SEC GPC for dextran based colloids

[fastislip]

Hello,

I am in the beginning stages of developing an assay for analyzing a colloid of approximately 750kda comprised of a core and a dextran coating. The individual dextran monomers that make up the coating are approximately 15 kda. What is critical is not necessarily the total 750kda colloid but the individual monomers that are present as independent species in the solution. Essentially as impurities or excipients. The trouble that we have had thus far with SEC/GPC is that the larger colloid containing the bound dextran is not well separated from the smaller individual dextran peak. Does anyone have a suggestion for improving separation? I was thinking about combining columns and want to avoid a filtration/sample prep step if possible.

I would be happy to share my outcome with the forum.

Many Thanks!
-Jim

[Gerhard Kratz]

Can you please give some more details about the column you tried - exclusion limit for dextrans, pore size, silica or polymer based, particle size. Size exclusion is "simple" - big molecules come out first, small molecules later. You are right, maybe a second column in series with a smaller pore size can help to get a better resolution.

[fastislip]

I was working off of an application note from phenomenex for a Polysep-GFC-P 4000 column with a separation ragne of 3k - 400k. It has a polymer based backbone. I was thinking of adding a 6000 column (range of 100K - 15M) to retain the larger colloid. The run times in the application notes are less than 20 min so I would assume a 40-50 minute method again with the focus of just the early eluting smaller monomers.

There has historically been quite a bit tried using GPC but we're talking more than 15 years ago and long before my time with poor records kept. My goal is to go into the project with little bias.

[Gerhard Kratz]

Hi Jim,
thanks for the information. I will be out of my office tomorrow and will come back to you over the weekend. Please check in the meantime the GFC/GPC/SEC information on the websites of Showa Denko, tosoh bioscience, and PolyLC for similar compounds. Maybe a silica based SEC column would deliver better results. You can also contact the company Ludger Ltd. in the UK via their website for more information. They are experts in Glycan analysis and are producing high MW dextran standards. It's late now in Germany and I have a meeting tomorrow morning. Good luck.

[fastislip]

Will do and thank you for the support. I will be spending 50% or so of my time on this project and will keep posting on the forum any updates.

Thanks!
-Jim

[Ken Tseng]

Besides the companies mentioned, you can also consider Agilent and Jordi Labs. They all have excellent GPC columns. Contact each companies (or their distributors) and describe what you want to achieve and they most likely will provide an adequate solution.

[Ting Wu]

Can you please give me some more details about the column you tried: silica based or polymer based? Is the mobile phase water based or organic solvent based?

[fastislip]

So far I haven't had a whole lot of time to explore. I purchased a Phenomenex Polysep GFC-P 3000 column and tried with seperately water as an eluant and then sodium hydroxide at .oo1 molar. The basic eluant shifted the peaks a few minutes later and sharpened them a bit but no improvement on separation. Actually both the free carbohydrate and the larger carbohydrate complex co eluted. My guess is because the larger complex is made up of multiple monomers of the same sized carbohydrate hydrogen bonded to a small metal oxide core. The interaction with the pores of the columns were probably not a whole lot different. Might try a weak ion pairing agent such as TFA. Any suggestions? If I don't have any luck with acidic mobile phase or changes to ionic strength I'll switch to an SEC column with some chemistry basis for separation.

Thoughts?

[Ting Wu]

I have one MW calibration data for dextran separation using Sepax SRT-300 SEC column. Please contact me twu@sepax-tech.com if you need. (MW range from 1 kD to 670 kD dextran standards)

[unmgvar]

could it be that a IEX method be more interesting?
it would also in the end separate based on size but because of net charge.
the less charge the faster the compounds would go out the column.

still one thing bothers.
the difference between the colloids and the dextrans should be huge so the 1 SEC column should not have problems separating.
maybe your dextran is recombinig and agregating? dextran can be also 150Kda and above.
if we look at the column you say you use then 750K is above the exclusion limit and so your product is flying out like a 400Ka with maybe other effects then the separation is bad to your smaller dextran

I think you should use a column that is 300A in pore size at least the Mw range is up to 1250KDa in general,

[fastislip]

I have been playing with ion exchange a bit on the side. I really want to use the PAD detector because it is so sensitive. In response to your comments I agree it seems illogical that the two aren't easily separating but I am new to SEC and there is likely some understanding that I am missing. Or I am sheering the dextran off the colloid. I have the next sized column on backorder with a range of 3kda to 400kda. It should provide for some additionally specificity but based on what I've seen out there with large and small Dextrans I am not super hopeful.

Part of my problem is the distributions are somewhat large think 3-40kda for the free dextran but still an order of magnitude smaller than the larger colloid.

This distribution has been really tough to control with the CarboPac PA1 column I have been trying using HPAC. It's much more fun to play with than SEC mainly because it's so much faster.

On an aside, I've already solved my problem using Field Flow Fractionation which is an interesting tool but it has proven to have limited robustness. We had a valve break before we could start validating hence my need for a chromatographic or column based separation.

[unmgvar]

basically you need to now that SEC chromatography is based on a given assumption:
you expect the size of your molecules to be in direct correlation to your volume size (Rh)
this assumption is many times correct and too often also wrong. the real way to do it is by knowing actual Rh and viscosity.
this is why modern precise SEC systems have a viscometer and use at least 2-3 angle LS detectors.
another thing that is very crucial im SEC is that the phase in the column be inert as much as possible to other interactions with your compounds
at rule of thumb there should be a separation between 1 size unit of your compound to a 2 size unit if no other in between species exist
so the 750Kda and the 40Kda should be very well resolved even if you are using the wrong column like you did now.
but it is not the case and I know that dextrans can actually come in bigger sizes then 40Kda, even up to 2000Kda, so maybe your are having agregates of the dextrans somehow?
now also dextrans are a compound that does branching, so the assumption of Rh to Mw is not precise.
only if you can keep your dextran chains short then you will ok with the called standard technique using only one concentration detector like RI

maybe you should also seek advice with people from Malvern or Whyatt about this.
also about the speed, if you really have such a huge difference between the dextran and your colloids then you could normally be able to increase flow rates in order to save time versus separation resolution.

[fastislip]

All great points and I very much appreciate the advice. As I mentioned initially, the project was to move extremely slowly as most of the work will be performed externally from my last post. I have enlisted Jordi Labs per the recommendation of this discussion and have had at this point some signs of success.

At this point in time I am departing from this project and would like to post our current results for the benefit of this forum. Obviously I had a lot to learn taking in this project as everyone has been kind to help me with.

Our final system settled on a Jordi x-Stream Water 1000000A 250x10mm column with water as an eluant. Column temp = 35C and a flow rate of 1ml/min. We were expecting much better resolution based on the expected size of the two compounds we are trying to separate but with a single column we only have the start of two peaks. We expect to need a series of columns (hopefully only 2) in order to achieve baseline separation. This is to be determined but it is clear that the separation is not taking place based on size alone.

Thanks again for the help!

[unmgvar]

remember the rule of thumb with SEC and very high MWs
almost no diffusion of big species, so you can use to your advantage going to lower flow rates in order to seek better separation between species.
I have to admit that I am amazed by technology, here
a 10u pore on a 5u dp column
and why go for a dimension of 10X250mm and not a 7.8 even 4.6 by 300mm and improve separation by almost 20%?
from your own details of MW, it should be better to use a 300A size and go for a 3u column and get a better separation.
or the expected MWs you told us were a mistake
remember that if you are not using the correct column for your species then you get bad separation, either because your compounds are too big and in the exclusion limit or also the oposite
from my experience i find it hard to trust your system when you seek species of 15Kda

you also point out that the separation is not only size based,
if this is not an important issue then forget of SEC and move to another system, if it is an issue then you have to change column type.
in SEC the issue is mostly the type of "bone structure" used, for bio sec, columns are mostly made of Si and covered with films to block interactions, and we know today that with some compounds it is not enough and new modifications have appeared to handle this.

[fastislip]

I question our size measurements as well. Thank you again for your help!