Chromatography Forum

I have seen recenty in a UPLC impurity method that has chromatographic conditions:
Flow: 0.3 ml/min
Column temperature:65 celcius
Wavelenght: 250 nm
Buffer: 6,25 g SDS and 2,73 g monobasic sodium phosphate dissolved in 500 ml water.Ph is adjusted to 6,8 with %2 NaOH.
Mobile Phaes: Buffer: ACN:MeOH (55:25:25) mixed by volume, and pH is adjusted to 4,2 with phosphoric acid.
Run time: 45 minutes.

You see the usage of high amount of SDS. After 10 injection , one has to clean the column. Thus one analysis is so much time consuming eventhough it is UPLC method.
In my opinion, SDS should be replaced with ion pair agent like Heptane sulfonic acid. Also mobil phase's pH is adjusted to 4,2 after mixing ACN and MeOH. Such a pH adjustment prone to errors since pH adjustment is not measured in aqueous medium.

Am I right about my comment? I want to know your comments on the methods.

The use of Heptane sulfonic acid in place of SDS will decrease your resolution and might not be anymore adequate for your separation (but maybe it is, you need to do the experiment). Equilibration time will indeed go down...

From the moment that you adjust the pH the same way every time, the method will be reproducible and that is what is all about...

I am not too keen on the method, and if this were a method that I would be given, I would argue to redevelop the method.

With SDS, it takes time for the column to equilibrate with the reagent. It can easily be hours. The fact that you need to wash the SDS off the column after 10 runs might suggest that at 10 runs, the column is finally equilibrated with SDS, but the method does not work any more. This is not good.

Also, a pH adjustment after the addition of organic solvent is problematic. I assume that this is done, because if you would do it in water, the SDS would precipitate. I can't tell if the condition that is used is at the buffer pKa of phosphoric acid in this mobile phase condition. It could very well be OK, since the pK would go to a higher value with the addition of organic, and you have plnety of organic in the mobile phase. My suggestion would be to use a predetermined amount of phosphoric acid rather than measuring the pH after the addition of the organic solvent.

The use of another ion-pairing reagent might speed up equilibration, but it is not clear at all if you will get to the same point as with the SDS. On the other hand, since you appear to never reach equilibrium with SDS, might as well try something new.

One injection runs 45 minutes. 45 minutes run? So where is the benefit of UPLC. UPLC was supposed to shorten analysis time. The method is validated. But it is not user friendly.
Do you use often SDS in mobile phases?

I suppose the method could use some improvement...

The method seems like it leaves a lot to be desired but you can never tell unless you know more about the number and nature of the compounds that are separated... maybe there is some merit for the present method...

In agreeing w/ Uwe, I'd also like to add that I have found SDS, no matter the source, to be among the dirtiest thing I've ever had to add to any mobile phase. If you haven't been filtering it much, I'd suggest that you make it the first addition to the aqueous portion, thoroughly filter it, then go on w/ the rest of the MP prep. I think you will be surprised at the look of the filter membrane after passing your SDS solution through it.

The question you need to ask the original method developers is: what prompted them to use SDS in the 1st place.

I'm guessing it is because you have basic analytes? Could you start with lower organic to obtain adequate retention perhaps?