Chromatography Forum

Hi guys,

I have a method. Not created by any of our team which requires the use of Indirect UV detection at the wavelength of 240nm.

Now i don't have any knowledge on Indirect-UV. can anyone give me a quick outline or guide to what it is?

Can my agilent 1100/1200 equipped with DAD run this type of method?

Column required is a IC-Pak A HR, 6 μm, 75 x 4.6 mm or equivalent . All suitable with our systems?

Cheers again guys..!

Mobile phase contains a UV-active modifier, and when your ion exhanges with it the UV signal drops, so you get a negative peak. We don't use it, we use conductivity detector for ions.

So this mean...?!

I can use the normal set -up and DAD detector that we currently have?!

Thanks for the reply by the way

Yes, you can use the DAD. The major difference is that your peaks will be negative (you're looking at a decrease in absorbance), so you will have to flip the polarity.

Some CDSs include an option to use indirect absorbance such as EZChrom and 32Karat. You might want to check to see if this is possible

There are some nice papers written back in the 80's (I think Jack Crommen worked a lot on it).

Not an easy route, very susceptible to get "system peaks".

Hi guys,

I have a method. Not created by any of our team which requires the use of Indirect UV detection at the wavelength of 240nm.

Now i don't have any knowledge on Indirect-UV. can anyone give me a quick outline or guide to what it is?

Can my agilent 1100/1200 equipped with DAD run this type of method?

Column required is a IC-Pak A HR, 6 μm, 75 x 4.6 mm or equivalent . All suitable with our systems?

Cheers again guys..!

Indirect UV usualy means using another standard the apear at different retantion time with a known absortivity ratio to the analyte you are looking to analyze quatitatively.
A good example of it is using Vitamin A acetate to quatitate Vitamin A Palmitate.
USP standard for Vitamin A palmitate is not available.
I hope this is understood.

Hi guys,

I have a method. Not created by any of our team which requires the use of Indirect UV detection at the wavelength of 240nm.

Now i don't have any knowledge on Indirect-UV. can anyone give me a quick outline or guide to what it is?

Can my agilent 1100/1200 equipped with DAD run this type of method?

Column required is a IC-Pak A HR, 6 μm, 75 x 4.6 mm or equivalent . All suitable with our systems?

Cheers again guys..!

Indirect UV usualy means using another standard the apear at different retantion time with a known absortivity ratio to the analyte you are looking to analyze quatitatively.
A good example of it is using Vitamin A acetate to quatitate Vitamin A Palmitate.
USP standard for Vitamin A palmitate is not available.
I hope this is understood.

Please, carefully read the original post and the replies before you post your opinion.

Indirect method of detection means, that substance is modyfied before detector:

1. derivatization
2. UV irridation
3. Ion-pair forming

Direct detection - native oryginal molecule is measured.
The most frequent is for fluorimetry, but UV or VIS also used for the sensitivity,
specifity, or separation parameters improving.

If you have complete method 'indirect' is only for you information, not for any
extra manipulate with detector.

Well, I always thought that this term was reserved for the technique that uses a light absorbing mobile phase for analytes which don´t absorb light. When the analyte comes through the mobile phase is diluted, the absorbance goes down, a negative peak results, as mentioned before. I never used that, seen it many times when shorter wavelength were used.

HW is correct. The idea is that a non-absorbing analyte displaces (or replaces) an absorbing component of the mobile phase.

Some examples:

http://www.plantphysiol.org/content/86/2/615.full.pdf

http://www.chem.ucalgary.ca/courses/f10 ... nC.doc.pdf

http://www.mendeley.com/research/indire ... age-forms/