Method development - where should I start?

[Askelaad]

Hi

I have this terpolymer (Mw~1000 )

a) Maleic acid
b) Ethyl acrylate
c) Vinyl acetete

And I really don't know which column I should use. (detector is UV 210 nm, matrice is sea water)

I have tried C-18, Hilic and I think about trying GPC, but really, i am tired of trying without understanding what i am doing. I get some peaks, a big one, and a couple of small ones, and i can't explain what is what. I hate that.

So, What clues can i get from the molecular formula that can help me to understand which column/eluent I should choose, and what is going to show on the chromatogram?

[DSP007]

Polimer structure is unregular , molecular mass unstabile ?
Its normal , no mistake chemistry (c, Boss) - for chromatography separation you have a mix of any polymers.

For example - abc , aba, cbc , abcb , abab, cbcb , bcb, bab.

[Askelaad]

Polimer structure is unregular , molecular mass unstabile ?
Its normal , no mistake chemistry (c, Boss) - for chromatography separation you have a mix of any polymers.

For example - abc , aba, cbc , abcb , abab, cbcb , bcb, bab.

I see, does it mean that I shouldn't care about selectivity, integrate all the peaks and count them as one?

[DSP007]

And that's, as commander orders. (c, translate)

Correct - identify abc and "others brothers" and calculate by individual components of mix.

[Askelaad]

Ok, thank you for your help so far! (although i didn't understand the first line of your last reply )

Now, I read somewhere that I should think upon which characteristic my separation is going to happen: polarity, electrical charge or molecular size.

well, I have a pretty polar molecule, thats for sure (or, is it?), but it's also an anionic polymer, right? and a polymer as well. So is there a way - besides more testing - to know which way is going to be the best?

[Mattias]

As DSP007 wrote, you will have huge number of different molecules if you can have a random mix of all three monomers. You will never be able to separate them all.

Is it an assay method for the polymermix that you want to develop? What other compounds can be present in your sample?

[Askelaad]

Not being able to separate all the different molecules of the mix is not a big issue. the matrix of my sample is produced water and besides the usual seawater salts, i don't expect to find anything else. I just want to quantify the whole "mix"

Do you think GPC would be a better idea than RP? The problem yhat I have with RP is that all the peaks elute very soon, (i use 5% ACN / phosphate buffer) and it seems that a couple of peaks elute in the dip.

[DSP007]

GPC is its- http://en.wikipedia.org/wiki/GPC_Biotech or its http://en.wikipedia.org/wiki/Gel_permea ... matography ?
If secondary - its normal. In you separation dimension (mass and form) of molecula should have big role.
In C18 80-130A you realize first variant of exclusion chromatography - water sorbtion in silicagel porous , but big polymer moleculs rides (Jump) through the (above, jump on) the hole (porous)
In gel sorbent you realize second variant of exclusion chromatography - small water moleculs freely flow through the gel , but big polimer stop in gel and sorbtion.Everything it is easier for a camel to go through the eye of a needle" (c Biblia, Matt 19 )
Secondary variant - is a best variant.

How to identify polymers - the best way to spend their directed synthesis. But it's hard to hand.

[Vlad Orlovsky]

Askelaad,

Don't worry about "Ok, thank you for your help so far! (although i didn't understand the first line of your last reply0", because DSP007 always quotes and translates Russian catch phrases and proverbs which mean nothing to people here unless they understand Russian. Proverbs do not translate directly from one language to another....and I am sure that 95% here has no idea who Lenin (mentioned in another post) is and another 5% have only negative impression of him (Lenin was one of the first Communist leaders back at the beginning of last century). There are equivalents for cited "references" but you need to know them....so pay attention only to scientific parts of posts

[Mattias]

Not being able to separate all the different molecules of the mix is not a big issue. the matrix of my sample is produced water and besides the usual seawater salts, i don't expect to find anything else. I just want to quantify the whole "mix"

Do you think GPC would be a better idea than RP? The problem yhat I have with RP is that all the peaks elute very soon, (i use 5% ACN / phosphate buffer) and it seems that a couple of peaks elute in the dip.

I am not sure if you have any free acid groups left in your molecules, but if so, you should get better retention at low pH in RP. You could try to run in 0.2% phosphoric acid and do a quick acetonitrile gradient (you want as little separation as possible in this case).

I would only go to SEC as the last option (expensive columns, long run times, only wide diameters availiable, polymers will elute with every other small molecules in your sample etc.).