Chromatography Forum

Hi,

I work in the cosmetic proteins industry and I have synthesized a number of proteins that I need the molecular weight distribution on.

In the past, we have used the Tosoh TSK G2000SW XL GEL and TSK G4000SWXL columns but these columns do give accuate results. For example, if I run glycine as a standard I get peaks after where glycine should be and in theory this should be impossible.

Having read literature I believe our current columns are somewhat dated and there are newer, better columns out there. However, I am not sure which columns to choose that would be most appropriate for the work we do.

Information about the products
Type: Proteins (Biopolymers)
Typical molecular weights: 1000 Da - 20000 Da. (This may require more than one column)
Physical form: All liquids
Protein structure: Most proteins will be linear. Though we do have some branched copolymers.
All proteins are aqueous based.

Regards

Gavin

Hi, Gavin -

Our company's PolyHYDROXYETHYL A column with 200-Å pores covers this range nicely. I wrote a book chapter on SEC of solutes in this range in 1999. It's in the Literature section of our Web site; here's a link:
http://www.polylc.com/downloads/SEC_boo ... r_ver1.pdf

Andy Alpert
PolyLC Inc.
aalpert@polylc.com

SEC is based on the hydrodynamic volume not on MW so you should use standards which behave similar to your proteins on on the column. Glycine is very small in comparision to your proteins.

Hi,

I work in the cosmetic proteins industry and I have synthesized a number of proteins that I need the molecular weight distribution on.

In the past, we have used the Tosoh TSK G2000SW XL GEL and TSK G4000SWXL columns but these columns do give accuate results. For example, if I run glycine as a standard I get peaks after where glycine should be and in theory this should be impossible.

Having read literature I believe our current columns are somewhat dated and there are newer, better columns out there. However, I am not sure which columns to choose that would be most appropriate for the work we do.

Information about the products
Type: Proteins (Biopolymers)
Typical molecular weights: 1000 Da - 20000 Da. (This may require more than one column)
Physical form: All liquids
Protein structure: Most proteins will be linear. Though we do have some branched copolymers.
All proteins are aqueous based.

Regards

Gavin

It is very common to have reversed-phase properties in SEC-columns. This can explain why you see peaks after glycine (BTW, do you see glycine in UV?). Add some organic to the mobile phase, if you haven't done that already.

I'm afraid I must disagree. As is addressed in the book chapter I cited in my previous post, amino acids differ considerably in their perceived size in SEC. The more polar they are, the larger is their sphere of hydration, which determines elution in SEC. One can get several baseline-resolved peaks for some amino acids that elute after glycine using the conditions described in the chapter. Trytophan has the highest molecular weight of any amino acid but elutes last in SEC since it's so hydrophobic.

Is there really a SEC column on the market that can separate amino acids, solely by size?

Anyhow, more hydrophobic peptides/proteins will elute later than they should. This can be overcome by addition of e.g. acetonitrile to the mobile phase.

Yes! Again, take a look at my book chapter on SEC of small solutes (it's a free download from our Web site: www.polylc.com). With a 60-Å pore column of PolyHYDROXYETHYL A eluted with 50 mM formic acid or some other mildly denaturing mobile phase, we can get seven baseline-resolved peaks from the set of 20 amino acids, eluting in order of decreasing polarity. That's because the more polar the amino acid, the larger is its sphere of hydration. None of them elute past the Vt peak, so there's no active hydrophobic interaction occurring. This contrasts with highly crosslinked Superdex and Sepharose columns, which do actively retain amino acids and peptides with aromatic residues.

I have to read your application, but at a first glance it appears as you are running some kind of ion-exchange rather than size-exclusion.

It is late in Europe now, and I will have a look tomorrow.

EDIT> I looks like SEC to me! I have never seen any SEC that works in this low molecular weight range before. Also interesting that you can change the range by changing mobile phase.

Andy,

Thanks for your help on this issue.

I have since purchased the column you recommend and it arrived this week.

Before I start using it can you tell me the method that you typically use:
- Flow rate
- Injection volume
- Running time (minutes)
- Mobile phase with specific quantities in g (for 1 litre)
- A set of standards I should use if I expect the MW to be ~2000 Da

Regards

Gavin

Gavin, we have no record of selling a column of this sort in the time frame you describe. Possibly you bought it through a distributor, in which case we would still have no idea what you got. Better contact me offlist to tell me what you bought so I can advise you per these questions.

Andy Alpert
410-992-5400
aalpert@polylc.com