Chromatography Forum

Hi all,

I am analysing b-carotene in watermelon peel and rind. However, the chromatograms of pure standards of b-carotene and b-apo-8'-carotenal (internal standard) showed two peaks instead of one. Why is that? Is it possible that the standards were degraded? And how do I obtain the calibration curve in such case?

Thank you.

I'd check that other compounds show a single peak. I'm wondering if there is a column or injection problem rather than a compound issue.

Are the peaks of the same area and shape? You may have an issue with either the choice of the standard or a mismatch between injection media and mobile phase.

Is your injection media matched to your mobile phase (i.e. same composition)?

Is your standard all-trans beta carotene or is it a mix of cis/trans forms? Depending on your column bed you may have separate the forms.

Hi all,

I am analysing b-carotene in watermelon peel and rind. However, the chromatograms of pure standards of b-carotene and b-apo-8'-carotenal (internal standard) showed two peaks instead of one. Why is that? Is it possible that the standards were degraded? And how do I obtain the calibration curve in such case?

Thank you.

why is it necessary to have your std and i.s elute at the same retention time?

I'm quite sure there are at least 2 isomeres but perhaeps even more.

It's also possible to have other carotene with the beta.

Try to have more information from the manufacturer of your standard about isomere.

For the quantification, it depends on the definition of the product. For exemple you can use the sum of cis and trans beta carotene if the specification of the product is based on the total beta carotene.

The standard should not be degraded if stored as required by the supplier and if your solutions are freshly prepared before injections. Beta-carotene is an anti-oxidyzing agent so it can oxidyse with time if exposed to oxygene.

What column are you using?
I found the best results for separation of carotenoids came from a C-30 column.
Plus, there is the possibility the beta carotene is degrading as it really needs to be stored at lower temperatures than a typical freezer can provide.
If you have purchased natural beta-carotene and are using a C-18 column, it may just be alpha carotene, as they tend to coelute.

Hello there
Separation on column C30 you can see almost all the enantiomers of b-carotene and a-carotene! it depends of the composition of your stds
anyway if you wish to analyze total carotene, you could analyze with c18 column instead!!!
if you need some help I'm working with carotenoids, and perhaps I can see your chromatograms and more or less help you.

Polo