Fixing Wavelength

[chrom]

hi,

While fixing wavelength in one of reaction monitoring by RP-HPLC, the following things i observed.

1. Starting material is showing its wavelength max at 248.
2. Intermediate is showing its wavelength max at 280.
3. Final product is showing its wavelength max at 228.

My doubt is how i have to select the wavelength.

Thanks in advance for valuable suggestions.

[Consumer Products Guy]

(1) Do you have multiple wavelenght detector, then monitor all three; but sounds like you don't have this.

(2) Can you program your detector to change wavelengths at set times, like Agilent VWD?

(3) Can you establish/demonstrate linearity (for example of your starting material) at the finished product ideal wavength and then use that wavelength?

[Jackus]

Hello,
you need not acquire data at wavelength with maximum response. Generally it is better to find some plateau with reasonable response because some change in WL accuracy has not so high impact to your results.
If you have DAD detector (with Chemstation), you are lucky man. Collect a spectra in a run with all three compounds and select Isoabsorbance plot from the menu. Here you can see your chromatograms only with selecting of desired WL.

[mbicking]

What information do you want to get from the chromatogram? Usually the absorbance spectra have broad absorbance bands, so you can select a wavelength where all compounds absorb to some extent. Then you will see all three compounds, but their response will not be as large compared to using the maximum for each. However, you probably do not have sensitivity problems so this is not important.

But, remember that you cannot simply measure the percent peak area for each compound and determine how much is present. If you have pure standards for each, you can calibrate your system at whatever wavelength you choose, and then get this information. Otherwise, you can only determine the relative change for each component.

Of course, if you have a diode array or multi-wavelength detector, you could monitor all three wavelength maxima.