Chromatography Forum

Hi All,
Has anyone ever attempted development of a method to assay HPMC using an RI detector via size exclusion. Lots of stuff out there using CAD detector but trying something new with RI detector and size exclusion column. Please let me know if you have any thoughts and thanks again.

Did you saw that:??
J Chromatogr A. 2011 Aug 26;1218(34):5787-93. doi: 10.1016/j.chroma.2011.04.076. Epub 2011 May 6.

Characterization of hydroxypropylmethylcellulose (HPMC) using comprehensive two-dimensional liquid chromatography.

Greiderer A1, Steeneken L, Aalbers T, Vivó-Truyols G, Schoenmakers P.

Hi.
Are you successful with your adventure with Hypromellose?
We have been testing for this polymer in our Eye drop product.
We use 3 columns in series.
Good luck.
Alfred

Hi Alfred 88,
Making some progress but not there yet. A couple of questions:
The use of 3 columns in a series, is that to improve resolution? I know SEC isnt the best technique to resolve components that are similar in size (Da)
What is the size in daltons of your HPMC?
Have you determined if HPMC is retained and not excluded on your column?
Can you disclose the range of your column in terms of Daltons ie what are the exclusion ranges?
Can you speak more about your method?
Thanks again.

Dear sahburn.
I try to answer your questions below.
The use of the 3 columns is to achieve the separation (resolution). The MW ave. of HPMC is about 1 Million Daltons (off my head).
We completed the validation for the test, and confirmed that HPMC is retained (not excluded).
I cannot reveal the range of the columns, or the vendors/MFGs at this time. (Besides, I am at home with no access to SOP).
The assay/test is performed on a Shimadzu HPLC with 2 pumps, one autosampler, one RID and one UVD. The CRM (standard) is from USP. The product is an Eye drop that we have been selling for ~10 years in the US market (plus Canada). The method is proprietary (I am the author).
I hope that you will succeed. Best wish for you.
Alfred

Hi Alfred,
Thank you for the response. I just want to ask a couple more questions but directly related to the chemistry and not specific to your method.
1) If the molecular weight of HPMC is about 1 million Daltons why not use a column that can accommodate weights greater than 1 million ie range of 100,000 to 2 million? Those columns exist.
I know some polymers aren"t straight but form bends or kinks as the unit chain increase (like what happens to a bamboo pole as it extends....it bends) could that be the reason why three columns are used in tandem rather than one column that has a large molecular weight range. ie even though the molecular weight of the column is acceptable for he polymer there are other forces acting that causes the polymer to be excluded from the column.
2) While buffers are used to interact with the column so that no secondary forces interacts with the polymer (so that separation is purely exclusion), could the buffer play other roles?

Thanks much and have a great day.

Ahh the polyelectrolyte effect.

When you say the need to use a series of column to aid in resolution it is because you have other polymers in solution and the three columns will help in resolving these peaks. If you only had HPMC in the formulation then one column would be enough to quantify HPMC in the product? Thanks again.