gelatin assay by SEC

[Johanna]

Hi,

I am working on gelatin and I used to assay it by BCA until had too many excipients incorporated in formulations which were interfering with the assay therefore I am moving on to using SEC. I am interested in how much gelatin I have in a sample (not individual MW's). I've got column C18 300A, which I am hoping to use along side with RP-HPLC. Has anyone used SEC for gelatin before? And if so has anyone got any suggestions what to consider as MP?

Thanks.

[jrussell]

Hi Johanna,

I currently use SEC to measure the average molecular weight of gelatine. I am not aware of using SEC to measure/quantify the amount of gelatine in a certain product. When I want to know the amount of gelatine in a formulation I measure the amount of 4-hydroxyproline present. This amino acid is very specific to collagenous protein, so it is a fairly accurate indicator of gelatine content.

There is a fair amount of literature on the subject, but basically chloramine-T is used to oxidize 4-hydroxyproline into a pyrrole-like compound, this is condensed with p-dimethylamino benzaldehyde (Ehrlich's reagent) to form a reddish compound, the absorbance is read in the visible region (about 560nm, but this can vary somewhat). Because the amount of 4-hydroxyproline present in gelatine is more or less fixed (depending on collagen source: bovine, porcine, poultry, fish, etc.), a constant is factored into the calculation made from the absorbance regression of hydroxyproline standards, and this will give you a good estimate of the gelatine content. It is not a very sexy procedure, but it works.

I am not very knowledgable in SEC techniques, I am a 1st year chemistry graduate student and there are a lot of things I don't know about the subject, so you may have some luck using SEC. But, I also work for a gelatine manufacturer so I know that the above method will work with some tailoring to your appplication. I hope this helps. Good Luck.

JRussell

[Johanna]

Hi JRussell,

Thank you very much for your reply and the information. I'll have a look into the literature too and hopefully I'll get it sorted

Kind regards,

Johanna

[JesseG]

I have some similar questions. I'm trying to calulate MW of soluble collagen via GPC/SEC. No easy task but I found a few papers for Gelatin which doesn't really help me as I need to characterize before it turns to gelatin.
Meyer & Morgenstern, Biomacromolecules, 2003, 4, 1727-1732. and Oxford, et al., Journal of Biological Chemistry, Vol. 279, Issue of March 19, pp10939-10945, 2004.

I have dug up some papers using SDS Page and Circular Dichromism also.