately, for UV/VIS characterization. Use a column compartment to maintain analysis temperature. Select a column and mobile phase that can retain both compounds well. The two compounds are very different, but one advantage in analysis is that their UV absorbance is also extremely different so be sure to use a scanning diode-array detector (required for most types of HPLC method development) to store their spectra for comparison and help in qualitative ID. *Different extinction coefficients = different response. Follow the basic guidelines of HPLC method development (get some training and help), retain, separate, resolve and elute. *There are plenty of free articles on the web to use as examples for each compound (keyword search) so compare those methods to see if one would be complimentary to both compounds. Learn to use the web (it is a tool) to conduct research and explore options on your own.

Dear Robert Mosley

I worked on this device in the USA

It's a great device.

You can change the wavelength after completing the scan

Even when the device is turned off

Just use the program and change the wavelength until you get the wavelength that shows the peaks appropriately

Greetings.

see this picture Robert Mosley William Letter Dhifaf Zeki Zahraa Kamil Kadhim Lawi Maysaa Adil Hadi

Nasar: the "document" you posted is not relevant to this discussion. There is no "device" and no information provided. It is just a chromatogram. Single or multi-wavelength UV/VIS detectors are well know in this field and used by most chromatographers (they generate chromatograms like you have). If you are interested in learning how to perform HPLC method development to qualitatively ID different UV/VIS active compounds, please include a scanning diode array detector in your HPLC system and use it to take spectral scans of each retained compound (that is what this discussion is about) to aid you in ID. This is usually performed during the method development process and should be included in anyone's training if learning HPLC method development (learning how to use a scanning DAD / PDA). Perhaps someone at your school (or another school) can assist you?

Dear William

I respect your objection to the researchers' answers in any way, however, I am followed you.

I think you did not understand my answer and the document (that was@ just for example).

The answer was intended to work on these two compounds using the WATERS HPLC. What distinguishes the agilent company from that it can choose the wavelength that shows the two compounds well .......

And through my work. The appropriate wavelength can be chosen to show the two compounds even after the examination has ended simply without the complication of the answer

This is through my work.

I respect your objection to my answer and respect your answers no matter what William Letter

Best regsrds

Naser: Your initial answer had no context or information. Your follow-up has provided context, but I strongly disagree with what you suggest as a possible solution.

The brand of HPLC is not important as any of the Waters, Agilent (or other quality brand) of scanning DAD units can find and show multi-wavelength spectra for different compounds through their software (CDS). When two different compounds are present in the sample, with different absorption maximum and spectra, the HPLC system should be set-up to collect and store the two wavelengths separately and not use a compromise signal. This preserves Method SELECTIVITY, a basic HPLC fundamental guideline. That is one of the key advantages to using a scanning diode array detector and what we teach as the process of modern HPLC method development (multi-wavelength acquisition is selected AFTER the use of the scanning DAD feature has revealed where the absorption maximums are for each compound). For sample with chromophores, HPLC method development should always include a scanning diode-array-detector for the 3rd dimension of data it offers (UV/VIS spectral data). This 3D data is used to help qualify compound ID's (an additional orthogonal dimension), keep track of different samples, improve the chances that other compounds, if present, can be detected to improve the quality of the data collected. For maximum value, the scanning mode of the DAD detector should be turned 'ON' all of the time during the analysis.

For two compounds, it is not wise to select a different HPLC wavelength which is based on a compromise between two separate spectral profiles. Anytime an impurity is present in the sample, there is a strong chance you may not 'see' it or it will contribute in a positive or negative change to the spectra, resulting in inaccurate determinations of purity or concentration. Such methods would be considered in-valid by most regulatory authorities. We want to avoid this. It is specifically because of this concern that we do not use "compromise" wavelengths in HPLC method development, but instead select the best spectral apex maximum values (with appropriate bandwidth value) for each compound. This allows for independent compound calibration at the appropriate wavelength leading to the best linearity, lowest noise (high S/N) and highest selectivity (via: compound multi-level calibration). Most CDS packages allow the user to easily input the multi-level calibration data for each compound, at the chosen wavelength, to make identification more accurate.

"Proper Wavelength Selection for HPLC Method Development (or Purity Determination)"; https://hplctips.blogspot.com/2013/08/wavelength-selection-for-method.html

"UV / VIS, VWD, DAD, PDA HPLC DETECTOR SIGNAL BANDWIDTH (bw)"; https://hplctips.blogspot.com/2011/09/uv-vis-hplc-detector-signal-bandwidth.html

Additional to Williams great advice, you could try gradient instead of isocratic elution. This might improve the peakshape of Daunomycin and thus allow for detection at lower concentrations.