S/N response are generally considered as a basis criteria for the wavelength selection. In some cases researchers, in partice they take UV spectrum even though they are using HPLC with DAD Dectector. ...Is required or any scientific
Justification for it ?
Through UV we will get the spectrum and maximum absorbance of a sample, so that we can select the suitable wavelength. But in PDA with HPLC you will get the 'Maxplot' where the sample shows the maximum absorbance for each component (all peaks). That is the main advantage of the PDA detector. Here we will also get the λmax for all individual components of the sample at a time.
The λmax is the detection wavelength of any particular compound, After seleting the wavelength, we need to calculate the relative response factor for all the other impurities (at least known) as they might have different λmax.
Otherwise the wavelength needs to be select where we will get the similar purity of all the individual peaks, similar to that of 'Maxplot'.
Markus Sir
ya I agreed with your opinion using both is not required. ...If they have HPLC with PDA Dectector,using Uv spectrum as a preliminary step is not required.Although both give same information,better to go with PDA in HPLC. ....
You can take the column offline. Inject about 5 mL of the API in solvent so the PDA cell is full and scan. Select the wavelength maximum.
Diode Array HPLC Detectors
Diode Array HPLC Detectors are most commonly used to record the ultraviolet and visible (UV-vis) absorption spectra of samples that are passing through a high-pressure liquid chromatograph. This enables qualitative information to be gathered about the samples. Applications for Diode Array HPLC Detectors include pharmaceuticals and life sciences, chemistry, energy and petrochemical industry, environment and agriculture. An advantage to diode array HPLC detectors is the ability to select the best wavelength for analysis. Some features to consider when choosing a diode array HPLC detector include resolution, wavelength range, NIR (Near InfRed) ranges, low noise, baseline stability, peak integration and an interchangeable flow cell design. Some vendors also offer detection done with a configurable fiber optic light path. Be sure to inquire about features that may be an advantage in your application field.
Through UV we will get the spectrum and maximum absorbance of a sample, so that we can select the suitable wavelength. But in PDA with HPLC you will get the 'Maxplot' where the sample shows the maximum absorbance for each component (all peaks). That is the main advantage of the PDA detector. Here we will also get the λmax for all individual components of the sample at a time.
The λmax is the detection wavelength of any particular compound, After seleting the wavelength, we need to calculate the relative response factor for all the other impurities (at least known) as they might have different λmax.
Otherwise the wavelength needs to be select where we will get the similar purity of all the individual peaks, similar to that of 'Maxplot'.
It is NOT just S/N ratios that are used to select a wavelength in HPLC for analysis. It's spectral position is also very important. This is why a scanning diode-array detector is used (if the solution was 100% pure, then a conventional spectrophotometer could be used).
For HPLC, we first separate and then analyze the sample peak using a DAD (aka: PDA) to determine the maximum peak wavelength in which we can reliably use to detect the sample with. We use the full spectral data obtained for each peak. Ideally, the wavelength selected will have little to no interference from other peak sample wavelengths and provide a strong signal with a narrow bandwidth. This is called the lambda max wavelength, "λmax". Each compound will have one or more. The UV Spectra obtained for each peak can then be used to compare peaks with different or even similar retention times. The differences in their spectra can tell us something about their chemical bonds and structure. Also, the spectral data is very useful for ruling out and troubleshooting the ID of peaks seen in the chromatogram (Note: Identical spectra does not equal identical samples as it is a null test of sorts. Different spectra = different samples and identical spectra = maybe the same/maybe not) ). For example: True enantiomers of the same racemate must have identical spectra or they are not enantiomers. Spectra of intermediates or reaction products can often be ID'd quickly by their spectra when following reaction chemistries by HPLC. Spectral data is very useful and why we must use a scanning DAD/PDA for all method development (for samples with chromophores). The DAD provides you with a tool to "see" many compounds during the development of the method for accuracy and also later on for QC and validation. Use of a single-wavelength detector can provide very misleading data as it will show a "peak" at a particular set wavelength, but that same peak will probably appear at many other wavelengths too. Please refer to link:
http://hplctips.blogspot.com/2013/08/wavelength-selection-for-method.html
This may be of related interest too:
"Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer".
http://hplctips.blogspot.com/2013/09/method-development-hint-use-your-hplc.html
I used to scan with two wavelengths, one- the lambda max, the other- the wavelength of another spectrum crest of the std, and calculate both independently.
Different results usually mean an impure sample peak.
No need to take spectra first if you are using diode array detector. But taking the uv vis spectra on spectrophotomter can give you idea about the best wavelengths for selecting in HPLC.
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