Chromatography Forum

Dear Community,

I have recently began performing aquaeous GPC/SEC analysis using LS/VISC/DRI on some PEG analytes which are terminated with a maleimide group. The molar masses are ranged from 10K to 40K and unfortunately for the lower molar mass analytes, I am getting 2 very distinct and separate peaks. At higher molar masses this does not occur.

We assumed adsorption was occurring with the aqueous column, so we added a small amount of buffer (0.05M ammonium formate) to prevent interactions...with no avail. Should higher salt concentration be added?

Do you suppose the malemide end groups are causing this adsorption and therefore, separation of peaks of lower molar mass samples?

Should I add an organic modifier? (I am hoping to avoid this due to changes in RI, dn/dc, Mark-Howink coeff's, etc.)

I have looked through some of Chi-san Wu's books (which were very helpful) but I am not confident of what effects may be going on.

Any help/advice would be much appreciated.


Dustin

I doubt that you will have hydrophobic interaction with PEGs (assuming you use a standard packing for aqueous SEC). My first guess is that the difficulties are caused by ionic features of your analytes. The standard approach is to use NaNO3, 0.1 M. If this does not help, inclrease the concentration to 0.5 M.

I would try this first. Also, do you have normal behaviour with PEG standards?

We get normal behavior from the PEG standards from the oligomer to the 1 million range, and yes it is a standard aqueous SEC column. We did try using NaNO3 at 0.1 M and 0.2 M but we still get splitting of peaks (and it looks as if the peaks may have swapped places).

I was leary of increasing the concentration too high (i.e. above 0.2 M) in case of desalting on the column. But actually, I'm not sure how much NaNO3 can be used before this becomes an issue. Several colleagues suggested not going above 0.2 M due to its possible effects on the system.

Well, if indeed the location of the peaks is changing, then ionic effects play a role.

There is little concern about the column in high salt solution (assuming that you are using a silica-based SEC column). The instrument is a bit more of a concern: you need to look at the wash solvents that are used to wash whatever is washed in your instrument: needle wash, injector loops, pump seals... If you can prevent crystal formation, and if the column is a silica-based column, you can go as high as you want with the salt concentration.

Thank you much for the advice. I will see about increasing salt concentration to higher amounts.

hi
sorry for little off-way question...
i am trying to optimize mobile phase for SEC/GFC method for PEG could any one suggest me suitable mobile phase i can try..??
thanks a lot

Rick,

You can use pure water and see if that works. If it doesn't (as in my case) we eventually had to use a 75:25 water/methanol mixture with 0.1M NaNO3 added. This allowed the separation of PEG's between 10K and 40K g/mol.

For our samples above 30K, using 0.1M NaNO3 was enough to do the separation.

You can use reversed phase for low mwt PEGs.
Below are PEG applications on Cadenza CW-C18:

http://www.silvertonesciences.com/files/TI380E.pdf
http://www.silvertonesciences.com/files/TI381E.pdf

hi everyone..
just diverting from the post little bit...

which type of GPC column due you think works best for PEG,i.e. silica based or polymer based??
wht your personal experience say??