Madam, it seems that you have somewhere forgotten your basic biochemistry. You can NEVER detect Vit D2  and Vit A together in HPLC (as they have different lambda max in UV wavelength) and that too from Milk samples. 

 Thanks you sir for enlightening the knowledge.

Dear Veena,

Please find attached, an application note for the HPLC analysis of fat soluble vitamins, using UV/Visible detection.  Sample pre-treatment, including saponification is required, prior to introduction to the HPLC system

For multicomponent analyses of UV/Visible chromophores, one uses either:-

• A compromise wavelength, where a wavelength is selected, which is a compromise for all of the analytes of interest.  As each analyte has its own calibration curve, quantification should not be a problem.  There may be a loss of sensitivity for some analytes.
• A multi wavelength channel UV/Visible detector, where a number of chromatograms at different pre-selected wavelengths, are obtained from a single injection.
• Programming a single wavelength UV/Visible detector, to change the wavelength of single chromatograms at pre-set elution times.

Kind regards,

Ade Kujore

Marketing

Cecil Instruments Limited

Milton Technical Centre, Cambridge CB24 6AZ

United Kingdom

email:- [email protected]

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web site:- www.cecilinstruments.com

Sorry Veena,

I forgot to add the application note.

Kind regards,

Ade

Dear Ade Kujore,

Thanks for sharing the information.