Betacarotene Analysis by HPLC

[chemron]

Hi,

I was able to mae a separation in ACN:MeOH:EtOAc but in 1 ppm standard only. If I will try run it again in other standards it shows more peak again and no defined retention time. I suspect my baseline wasnt yet stable so i have to tried it again. I cant focus on this because I am doing another works also aside from betacarotene.

If the standard is already expired, it will still work right?

[Bruce Hamilton]

If the standard is already expired, it will still work right?

It depends on storage of that standard. The expiry date is based on the recommended storage conditions. If you followed their guidance the expiry date is valid, and you can't assume that the beta-carotene value will be correct after that date. If you stored the standard in more benign conditions ( colder, darker, inert gas blanket etc etc ) then an expired standard may be OK for development work.

Carotenoids are well know for degrading and producing multiple peaks, So i would want access to a known good standard to check your working standard against - to avoid the confusion you are already experiencing.

If your method will be used to certify the content of a product, you may want to find out the details of any methods preferred by the regulatory authority.

I personally think you are reinventing the wheel, as the Journal of Food Science and chromatographic journals have plenty of papers on analysis of beta-carotene in foods. C18, or even C30, columns can be used for reverse phase methods, methanol is often the preferred major mobile phase component, and good column temperature is critical for consistent retention.

There are several comprehensive reviews available, eg "Carotenoid reversed-phase high-performance liquid chromatography methods: reference compendium." N.E.Craft. Methods Enzymol 213:185-205, 1992.

A simple literature search will provide plenty of details on separations, and especially the necessary careful sample preparation and analytical conditions for beta-carotene in foods and drinks.

If you're struggling with the analysis of vitamins, I highly recommend the book " Modern Chromatographic Analysis of Vitamins " by Andre P. De Leenheer, Willy E. Lambert, Jan F. Van Bocxlaer. 3rd Edition ( 2000 ) Marcel Dekker ISBN 0-8247-0316-2. Chapter 1 covers Viatmin A and carotenoids.

Bruce Hamilton

[bisnettrj2]

"I was able to mae a separation in ACN:MeOH:EtOAc but in 1 ppm standard only" - I'm inferring from the rest of your post that a higher standard gives extra peaks? Or am I inferring incorrectly?

Were it me, I would get a new standard and store it carefully, and shoot comparable concentrations to what you've already shot, to see if the extra peaks are caused by a degraded standard or something in the system.

And Bruce, to me, seems like a wealth of information - take his advice and do a literature search for what you are trying to do. Stand on the shoulders of the giants that have come before you. There is no reason to re-do the work others have already done for you.