Hello!
I am trying to fractionate a complex mixtures of very small components (it contains peptides among other molecules, less than 1kDa). I am using the cation exchanger monoS at pH4 and I see no retention. I thought of salts in the sample, I diluted it in 100 volumes of the starting buffer
and I saw no retention either.
Mono S is used for proteins. Does the pore size (or other thing not taken into account) influence retention ?
Thank you in advance.

I think the most important things that you have to consider are the optimum range of pH for your column that perform as a WCX or SCX. For an example, some columns behave as a SCX at one pH range and WCX at above that pH range.
The other thing is, study the peptide chemistry of your peptide; check how many aa of + and - side chains in your peptide. What is the pI of your peptide?
Most of the IEX column have some kind of hydrophobic interactions too (mix mode) so you have to adjuts the organic percentage accordingly.

I think basically you have to find the proper pH for the Mphase, proper salt grdient and the ACN (if it is your Organic)!!.I am not familiar with your MonoS CEX column.

Hope this helps

Ananda

I think the most important things that you have to consider are the optimum range of pH for your column that perform as a WCX or SCX. For an example, some columns behave as a SCX at one pH range and WCX at above that pH range.
The other thing is, study the peptide chemistry of your peptide; check how many aa with + and - side chains in your peptide. What is the pI of your peptide?
Most of the IEX column have some kind of hydrophobic interactions too (mix mode) so you have to adjust the organic percentage accordingly.

I think basically you have to find the proper pH for the Mphase, proper salt grdient and the ACN (if it is your Organic)!!.I am not familiar with your MonoS CEX column.

Hope this helps

Ananda

The monoS column is a 10um porous strong cation exchange bead, with a methyl sulfonate group. There is, therefore, no effect of pH with charge density of the stationary phase at any pH you would realistically work at.

You must use a conductivity and a pH meter to check your sample before loading onto the column. There is no point assuming either is, or is not the cause of non-bind.

You must also have an idea about the charge behaviour of your peptides. Why assume that they will have a net positive charge at pH4. Even if they do have a net positive charge at this pH, it may not be enough to bind the column, and especially with complex protiens, it is localised areas of specific charge that can be the cause of binding i.e. just because a polymer has a net positive charge, doesn't mean it will bind to the monoS.

The monoS bead has a pore size suitable for large molecules but I have used it to separate small postively charged species with success.

Are you certain that you have non-bind of all your solutes. What is 100%B, have you measured its pH and conductivity. What about A as well.

When working with such smaller molecules that will have less charge possiblilities, you may have to be very careful with salt concentration to get a bind.

Give the Amersham technical support line a call.

Thank you Ananda.
Mono S is a strong cation exchanger and it works well at pH 4. In the sample there are basic peptides among other molecules, I think they can be retained at low ion strength. These molecules are very hydrophilic and most of them are not retained in C18 vydac, I have discarded hydrophobic interactions.

Thank you mgb.
I will check the sample.
The buffers are
A:ammonium acetate 10mM pH 4
B:ammonium acetate 1M pH 4.
They were checked.

I ran complete gradients from A to B and I saw no peaks. The non-retained fraction is huge.

Do you have a standard test sample to show that
1) the column is not dead and
2) the mobile phases work as intended to allow a separation by conductivity / ammonium cation displacement. Have you had success with this mobile phase pairing before?

How do you know what is in the flow through?

I have a few suggestions for you.

Since it is a Strong cation exchange column, use a stronger salt than ammonium acetate. Use Na2PO4 (10-20mM) and 1MKCl; probably 0to100%B in 60 min at the begining. I think that is where your problem is because if it is a strong cation binding, you need a stronger cation than NH4+. It is OK to add 5-10% ACN to your mobile phase too if your column allows to use organic (check manufacturerrs specs).

I think this should work for you.

Cheers!

Ananda S.

Thank you mgb.
I do not have a standard test sample but I purify proteins with this column and it works.

I had not used these buffer before. I have heard about successful gradients from water to ammonium acetate 1M pH 5.2 in a strong cation exchanger.

My purpose is to fractionate using volatile buffers. After chromatography I am going to freeze-dry the samples. The purpose of using low pH and ion strength is to guaranty the best retention.

Thank you Ananda.

I think it may be best to focus on the fact that nothing binds. Not a thing.

Either one or a combination of pH and conductivity could be the problem.

Do you have inline pH and conductivity meters?

Is the on column conductivity and pH at a suitable 100%A level before the sample is loaded.

Is there a pH or conductivity spike or trough when the sample is loading.

Thank you mgb.
I don't have pH meter inline but a conductivity
meter I do have.
The column is equilibrated with more than 10 volumes of buffer A. The capillary tubings of conductivity and UV detectors are well equilibrated in 100% A.
But when the sample is loading conductivity increases. Maybe it still contains amonium acetate after the freeze-drying step. The ammonium acetate is introduced in a previous chromatography step (gel filtration). After this I freeze-dry the sample and apply it in IEX.

If you do your gel filtration at 10mM NH4OAc pH4, what happens if you load this desalted material directly onto your cation exchange column.

Let's go back to the earlier question by mgb: how do you know that your peptides are positively charged at your pH? most peptides may be neutral. I suggest you go to pH 2.

Thank you all for your suggestions: mgb, Ananda and Anonymous.

I think the suggestions above are a good starting point but would like to add a few more comments.

First, to clarify some of the comments above: the relative elution strength of cations is: Li is less than H which is less than Na which is less than NH4 which is approximately equal to K for sulfonic acid resins so if you are observing insufficient retention you should use sodium or better still lithium based eluent systems.

Second, as mentioned above you need to make sure that you maximize the ionization of your peptide since your problem is insufficient retention. You need a pH low enough to completely protonate all acidic amino acids in your peptide. As mentioned above, this may require pH values as low as 2.

Third, your problem might be that the compound is very weakly retained simply because the net charge can never be greater than 1 (for example). If you're peptide has only one ionizable cationic group, even if you fully ionize that group you will still need to use a fairly dilute eluent in order to achieve retention. In this case, perhaps you'd be better off doing this separation by reversed phase or anion exchange.

Thank you Chris for your advices. I will let you know as soon as a have results.