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Understanding pH Buffers. Which one to use, and at what concentration.

The importance of pH in HPLC.


For some separations the effect of changing pH is minimal. However for acids and bases, the effect of even a very small change in pH is very significant. Changing the pH changes the degree of ionisation of molecules in solution. It thus affects their polarity, and as a consequence it changes their retention times in an HPLC separation.


Even more of a problem is that it changes the retention times of different components in the sample mixture to different extents. Hence it affects the degree of selectivity. It can mean that peaks become further apart, but it can also mean that at certain pH’s they co-elute, and then the peak elution order will change.


It is therefore important that for this type of separation, the pH is controlled using a pH buffer.



One of the most important things to know about a buffer is its pKa. The pKa (or Dissociation Constant, or Ionisation Constant) is a measure of the strength of an acid or base, and allows us to determine the degree of ionisation at a given pH.


Strong acids and strong bases are those which are fully ionised within the pH range 0-14, and weak acids and weak bases are those which are incompletely ioniosed within the pH range 0-14.


For acids, the Equilibrium Coefficient for the neutral and charged forms is:


Ka = { H+ } { A- } / { H }


The pKa is given by:


pKa = -log10 ( Ka )


At a given temperature these are Thermodynamic Ionisation constants, which are independent of concentration. Since log 1 = 0,


the pKa corresponds to the pH at which the concentration of ionised and neutral forms are equal.


Note that pKa’s are temperature dependent. It is normal to quote the pKa at 25oC.

The example below shows an acid with a pKa of 8.0:



The example below shows a base with a pKa of 8.0:



The example below shows a zwitterion with pKa base at 5.6 and acid at 7.0:




Which buffer to use at which pH?


A pH Buffer should be used within +/- 1pH unit of its pKa. When used in this manner, the buffer will change its level of ionisation to counter any attempt to change the pH.


Many HPLC methods are designed with inappropriate buffers, often a long way from the pKa. In this situation, the only help given by the buffer in resisting pH change arises because its concentration is very much higher than that of the sample.


The table on the following page gives a list of buffers (downloaded from the internet!) showing their pKa and the pH range within which they should be used.


Procedure for buffer selection


Set up the HPLC method and run trials at different pH’s to establish the effect of pH change. Establish the optimum pH for the separation.


Using the table, obtain a list of suitable buffers with their pKa within 1 pH unit of the desired pH.


If using UV or fluorescence detection, check the UV cut-off of the buffer to ensure that it does not conflict with the detection wavelength being used.




pH Range

UV Cutoff (A > 0.5)

1-methylpiperidine HCI/1-methylpiperidine


9.1 - 11.1

215 nm (10 mM)

Ammonium acetate



3.8 - 5.8

8.2 - 10.2

205 (10mM)  

Ammonium formate



2.8 - 4.8

8.2 - 10.2


Ammonium hydroxide./ammonia


8.2 - 10.2

200 nm (10mM)

Bis-tris propane HCI/Bis-tris propane


5.8 - 7.8

215 nm (10mM)

Bis-tris propane HCI/Bis-tris propane


8.0 - 10.0

225 nm (10mM)

Borate (H3BO3/Na2B4O7 10 H2O )


8.2 - 10.2


Diethylamine HCI/diethylamine


9.5 - 11.5


Glycine HCI/glycine


8.8 - 10.8


KH2PO4/ K2PO4/


6.2 - 8.2

<200 nm (0.1%)

KH2PO4/phosphoric acid


1.1 - 3.1

<200 nm (0.1%)

Potassium acetate/acetic acid


3.8 - 5.8

210 nm (10mM)

Potassium formate/formic acid


2.8 - 4.8

210 nm (10 mM)

Pyrollidine HCI/pyrollidine


10.3 - 12.3


Triethylamine HCI/triethylamine


10.0 - 12.0

<200 nm (10 mM)

Trifluoroacetic acid

< 2

1.5 - 2.5

210 nm (0.1%)

Tri-K-Citrate/hydrochloric acid 1


2.1 - 4.1

230 nm (10mM)

Tri-K-Citrate/hydrochloric acid 2


3.7 - 5.7

230 nm (10mM)

Tri-K-Citrate/hydrochloric acid 3


4.4 - 6.4

230 nm (10mM)

Tris HCI/Tris


7.3 - 9.3

205 nm (10 mM)

Buffer Concentration

The buffer concentration is important for three reasons.

Firstly, selectivity is affected by buffer concentration. As the concentration increases, the faster that polar species are eluted. However the rate at which their elution time decreases may be different for different sample components. Hence by changing the buffer concentration, peaks may be caused to co-elute. Conversely, it also follows that co-eluting peaks may be resolved at a different buffer concentration.

If the buffer concentration is too low, it will not be able to act as a buffer. Hnece the pH will no longer be held at the required level, and as a consequence, results may be different from one day to the next. Generally speaking a buffer should be present at at least 0.005M.

If the buffer concentration is too high, the eluent solution becomes viscous (and hence the back-pressure becomes unacceptable), a silica based column packing will tend to dissolve, even below pH7, and solubility of the buffer becomes an issue when mixed with an organic solvent, making eluents hard to make up and gradients a risky business. Normally 0.1M would be the maximum.

If in doubt, use about 0.05M. Provided that selectivity is not an issue, that should be fine. If peaks are co-eluting, try 0.1M and 0.05M.