Chromatography Forum

Hi all..

I have difficulties in analysing serum containing peptide (as analyte) and Dow Corning 9509 silicone elastomer as one of matrix components. The silicone and my analyte has same retention time, so I always get higher recovery. I use Acetonitrile : water gradient for my mobile phase.

I have tried to separate it, but it doesn't work until now. So I make a linearity of matrix, and calculate my analyte recovery manually. Am i allowed to do that?

Or maybe some of you have experience with silicone elastomer? Please reply me then. Thanks a lot.

No, one way or another, you have to discriminate between them.

That said, those are chemically *very* different. How are you detecting? What pH are you running in your mobile phase? what kind of column?

I use Zorbax Eclipse XDB C-8 column (Agilent), and detection with
UV detector at 220 nm.

This is my mobile phase:
time %acetonitrile %water
0 25 75
4 55 45
5.5 55 45
6.5 25 75
The flow rate is 1 ml/minute at 30°C.

I didn't measure the ph..but i think it's about 6.

The silicone "elastomer" is not soluble. It is a suspension. Hopefully you are carefully filtering your sample before injection.

What you are seeing is not silicone, but one of the ingredients in the silicone product. An alcohol and several preservatives present. One of these components is probably causing your interference, but it is difficult to know which one.

Your main options are:
1. Change wavelength - 280 nm works for proteins and peptides, but absorbance is reduced compared to 220 nm.
2. Change column - find a reversed phase column with different selectivity - different C18 manufacturer, polar embedded phase, etc.

Adding a perfluorinated carboxcylic acid (trifluoroacetic or heptafluorobutyric) may change selectivity enough to resolve the two species, though 220 nm is a bit on the low side for working with these modifiers. Changing your gradient rate and endpoints and your "B-phase" (organic) composition can also have a significant effect upon selectivity. Changing column temperature can help as well. I'd exhaust these possibilities before putting in another column. I was just yesterday assaying for a trace polypeptide in a heavy silicone matrix and found that thinning it with dimethicone then doing a liquid-liquid extraction with water-ACN gave excellent recovery of the analyte (~99% theor) without interference.

Remember, proteins and polypeptides, if large enough, act completely differently on reversed phase columns than do most smaller compounds. In many cases they cannot be eluted under isocratic conditions; below a threshold organic level, they won't elute and above it, they'll elute in the void. If you're lucky and you can get your elastomer contaminant to elute earlier than your peptide under isocratic conditions, all youll have to do once the contaminant elutes is titrate in enough organic to elute your peptide and you're done. This doesn't apply for small polypeptides, though (like the one I'm working with....), so this little scrap of info may do you exactly no good.