HPLC Separation of Hyaluronic Acid

[akthmps]

I am looking for advice from other chemists who have experience (or suggestions) on how to perform HPLC on hyaluronic acid. I am wanting to analyze a solution of approximately 10mg/mL hyaluronic acid (1 MDa). I have setup a system with a Presto FF-C18 analytical column (150 x 4.6mm) in an Agilent LC connected to an ELSD. I can see the hyaluronic acid when I directly infuse the solution into the ELSD, however I can't seem to separate it on the column. The method involves a gradient elution with mobile phase A (10 mM formic acid) and B (acetonitrile) with a gradient of 3-10% acetonitrile over 30 minutes. The column flow is 0.5mL/min, and ELSD has been set with spray chamber at 50C and drift tube at 100C. Since I see the acid on the ELSD when bypassing the column, it appears the problem is with the column or mobile phase setup.

In any event, I'd love to hear suggestions from others. I appreciate any input!

[Vlad Orlovsky]

I have big doubts that your approach will work. Your compound is a complex mixture of polymers, something similar to alginic acid. Here is a method we developed for alginic acid which was validated in one of the companies:

http://www.sielc.com/Compound-Alginic-Acid.html

Separation is done in ion-exclusion/size exclusion mode. Please pay attention to a flow rate. regular flow rate for 4.6 mm column is 1 ml/min.

[akthmps]

Unfortunately, I don't have a Primesep C analytical column but I do have a Primesep D analytical column, 4.6 x 150mm, 5 um, 100 A. Any hope of getting this particular column to work for hyaluronic acid? If so, can you suggest any mobile phase revisions I should take into account?

Do you feel that the higher aqueous content of the mobile phase shown in your application note will lead to increased noise in the ELSD baseline? I'm guessing the 0.25mL/min should help compared to the 0.50mL/min I've been trying with the Presto column.

Thanks again for any suggestions.

[Vlad Orlovsky]

You need to go into ion-exclusion mode and your compound is acidic. Primesep D column is reversed-phase anion-exchange (cation-exclusion) column. You should not have any problem with amount of water in the mobile phase. Our columns are compatible with 100% water and 100% ACN.
You can try Primesep D column but I think that there is a chance that part of your compound will be stuck on the column due to strong anion-exchange interaction. This will cause modification of the column and reproducibility will become an issue. Do you have any of our RP/cation-exchange columns?
You can probably try your RP column at higher pH (above 6) which will cause residual silanols ionization and you will be using RP column as ion-exclusion column. Capacity might be problem, RP interaction might be a problem, etc.

My opinion is that in 99% of cases it is better to use right tool for the task, rather then try to use whatever you have. You would never try to dig foundation for the building with the shovel, right? You will get one of caterpillar excavators
http://www.catallday.com/e-series/?gcli ... MgodO1oA_A

P.S. this is true in US, some other countries with cheap labor can afford digging with shovels.

[upamaniah]

to dear akthmps,
check www.shodex.com, and search for your molecule "hyaluronic"
more over what is your aim of analysis with such a huge molecule and with such a column which can generally used for molecules less than 5000 MW??!!
for more details write to me vlvijayaragavan@inkarp.co.in
regards

[Coffo]

Hyaluronic acid is normally analyzed by SEC but the mobile phase conditions (buffer, salt) would prevent the use of an ELSD.
You might be able to perform a reversed phase separation using the non-porous particle column you mention, but you'll need to go to much higher ACN content than 3-10% ... your gradient needs to go from 0 to 90% or 100% B to start with and then try refining the conditions to optimise the separation.

[max_planck]

I have big doubts that your approach will work. Your compound is a complex mixture of polymers, something similar to alginic acid. Here is a method we developed for alginic acid which was validated in one of the companies:

http://www.sielc.com/Compound-Alginic-Acid.html

Separation is done in ion-exclusion/size exclusion mode. Please pay attention to a flow rate. regular flow rate for 4.6 mm column is 1 ml/min.

Did someone try this method with hyaluronic acid instead of Alginic acid?

Would be interesting to know if this works. As far as I know is hyaluronic acid not UV-detectable due to the missing chromophore.

[max_planck]

Hyaluronic acid is normally analyzed by SEC but the mobile phase conditions (buffer, salt) would prevent the use of an ELSD.

Can't you use an RI-detector?