Chromatography Forum

Hello,
In our lab, we are using a method from the US pharmacopeia. the mobile phase is a mixture of buffer:ACN (53:47,v/v). The buffer is prepared by dissolving 6,9 g of sodium dihydrogen phosphate monhydrate in 1L of water, then the pH is adjusted to 2.5 with phosphoric acid, after that, we dissolve 12 g of sodium sodecyl suflate in 1L of this solution.
This mobile phase is used for the assay of a basic drug.
When we mix the buffer solution with the ACN, small particules (probably of the ion pairing reagent) are formed.
We suspected that the concentration of sodium sodecyl suflate is too high to be dissolved in a mobile phase with a high organic proportion (47%ACN), so we tried to adjust this concentration with respect to the limits adressed in the pharmacopeia (-10%, 10,8g/l) but we obtained the same results as for the initial concentration.
I ask you kindly, if you have any idea about this problem, to answer the following questions:
- is the concentration of sodium sodecyl suflate too high to be dissolved in the mobile phase
- if this is true, why the concentration is so high in an USP method
- can we use a mobile phase with particules in suspension without harming the column or the HPLC instrument (we tried a few injections, but the substance was highly retained in the column and we got a peak with bad shape only when we increased the proportion of ACN to 57%, Rentention time was 21 min instead of 7min )
Thank you for your help

You mentioned that when you mix buffer solution with ACN you see small particles; are you sure these are not very minute bubbles which form upon mixing ACN and aqueous solutions? The bubbles are so small that they indeed look like a ppt in the beginning. Keep in mind that there is no ion-pairs in solution.

(1) Try sonication and see if the mobile phase becomes perfectly clear. Mobile phases containing particles will ruin the pump seal (act like abrasive sand on polymer seals) and clog the column head

(2) Your salt/ surfactant concentrations seem to be too high. Please check the solubility of sodium dodecyl sulfate in acetonitrile.

Given that your are indeed getting incorrect retention chances are that there is something wrong with the mobile phase.

I 100% agree with Farooq,

Particles in MP are not good for the instrument and the column.
Are you sure that you see particles? Or do you see small gas bubbles?
USP methods are mostly very challenging. Good luck

As an addition: I hope you're adding ACN to the buffer solution, not the other way round, right?
Could you provide which USP monograph we're talking about?
Concenring retention time differences, are you using the exact column specified in the monograph or some "equivalent" column?

Yes, I am adding ACN to the buffer solution and I am sure that the small particules are not bubbles because after mixing ACN and the buffer, the mobile phase is sonicated.
We tried to prepare different mobile phases from different buffers:
- buffer containing 12g/L of sodium dodecyl sulfate (as in the monograph), 10.8, 8.0, 6.0 and 3.0g/L
The mobile phases were clear for concentrations of SDS equal or less than 8g/L, we are also planning to try concentrations between 8.0 and 10.8 g/L
Moreover, we prepared the mobile phase with an adjustment of ACN proportion, original buffer:ACN (63:37, v/v), however, we obtained an unclear solution.

The USP monograph is olanzapine drug substance assay. The same method is used in the British pharmacopeia.
We are using Zorbax C8 column while the column specified in the monograph is Zorbax RX C 8.
Thank you very much.