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Basic Analytes and Ion Suppression

Unlike the dissociated (ionized) acids, which when charged, elute rapidly from the column, protonated bases often have long retention times and poor peak shape.  This retention behavior is due to the interaction with residual silanol species on the silica surface.

Separations of basic compounds, however, are not were not traditionally carried out under ion suppression conditions as the analyst would have to raise the pH of the mobile phase to produce the neutral molecule. High pH mobile phases can damage traditional silica based RP-HPLC columns (working pH range 2.5-7).  However, columns which are specifically designed to operate with high pH conditions (working pH range 1-12) are available allowing the analysis of basic analytes using ion suppression.  Traditionally the analysis of weak bases has been carried out at low pH; essentially because the surface silanol species are non-ionized (pKa approximately 3.5) and peak tailing is improved
(Figure 1).

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Figure 1: The effect of mobile phase pH on the extent of surface silanol ionization.

Key Learning Points

  • The extent of ionization of the surface silanol groups directly influences the peak shape of basic species – non-ionized silanol groups result in the least peak tailing, whilst the ionized form results in the worst peak shape
  • At pH 3.5 the surface silanol groups are 50% ionized/50% non-ionized
  • Lowering the mobile phase pH causes the acidic silanol groups to become less ionized – below pH 1.5 the silanol groups are approximately 100% non-ionized and peak tailing will be reduced (care should be taken at this extreme mobile phase pH as the bonded phase may be degraded)
  • By raising the pH to 5.8 the silanol groups will be 100% ionized and peak tailing will be at its worst with respect to secondary silanol retention

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically), highly surface active base such as triethylamine (TEA), piperazine, N,N,N’,N’-tetramethylethylenediamine (TMEDA), or dimethyloctylamine (DMOA) (Figure 2).  These bases interact with the surface silanol species in preference to the analyte molecule and are called ‘sacrificial bases’.  They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times.

Figure 2: Sacrificial bases.
 
Figure 3: Improvement in basic analyte peak shape at low pH due to reduced surface silanol interaction.

 

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Figure 4: Improving peak shape for ionizable analytes using triethylamine as a mobile phase modifier.
 

It is possible to improve the peak shape of basic analytes using a sacrificial base.  In this example the amphetamine molecule (pKa = 10.01), exhibits very bad tailing with no triethylamine added (pKa = 10.21), in a mobile phase of pH 7.1.  As the concentration of sacrificial base is increased, the peak shape steadily improves.  The curve representing peak tailing against TEA concentration in the mobile phase plateaus at approximately 10 mM, indicating almost total coverage of the reactive silanol groups (Figure 4).

There is often a necessity to improve peak asymmetry (tailing) when analyzing bases as many are ionized at pH values used with traditional silica columns.  HPLC column manufacturers produce columns which can be used to analyze basic analytes; these columns will either by produced from Type B silica, which has fewer surface active silanols, or will have been endcapped to reduce the number of silanol groups available for the analyte to interact with.

It should be noted that columns are available that can be used at high pH to analyze the ion-suppressed base
(non-ionized).

For more information see the CHROMacademy module - Column Chemistry.

 

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically), highly surface active base such as triethylamine (TEA), piperazine, N,N,N’,N’-tetramethylethylenediamine (TMEDA), or dimethyloctylamine (DMOA).  These bases interact with the surface silanol species in preference to the analyte molecule and are called ‘sacrificial bases’.  They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times.  The interaction of TEA with the silica surface is shown in Figure 5.

Figure 5: Use of a sacrificial base.

The use of both triethylamine and high mobile phase pH has been used to improve peak shape for the separation of antihistamine analytes (Figure 6).  At high mobile phase pH values (i.e. 2 pH units above the pKa of the analyte) the analyte will be non-ionized (neutral) and there will be no interaction with the silica surface silanol groups, reducing any peak tailing.  When a mixture of analytes with varying pKa values (i.e. the separation of the antihistamines, Figure 6) are analyzed the use of increased mobile phase pH may not render all analytes neutral, therefore, TEA (or another sacrificial base) can also be used to interact with the surface silanol species and improve peak shape. 

The column used in this particular separation has a bidentate ligand structure which acts to protect the silica surface from hydrolysis at high pH.  Most manufacturers have columns that can be used at extremes of pH.

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Figure 6: Improving peak shape for ionizable analytes using high pH and triethylamine as a mobile phase modifier.
 

Ion pair reagents can be used when other methods such as reversed phase and ion suppression techniques have not been successful.  Samples containing both anionic and cationic components have one type ‘masked’ by the ion pair reagent and the other suppressed by pH.  This is a useful technique if the pKa of sample analytes are not similar.

For example:

  • Tetrabutylammonium phosphate +N(C4H9)4 at pH 7.5 forms strong ion pair with acids, the pH suppresses weak base ions (for bases with pKa ~5.5)

  • Alkylsulfonic acids -SO3(CH2)nCH3 (n = 4-7) at pH 3.5 forms ion pairs with bases and weak acids (with pKa ~5.5) are suppressed by pH

  • Trifluoroacetic acid (TFA) forms ion pairs with bases

The mechanisms by which ion pair reagents are thought to operate by are: 
1) The analyte is paired in solution with the ion pair reagent and the neutral complex undergoes partition interactions with the stationary phase or
2) The ion pair reagent populates the hydrophobic surface and the analyte molecules undergo an ion exchange reaction.  This method is generally accepted as the primary mode of ion pair retention.

Some disadvantages of ion pair reagents are the long equilibration times that are required (> 100 column volumes).  Ion pair reagents are notoriously difficult to remove from the column, therefore, it is recommended that a guard column or dedicated column is used for ion pair applications.  Ion pair reagents, along with irreversibly modifying the stationary phase, will drastically reduce the column lifetime.  Finally, ion pair reagents are not generally suitable for LC-MS work as they will suppress ion formation and reduce sensitivity.  There are, however, alternative approaches that can be used with LC-MS such as HILIC and mixed mode stationary phases.

Further information on:

Ion Pair Chromatography

HILIC and Mixed Mode Chromatography

 

 

 
 
 
 
 
 
 
 
 
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