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HPLC Method Confusion

Following a published method is great when it works, however, what happens when the chromatography fails and you don’t understand why there are certain components in the mobile phase?

Or when you just look at a method and don’t understand it? One of our CHROMacademy users was lucky enough to find a literature LC-MS method to separate their analytes; however, the mobile phase composition didn’t entirely make sense to them.

The method worked, nevertheless, their natural scientific curiosity got the better of them and they wanted answers. The mobile phase used was acetonitrile/water (15/18 v/v), 1 mM ammonium acetate, and 0.1% formic acid.

This mobile phase composition just does not seem to make sense at all. This was an isocratic separation but our user also had some further questions about gradient HPLC.

Initially all looks well; however, the following questions were raised:

  1. Why were two different types of buffer used for the LC separation?
  2. Why is there ammonium acetate in one mobile phase and formic acid in another?
  3. In gradient HPLC should the formic acid be in the aqueous or organic phase or in both?



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The CHROMacademy team posed the following answers.

Technically the ammonium acetate and formic acid are not acting as a buffer but will be affecting the pH of the mobile phase. A buffer is used to resist small changes in pH and must be made from the acid and corresponding salt, i.e. ammonium acetate and acetic acid or ammonium formate and formic acid.

Therefore, in this case it may have been that the initial user of this method wanted to buffer the system and, hence, chose ammonium acetate and formic acid; however, as mentioned previously this is not a buffer as the correct choice of salt and acid would be ammonium acetate and acetic acid or ammonium formate and formic acid. This may have just been a mistake during development but the method worked, or perhaps the pH was lowered using ammonium acetate and formic acid was added to aid ionization of the analytes, however, formic acid alone would have done this.

Another point to note is the concentration (1 M) of the “buffering” component in the mobile phase.  There are cases where analyte retention in reversed phase HPLC is affected by buffer concentration.  These cases are usually confined to situations where there are ion exchange interactions taking place between basic solutes and acidic silanols on the surface of the silica stationary phase support (separation of basic compounds) using reversed phase stationary phases that have significant silanol activity with mobile phase pH > 3.  Increasing the concentration of the mobile phase buffer, and thereby increasing the ionic strength of the mobile phase, will sometimes suppress this ion exchange interaction and reduce this ‘secondary retention’ effect.  

It should be noted that as the buffer concentration is increased the mobile phase is made more polar (ionic).  This can affect analytes in differing ways depending on their chemistry; some analytes may experience reduced retention while some will exhibit slightly increased retention.  The main objective is to use an appropriate buffer (i.e. buffer pKa vs. mobile phase pH) in the correct concentration to have sufficient buffering capacity to overcome peak shape and retention time irreproducibility.

Buffer capacity is a measure of the efficiency of a buffer to resist small changes in pH.  Buffer capacity (β) can be expressed as the amount of strong acid or base (in gram equivalents) that is required to change 1 liter of a buffer solution by one pH unit.

ΔB = gram equivalent of strong acid/base to change the pH of 1 liter of buffer solution by 1 pH unit
ΔpH = the pH change caused by the addition of the strong acid/base
The buffer capacity depends on two factor:

  1. The ratio of the salt to the acid or base.  The buffer capacity is optimal when the ratio is 1:1 i.e. when pH = pKa
  2. Total buffer concentration.  It will take more acid or base to deplete a 0.5 M buffer than a 0.05 M buffer

The relationship between buffer capacity and concentration is given by the Van Slyke Equation.

Where: C = the total buffer concentration (i.e. the sum of the molar concentrations of acid and salt).


Figure1: Buffer capacity for a buffer pKa = 7 as percentage of the maximum buffering capacity.


The ammonium acetate and formic acid in the mobile phase will reduce the pH (acidic pH) which will affect the ionization state of the analytes and of any exposed free silanol groups on the silica surface. 

Changing the ionization state of the silanol groups on the silica surface can be particularly useful when working with basic analytes, which under reversed phase conditions can exhibit peak tailing. 

The peak tailing is due to the protonated basic analyte interacting with the acidic silanol species (pKa ≈ 3.5, Figure 2) on the surface, which at pH values greater than 3.5 will be deprotonated and negatively charged and can, hence, interact with positively charged basic analytes (this is the secondary interaction which causes peak tailing). 


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Figure 2: The effect of mobile phase pH on the extent of surface silanol ionization.


Therefore, at a low pH even though basic analytes may be protonated (depending on their pKa) and positively charged the surface silanols will also be protonated (neutral) and unable to interact. If there are acidic analytes in solution they will also be non-ionized and protonated ensuring good retention under reversed phase conditions.


Figure 3: Improvement in basic analyte peak shape at low pH due to reduced surface silanol interaction.


Another possible role of the formic acid is that it has been shown to aid ionization of analytes in LC-MS applications.

It has also been noted in the literature that the solubility of ammonium acetate with acetonitrile can be poor and the addition of acid can aid in solubility - this would particularly affect gradient analyses where the percent of organic will increase during the run.1-3

Furthermore, when choosing any additive that will be used in LC-MS applications they must be volatile which is why in this case ammonium acetate and formic acid would be appropriate. Involatile additives will cause fouling of the MS ion source and, furthermore, some additives will cause ion suppression of analytes (TFA in particular). Phosphate buffers are particularly problematic in LC-MS.

Regarding the question about gradient methods and which mobile phase component should contain acid; having acid in only one or in both eluent components are both viable options.
However, the role of the acid in a method must be considered. If the acid is purely lowering the pH then it does not matter if it is only in A or in both A and B as long as there is sufficient acid in the final mobile phase composition to achieve the required pH. For example, if there is 0.1% formic acid in solvent A (pH = 2.68) initially and only pure MeCN as solvent B, during a gradient run this becomes 0.01% formic acid (pH 3.22) with a 10:90 ratio of aqueous/MeCN. If there are analytes that are particularly sensitive to changes in pH this could have a detrimental effect on chromatography. Furthermore, if the acid is being used to lower the pH and negate secondary interactions of basic analytes with surface silanol groups by ensuring that they are non-ionized, then the change in pH to 3.22, which is close to the pKa of a silanol group, will mean that these groups will be 50% ionized and there is an increased chance of interaction with positively charged analytes which will subsequently lead to peak tailing.

Adding formic acid to only B is significant when the ionic strength (I) of the mobile phase is important.

zi = charge of ionic species i

mi = molality of ionic species

Ionic strength is important in separations where ionic or electrostatic interactions are present. This would be relevant in ion-exchange, HILIC (hydrophobic interaction chromatography), and mixed-mode separations whereby reversed phase interactions are also combined with ion-exchange. The ionic strength of the mobile phase plays a major role in the retention of any ion-exchange separation. Eluents with high ionic strength facilitate analyte desorption and are used to elute species from the column (Figure 4).


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Figure 4: Elution considerations in ion chromatography.


Analyte retention in HILIC is due to the combination of partition and also commonly electrostatic processes (Figure 5). 


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Figure 5: Hydrophilic interaction chromatography (HILIC) mechanism.


For both of the cases outlined so far, as long as the mobile phase has been prepared in a consistent manner, there should be no problem with chromatography.

When the acid is being utilized as an ion pair reagent (i.e. trifluoroacetic acid, heptafluorobutyric acid, or hexanesulfonic acid) it is advisable to keep the concentration constant as small changes in concentration can cause significant changes in selectivity. Therefore, it would be necessary to have the ion pair reagent in both A and B solvents.

Often a better and more robust separation will be achieved with the acid in both A and B as there will be no changes in concentration. However, when reporting methods it is important to state clearly how the mobile phase has been prepared so that others can reproduce the chromatography reliably.

In regards to the question submitted by the CHROMacademy user, a similar explanation could be applied. It may have been the case that having both ammonium acetate and formic acid in A and B was required in order to maintain the ionic strength or concentration of one of the additives.

This could be seen as a cautionary tale to make sure that you think about the methods you are carrying out, question anything that does not make sense to you, and make sure to correctly and fully report how you prepared your mobile phase so that others can reliably reproduce your work.



  1. Leitner, A.; Emmert, J.; Boerner, K.; Lindner, W. Chromatographia, 2007, 65, 649-653.
  2. //
  3. //
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