Chromatography Forum

Hellow everybody .

i have some problem to fill my sequences in totalchrome software . i try to separate 10 food colors .every color has individual wavelength . how can i fill these wavelengths in the software ( detector window ) .

Hellow everybody .

i have some problem to fill my sequences in totalchrome software . i try to separate 10 food colors .every color has individual wavelength . how can i fill these wavelengths in the software ( detector window ) .

Do we need to guess what kind of detector you have (PDA or variable wavelength) ?

Thank you dblux for your response .

i have ( DAD ) detector . and inside the software ( Instrument control - detector window ) there are only 2 spaces to put wavelengths as following :-


wavelength (channel A ------- ) & ( channel B --------)

Bandwidth ( nm ) ------------- & ( ------------------ )

Reference wavelength( nm ) ( --------) & ( ------------------)

I guess you will use one channel eg. A.
Then you need to input the lowest and the highest wavelength of interest for your analites.
Bandwidth is the spectral resolution of your DAD and you may use full resolution or make the bandwidth wider.
Reference wavelength is the wavelength which you will find in your spectrum in the region where no analites elute.

You may always switch on software help and find the meaning of dialog windows fields.
Not too much, but hope that I helped you a little.
I'm confident that soon people more experienced in HPLC will sched more light on this.

If your DAD can only check two wavelengths it will be a tad more tricky. I would try to get a spectrum of each of the dyes on a UV-VIS by themselves in your mobile phase. The usual plan would be to monitor the wavelengths for where each dye has a maximum absorbance, with 2 channels and 10 dyes that becomes more tricky.

The wavelength monitored doesn't have to be a max, it is just easier to detect compounds if it is a max. Try to find 2 wavelengths which could be used to monitor all 10 of the compounds.

Hope that helps.

Tank you a lot dblux & itspip for your replays . and i put an image for the detector software window for more explanation . as you see the images . my method have 10 food dyes we will take 5 food dyes as example as following:
( E 104 @ 414 nm) & ( E 102 @ 426 nm) & ( E 110 @ 483 nm) & ( E 124 @ 510 nm) and ( E 127 @ 530 nm).
now how can i put the suitable wave length for 5 food dyes by this software . and i am sure this detector ( DAD) can detect more than 5 dyes . i mean can detect wavelengths . what is your opinions abut the wavelengths must put it in the space of :

wavelength (nm) (channel A ------- ) & ( channel B --------)
Bandwidth ( nm ) ------------- & ( ------------------ )
Reference wavelength( nm ) ( --------) & ( ------------------)

thank you so much again .

Usually DAD detector can aquire the whole spectra. I am not aware of your particular instrument, but the manual has an appendix entitled "Configuring Series 200 DAD for Acquiring Spectra" which recalls Turboscan 200 software.
http://www.samsi.no/Products/perkinelme ... ctorII.pdf

thank you dblux for your replay . but also i did not found any clear answer for my questions . so what is numbers will put it in the spaces in the detector image .( instrument control - detector window ) ?

I would probably use 426 and 510 as the wavelengths in channel A and B. Keeping the bandwidth at 20, someone correct me if I'm wrong, make it so that 500nm - 520nm will be monitored and labeled as 510nm.

The reference wavelength is a wavelength where there is no absorbance for your samples. I would check the spectra for your compounds and use a wavelength where there is minimal absorbance, probably around 600nm.

I would probably use 426 and 510 as the wavelengths in channel A and B. Keeping the bandwidth at 20, someone correct me if I'm wrong, make it so that 500nm - 520nm will be monitored and labeled as 510nm.

Thank you itspip for your response . i will try your sequences . and i will show you what happened with me .
thanx again .

Hi itspip .

i did your sequences as you told . but i got only one good separation ( tartrazine color (426 nm ) . the other colors very bad chromatograme . i can show you if you want . so do you have HPLC 200 serious ( perkin elmer )? .we can contact together more .
my test samples were : Tartrazine (426 nm) & Erythrosine (531nm) & Carmosine (522nm) all of them individual .

thanx again for your response .

Hi itspip .

i did your sequences as you told . but i got only one good separation ( tartrazine color (426 nm ) . the other colors very bad chromatograme . i can show you if you want . so do you have HPLC 200 serious ( perkin elmer )? .we can contact together more .
my test samples were : Tartrazine (426 nm) & Erythrosine (531nm) & Carmosine (522nm) all of them individual .

thanx again for your response .

I do not have a Perkin Elmer, my current instrument is an Agilent 1260. Since your DAD can only monitor 2 wavelengths I'm thinking that it is fairly old or it is a Variable Wavelength Detector with 2 channels.

If you have access to a UV-Vis, I would suggest obtaining spectra for each of the dyes and then determining which wavelengths to monitor.

The newer DADs have more channels to monitor and would make this a much simpler problem to resolve.

Good luck.