Insulin sodium sulphate buffer?

[apcul]

Hi all,

I am currently working on a insulin HPLC method.
The method is based on the USP monograph for insulin.

Can anyone please explain to me how this buffer works.
dissolve 28.4g of sodium sulphate in 1000ml of water, pipet 2.7ml of phosphoric acid into this solution, adjust if necessary with ethanolamine to pH 2.3 and mix.

Any comments greatly appreciated.

Mobile phase.

[danko]

In this particular case ethanolamine is not only a base for adjusting the pH to 2.3 (if needed) but a competing base in case there are some active silanols. Most relevant to the good old days for type A silica.
The funny thing is, if the pH is 2.3 from the start, you won’t have any ethanolamine in your eluent.
However, if you add too much phosphoric acid, you’ll end up with some ethanolamine, in addition to more phosphate. So, it’s a kind of art. Isn’t it?

Best Regards

[apcul]

thank you for the reply danko,
the ethanolamine is indeed strange.

The Human Insulin monograph also uses a sodium sulphate buffer.
I am more used to method development for small molecules.
I have never used a sulphate buffer before, sodium sulphate seems much more commonly used in protein analysis do you know why.
Why not use a phosphate buffer?
Why is phosphoric acid used with Sodium sulphate what is the reaction equation. what is the acid and the conjugate base?

sorry for all the questions i dont like magic mobile phases.

[HW Mueller]

I thought someone would take this who was more successful at RP protein anal than I was. We were not told but this looks like some sort of HIC is implied. Na2SO4 is not a buffer it is apparently there to raise the ionic strength and do some "salting out" of insulin. The buffering is done by phosphate here. Protonated amines as counterions are common in protein analysis, one can even buy such amine phosphates. I am not sure whether the amine is included only for the silanol interaction mentioned by Danko, or whether it interacts with the protein. With this sort of Na2SO4 concentration and with that pH there wouldn´t be much of a chance for silanol interaction anyway.
As far as the prep is concerned, if you do it by weight you can repeat it sufficiently enough.

[danko]

Hi Apcul,

Yes, you’re right – sulphate is largely utilized in protein separation context. It is important to mention though, that it is not related to its acidic/alkali properties, but rather to its water structuring ditto. It’s a longer story, but very shortly told, it forces the protein molecule to expose its hydrophobic domain/s to the surrounding environment, which consequently facilitates the retention on a reversed phase material. If it sounds interesting you can find out more about it by reading stuff like Hofmeister series etc.
The buffer thing in this particular case is more related to the phosphate part of the eluent. Adding some phosphoric acid, followed by adjusting the pH to 2.3 with ethanolamine results in the following ions 1) ethanolammonium 2) phosphate – which means you have a buffer (pKa 1 for phosphoric acid is about 2.2 so everything is looking fine).
The problem is then, what happens if the pH is 2.3 without adding any ethanolamine?
Btw, I’m not completely convinced that the pH is 2.3 anyway, because the pH is to be adjusted after addition of sodium sulphate. That means that the sodium ions in this high concentration influence the pH reading – it simply “cheatsâ€