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Calculating pH

  • Nancy Schwartz
  • #Chromatography Fundamentals
  • #Raptor LC Columns
  • #Analytical Columns
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  • #LC
  • #HPLC & UHPLC Columns
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I’m guessing that most of us that work in the lab would find it more convenient to measure a pH rather than to calculate a theoretical value.  But, at times, it can be very useful to make that calculation and get an idea of what to expect.  As far as chromatography is concerned, all columns have suggested operating ranges for pH, so it is important to know whether or not it is safe to inject your sample. Of course, this pertains more to reverse phase LC, because sample extracts usually do contain at least some water.  (In fact, pH can only be measured in the presence of water to allow for dissociation.)

Here is what you need to know to calculate pH:

  • For weak acids or weak bases, the pKa of the acid or compound in solution
  • The molarity (M) of the solution, which is defined as moles per liter
  • If concentration is only known in weight units/volume, you will need to know the molecular weight of the acid or compound.
  • If concentration is only known in terms of volume/ volume, you will need to know the density of the acid or compound.

For strong acids

There could be a need to calculate pH as well for strong bases, but acids are much more commonly used with reversed phase LC, so our discussion here will be limited to the acids. Before calculating the pH, first determine the molarity of your solution.

Example calculation:  0.2% TFA (in water), v/v or volume/volume

Density =1.49 g/mL, Molecular Weight =114 g/mole

Molarity =2mL/1 L x 1.49g/mL x 1 mole/114g= 0.026 moles/L =0.026M


Then, determine the molarity concentration of H+ ions from the dissociated compound in solution. This is needed because pH is defined as the negative log of the concentration of H+ ions in molarity, expressed as [H+].

In the example above, TFA is a strong acid, so is 100% dissociated. In this case, the calculation is easy because the molarity for H+ ions is the same as the molarity of the acid.  It is 0.026M.

So, the pH is calculated for the example like this:

pH=-log (0.026)= 1.6

A good reference that can walk you through the calculations for pH and related topics can be found here at Purdue University’s website:


For weak acids or bases 

The example above is for a strong acid. To do this calculation for a weak acid or base, you would need to know the Ka, which is the acid dissociation constant for the acid, or the pka, which is the negative log of Ka. For this discussion, we will focus primary on weak acids, using the following equations:

Ka = [H+] [A-]/[HA]

pKa = -log Ka

Let’s do an example here for a monoprotic weak acid, in this case, a 1M solution of acetic acid.  If you look up the pKa for acetic acid, you will find that it is 4.754. So, using the above equations, we calculate:

pH for weak acid


So, now we know that a 1 M acetic acid solution has a pH of 2.38.


The next question you have may pertain to actual limits that we recommend for our columns. The following table lists some general guidelines for HPLC:

LC pH ranges


If you compare the above ranges to the example we did earlier for TFA, you can see that a 0.2% TFA solution is a bit too acidic (pH= 1.6) for most of our HPLC columns. It is easy to see why we do not recommend TFA as a mobile phase modifier very often. Also, I always try to remind folks to check their mobile phase periodically, as mistakes in preparation could lead to column damage if it should go unnoticed.

On the other hand, our result for 1 M acetic acid solution is too close for comfort to the lower pH limit for all of our fully porous particle columns. However, it should be OK for any of our Raptor columns.  Again, it is a good practice to check the pH and be certain no mistakes were made. 1M is actually higher than you will usually see for HPLC mobile phases. Usually for this reason, acetic acid does not present too much cause for concern.

For guidance with GC columns please see the following FAQ and blog post:

Can I inject acids or bases onto my polysiloxane column? Will they damage the column?

Capillary GC Column Killers- Part 1

I hope that you have found the suggestions and information here useful. Thank you for reading.


Mon, Dec 03, 2018

Hi Justin, yes the molarity comes into play in the calculations. For a strong acid, the pH equals the negative log of the molarity. For weak acids, the molarity determines the [HA] concentration that is used in the equation. I hope that helps to clarify.

Fri, Jun 08, 2018

Just curious where the 1M comes into it? If the solution was 0.1M, that would have to change the pH wouldn't it?