Chromatography Forum

Hi ,

I have the following questions about the USP method for Insulin High Molecular Weight Protein (the separation of insulin hexamer, dimer, and monomer):

• 1. What is the source (or reference) for USP methods?
  2. Is this method a size exclusion (aka gel filtration) chromatography method? Please explain how separation of SEC differ from RP.
  3. Why is and what is the purpose of L-Arginine used in the mobile phase?
  4. The method recommends a USP L20 (Dihydroxypropane group bonded to porous silica particles, 5 to 10µm) packing column of 7.8mm x 30cm. Could anybody suggest a manufactured column with this specification? Please explain how this column difers from a C18 or C8.
  5. Does anybody know any other SEC (or GF) methods for Insulin?

Thanks
Winnie

1. What is the source (or reference) for USP methods?

In principle, the USP method is complete in and of itself. The reference is the USP method. These are available from the USP ( http://www.usp.org/ ). That should also point you to the original publication.

2. Is this method a size exclusion (aka gel filtration) chromatography method? Please explain how separation of SEC differ from RP.

From the column description I would guess that is a size exclusion method. A discussion of the differences between SEC and RP go far beyond the scope of a forum post. An excellent text that covers this and related topics is Basic HPLC and CE of Biomolecules by Cunico, Gooding, and Wehr. It's available from Amazon ( http://tinyurl.com/n4knp ) . The short answer is that SEC separates on the basis of size of the native protein, while RP separates on the basis of hydrophobicity of the denatured protein.

3. Why is and what is the purpose of L-Arginine used in the mobile phase?

I don't know for sure, but I suspect it's there to tie up any residual active sites on the stationary phase.

4. The method recommends a USP L20 (Dihydroxypropane group bonded to porous silica particles, 5 to 10µm) packing column of 7.8mm x 30cm. Could anybody suggest a manufactured column with this specification?

Search all of the usual suspects. These are more commonly known as "diol" columns. Check the original monograph to establish the appropriate pore size if it is an SEC separation.

Please explain how this column difers from a C18 or C8

The "lawyer's" answer is that the diol column has dihydroxypropyl groups bonded to the silica, while the C18 column has octadecyl groups and the C8 has octyl groups (strictly accurate, but not very useful). The chemist's answer is that the diol column is hydrophilic, while the C18 or C8 columns are hydrophobic. If you need more detail, get the book I suggested above.

5. Does anybody know any other SEC (or GF) methods for Insulin?

http://www.google.com/search?q=insulin+ ... 8&oe=utf-8

thanks
WW

Dear Tom:
Your response is good and complete. I just want to add a little to # 1.

USP methods are contributed by various authors who want to make their methods available to the public. Most of the time, USP (an organization) will solicit contributions for test methods on new substances. Even though references are not mentioned in USP/NF (the official book), users can request for reference(s) from the author of each monograph.

Alfred.

Dear Winnie,

We have methods for monomeric insulin which can be applied to the mixture. This is not SEC method but rather mixed mode. You can consider this as laternative approach.
Monomeric insulin has molecular weight around 6000 Daltons which brings dimer and hexamer to 12 kDa and 36 kDa. You can analyze this mixture in reverse phase conditions on Promix MP column.
Here is the link for separation of three insulins (Humulin, Humalog and Lantus):
http://www.sielc.com/compound_212.html

Also check the following brochures it shows method development for insulin:

http://www.sielc.com/pdf/Proteins%20and ... olumns.pdf

regards,

Vlad