Hyaluronic acid method optimization.

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

5 posts Page 1 of 1
Dear fellow Analysts,

I am currently trying to optimize an existing HPLC-method for the determination of Hyaluronic acid. The method uses a Shodex OH-Pak SB-806 HQ, 300 x 8.0 mm column which is based on size exclusion (SEC). The Hyaluronic acid peak comes at 10 minutes and has a width of 8 - 12.5 minutes which is a very broad peak. The problem is a very close negative peak at 13.5 minutes, followed by two more peaks. I was wondering if anyone can provide some ideas how to make the Hyaluronic acid peak come earlier, hence moving it away from the other peaks.

I have inserted a typical chromatogram below and the method details are as follows:

Column: as mentioned above
Guard column: Phenomenex Security Guard C18, 4 x 3mm
Flow rate: 0.8 ml/min
RI -Detection
Columntemp: 21C
Detectortemp: 35C
Run time: 25 min
Mode: Isocratic
Mobile phase: 11.7 g Sodium chloride + 0.1 g sodium azide, dissolved and diluted to 1000ml with water.
Sample diluent: same as mobile phase

Image

Many thanks in advance!

Max
why are you using a C-18 guard column for a SEC application? i would remove it
your peak is maybe very large because you have a very large population of this polymer.
the peaks at the end of the chormatogram can be from the salts you use

can you say the MW that you have of your compound?
it can be around several millions to 20 millions Da.
one way to move it closer to the beginning of the run is take a column with a lower exclusion limit but that will maybe give you false results if you are looking at calculating the MW and poly-dispersity
unmgvar wrote:
why are you using a C-18 guard column for a SEC application? i would remove it
your peak is maybe very large because you have a very large population of this polymer.
the peaks at the end of the chormatogram can be from the salts you use

can you say the MW that you have of your compound?
it can be around several millions to 20 millions Da.
one way to move it closer to the beginning of the run is take a column with a lower exclusion limit but that will maybe give you false results if you are looking at calculating the MW and poly-dispersity


The guard-column is actually not used anymore with this method. For some reason its still listed in the test procedure. The chromatogram I attached was already generated without a guard column. The compound has a molecular weight of about 1.2 - 3.0 x 10 to the power of 6.
your column is a 13u particle size column for 20 million Da exclusion limit
what you can do to improve on the chromatography and reduce RT is to use a 5u particle size column with 500A pore size
we like to use sepax these days for GPC, and such a column would have a 5 million Da exclusion limit
if you need to be on the safe side you can go for a 1000A and 7.5 millions Da exclusion limit

the 500A column should put the peaks closer to 6 minute RT
the peak shape should improve if you have a low population dispersity
the negative peak is the low Mw inclusion limit
maybe you can make it go away by changing the mobile phase if it is possible.
Hi, you can refer to the Phenomenex catalog for SEC columns, the chromatograms published are vey similar to yours.

Good luck
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