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How to Select a Buffer for your HPLC Mobile Phase?

Jan 21, 2022

There is a lot that goes into answering this question. 

 I usually give an hour-long lecture on the role of pH and buffers in HPLC mobile phases during my hands-on HPLC courses.

So first let me give you a big picture view and then dive into the most important details.

The first question to help us think is this:  Is pH important?

If your analyte is an acid or a base then pH is incredibly important.  You must set the pH correctly. 

If your analyte is neutral – like benzene, toluene or xylene – the pH has no effect at all.  It doesn’t matter what pH you set, so don’t worry about it.

So the first step is to determine what type of molecule your analytes are.  If they are acids and bases, worry about your buffer.  If they are neutral don’t worry about it. 

Here is the next question:  How do I choose the right pH?

If you have looked for the answer already you probably saw that there is quite a discussion on the topic.  Historically, there is a philosophical conversation about choosing the right pH of the HPLC buffer based on the pKa and the pKb of the analytes.  That gets really complicated, so let me give you my simple solution that works for about 90% of people.  I won’t claim 100%, but for 90% of people this works.

And please bear with me here:

If you are running organic acids; if your analytes are acids, then you want to put acid in the mobile phase.  ‘Acids for acids’ is the way that I remember it.

Why do I say that?  Let’s take benzoic acid as an example.

Benzoic acid can exist in two forms.  Neutral benzoic acid and ionized benzoate.  Sodium benzoate for example is an ionized form of benzoic acid.

The ionized version comes off early because it is very water soluble.  The non-ionized version comes off later.  That means that the two different forms of benzoic acid have two different retention times.  That is terrible, you don’t want to live with that!  So you must set the pH in order to keep it from ionizing.  So in this case, low pH will make benzoic acid 100% neutral.  Once it is 100% neutral, life is easy.

So here is the ‘official’ rule:  Set the pH two units below the pKa.  That’s the rule, but here is my easy answer:

Run everything at pH of 2!  Run acids, bases and neutrals at pH of 2.

Now you are thinking, ‘That is really too simple.’  It is too simple, but it works and let me explain why.

A pH of 2 is the lowest pH that we are supposed to use in HPLC.  You don’t want to go too much lower than pH of 2 because that damages the column.

So if I go to pH of 2 then I suppress every organic acid in the world (that could be suppressed).  When I say suppressed I mean that the acid will be in its neutral form rather than its ionized form.

If your pKa is 5 or 4, 6 or 4.5 – I’ve got you covered, pH of 2 is golden.

Now you could challenge me on that.  You could say, ‘What if I have a pKa of… 2!?’  Well you got me there. I would have to run a pH of 0 which I can’t do.

At pH of 2 I will suppress any organic acid that could be suppressed.  By the way, that is all the carboxylic acids – we’ve got them covered.  

So pH of 2, all the acids are happy.   All the neutrals don’t care – they are happy.  Now let’s talk about the bases.

If we follow the same philosophy we should run bases at a pH of 12.  In theory that is a good idea but in practice – please don’t do that! – it’s a terrible idea.  You will destroy your column in hours!  At pH of 12 you will dissolve the silica.  

So we have always had this dilemma:  How do we run bases?  They are challenging.  

Well here is my trick, and I admit it sounds backwards:

We run the bases at pH of 2 as well.  How does that work?  Yes, they are ionized, they are now positively charged.  In the olden days that was a problem because our columns were prone to having negative charges.  The negatively charged silanol groups would stick together with the positively charged ionized bases.  This would result in tailing peaks.

But now we can buy columns without negative sites.  When you buy a good base-deactivated column that means that there are no negative sites.  So the fact that the base is ionized and positively charged isn’t so bad anymore.  There is nothing on the column for it to stick to. 

So pH of 2 is a great starting point for all acids, all bases and all neutrals because it works 90% of the time.  That is why I use phosphoric acid in my mobile phase.  It makes all tailing go away and everything looks beautiful at pH of 2.

However, I will put some of you into a special category.

If you work at a pharmaceutical company you probably want to use a buffer.  That means that your mobile phase will always be within a defined pH range.  A buffer maintains the pH even when there are other sources of acidity or basicity.  For example if your source water has varying acidity, if carbon dioxide in the air dissolves into your mobile phase (normally adding to acidity) the buffer will buffer that and maintain the pH.

Why is that important for pharmaceutical folks?  Because you have the FDA looking over your shoulder and you really want to make sure that your mobile phase is the same today, tomorrow and a year from tomorrow.  That is where buffers come into play.

What kind of HPLC mobile phase buffers do we typically use?

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