Basic question mobile phase preparation

[dragosb]

Hello,

I have a basic question concerning mobile phase preparation.
I intend to use a gradient from 4 to 10% acetonitrile in a mobile phase with 10 mM sodium phosphate buffer.
How should I prepare the two?
Should I prepare first a solution ( say 2L ) 10 mM buffer and then in two different vials should I put the desired ammount of acetonitrile and buffer up to say 1L or shoud I put the ammount of buffer for 10 mM in a vial, and put water and acetonitrile up to 1L. Does it make any difference, because for me in the first case I would say that I have a dillution and that in the end I do not have 10 mM anymore.

Thanks for helping me with this question

[JMcK]

Are you developing this method, or are you trying to follow someone else's procedure? If you're following someone else's procedure I would contact them and ask for clarification, because the difference in mobile phase prep may be enough to shift peaks slightly and could cause some trouble.
If you are developing this method, and you're not sure of the gradient yet, use the 10 mM buffer as mobile phase A, and ACN as mobile phase B, and vary the gradient as needed to achieve suitable separation.
Once you decide on the conditions you can premix the mobile phases if desired. I would suggest for mobile A containing 4% ACN, mix 40 ml of ACN with 960 ml of buffer. This mixing is consistent with mixing that the gradient system uses, volume to volume, rather than volume to final volume.

[Andy Alpert]

We prepare such mobile phases by first preparing a stock solution of the salt. In your case, you might make a (filtered) 100 mM solution of the sodium phosphate. Then, for every liter of mobile phase, put 100 ml of the stock solution into a 1-L. volumetric flask. For the 4% ACN mobile phase, add an additional 896 ml of water to the flask. Then, add ACN to about 1 ml below the calibration mark. Invert the flask 8-9 x, then allow the contents to reach room temperature (the addition of ACN to water is endothermic, so the solution will cool. As it warms to room temperature, the volume of the contents will expand). Then, add ACN dropwise to the calibration mark. Invert 8-9x.


Adjustment of pH: Addition of ACN will shift dissociation constants and hence the pH. Accordingly, we always adjust the pH as needed at the stock solution stage and don't bother measuring or adjusting pH after addition of ACN. Your should specify these details when writing up your method. The important thing is not to get a specific pH in a water-ACN mixture, it's to specify the method you used so others can reproduce your results.

[DR]

Are you developing this method, or are you trying to follow someone else's procedure? If you're following someone else's procedure I would contact them and ask for clarification, because the difference in mobile phase prep may be enough to shift peaks slightly and could cause some trouble.
If you are developing this method, and you're not sure of the gradient yet, use the 10 mM buffer as mobile phase A, and ACN as mobile phase B, and vary the gradient as needed to achieve suitable separation.
Once you decide on the conditions you can premix the mobile phases if desired. I would suggest for mobile A containing 4% ACN, mix 40 ml of ACN with 960 ml of buffer. This mixing is consistent with mixing that the gradient system uses, volume to volume, rather than volume to final volume.

[juddc]

Alternately, if you have quaternary mixing capability, you can prepare perhaps a 50mM phosphate buffer at your desired pH, place that in one reservoir (A), put water in your second reservoir(B), and acetonitrile in your third reservoir(C), and keep the phosphate concentration constant by running a gradient from 20:76:4 to 20:70:10.

This would yield several advantages in my humble opinion: First, it would be more reproducible than premixing, second, you could vary your phosphate concentration if needed, third, you'd be able to rinse the phosphate from the system when you're done very easily (doing this is VERY important), and fourth, you'd have the equivalent of 5L of 10mM phosphate to work with if you had a long run. Topping up water is much less of a PITA and introduces less error than making 2 or three lots of buffer over a short time period. If you're running near a neutral pH, be careful of biological growth in your aqueous solutions, however. Don't run them for more than a few days.

Hope this helps!