Reverse Phase Buffer B

[Johte]

Maybe I'm daft or maybe I missed a detail somewhere. Most of knowledge comes from methods papers, reviews, and book chapters in my field of interest and if that matters it's nucleic acids. I only mention this because I've never read a pure HPLC theory book.

Anyway, buffer B is usually listed as ratio of A(aq):B(org). Occasionally the author will specifically detail that X L of A was mixed with Y L of B. So if you're using an A buffer of 10 mM ammonium acetate (AA) and you make your 50:50 buffer B by mixing equal volumes of A with acetonitrile, your buffer B's final composition 5 mM AA and 50% acetonitrile. A gradient from A to B is a gradient of increasing organic but also decreasing buffer. Is this intended?

My buffer preparation entails adding desired amount of AA stock to a volumetric flask and then proceed. For A, I fill to 90% with water, invert a few times, sonicate, then fill to to 100%. For B, I add enough water for the desired final % aqueous of B, slowly add my organic in portions whilst inverting to 90%, sonicate, and finish to 100%. Such that, both my A and B have same concentration of buffer and is not variable over the gradient.

Is this incorrect?

Thank you.

[Magpie]

I'll leave your question regarding buffer concentration to others.

However, your ratios of water to acetonitrile for your mobile phase may be not what you intend.

From your mobile phase preparation description for a 90:10 water:acetonitrile solution for say a theoretical total volume of 1L , it sounds like you are adding 900mL of water to a 1L volumetric flask and then filling to the mark with acetonitrile.

The problem with this method (if the recipe calls for a 90:10 v:v mixture) is that mixing water and acetonitrile is endothermic and will cause a reduction in volume. Thus you will end up with more acetonitrile than you intend. Thus you may notice your peaks elute sooner than expected.

If you need to prepare a 1L 90:10 mixture, you're better off measuring 900mL of aqueous and 100 mL of organic separately and then mixing in a separate container.

However if I'm not correctly interpreting how you're preparing your mobile phase, feel free to disregard this post.

[tom jupille]

Whether you are "correct" or not is irrelevant. What matters is that you are *consistent*. The best way to do that is to describe your mobile phase prep *explicitly*. The second best is to have an SOP in your lab that covers mobile phase prep.

As the previous post pointed out, the exact procedure does matter. Check this slide from our Making LC Methods Work course for an example (shameless plug, here's a link to the course http://lcresources.com/training/MMW.html ):