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Buffers for Reversed Phase HPLC

In order to control the retention of weak acids and bases, the pH of the mobile phase must be strictly controlled.  This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH.  Most analysts will use a buffer to resist small changes in pH that may occur within the HPLC system (i.e. at the head of column when sample diluent and mobile phase mix, or via evaporation of the organic solvent in a pre-mixed mobile phase, ingress of CO2 into the mobile phase etc.).

The definition of a buffer is a weak acid or base in co-solution with its salt – an example would be acetic acid (the weak acid) and sodium acetate (its salt).  A known weight of the salt is usually added to the mobile phase to achieve a known concentration, the weak acid or base is then added to the mobile phase (with stirring), until the desired pH is achieved.

Some common HPLC buffers, their pKa, working pH range, and UV cut off are detailed in Table 1.

Buffer

pKa

pH range

UV cut off (nm)

Phosphate

2.1
7.2
12.3

1.1-3.1
6.2-8.2
11.3-13.3

< 200

Acetate#

4.8

3.8-5.8

210 (10 mM)

Citrate

3.1
4.7
5.4

2.1-4.1
3.7-5.7
4.4-6.4

230

Carbonate

6.1
10.3

5.1-7.1
9.3-11.0

< 200

Formate#

3.8

2.8-4.8

210 (10 mM)

Ammonium bicarbonate

7.6

6.6-8.6

230

Borate

9.3

8.3-10.3

N/A

Table 1:  Common HPLC buffers. 8,9

The compounds detailed Table 2 are not buffers, however, addition of these compounds to a mobile will result in a change in pH.  Many of the compounds listed are used to make buffer solutions along with their corresponding salt, acid, or base, they are also used alone to alter the mobile phase pH (TFA in particular) so that surface silanol species will be non-ionized, and finally some will also be used as ion pair reagents.

Compound

pKa

Trifluoroacetic acid

0.3

Phosphoric acid

2.15
7.20
12.33

Citric acid

3.13
4.76
6.40

Formic acid

3.75

Acetic acid

4.76

Propionic acid

4.86

Carbonic acid

6.35
10.33

Tris

8.06

Boric acid

9.23

Ammonia

9.25

Glycine

9.78

Triethylamine

10.217

Pyrrolidine

11.27

Methanesulfonic acid

-1.617

Table 2: pKa values for common HPLC additives. 7-9

Users of LC-MS require volatile buffers (denoted in Table 1 as #) to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals.  The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects.

A particular buffer is only reliable in the pH ranges given – usually around 1 pH either side of the buffer pKa value (note some buffers have more than one ionizable functional group and, therefore, more than one pKa value).

The concentration of the buffer must be adequate, but not excessive.  In general, HPLC buffers range in concentration from 25 to 100 mM.  Buffers prepared below 10 mM can have very little buffering capacity and impact on chromatography, whilst those at high concentration (>50 mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (i.e. > 60% MeCN), which may damage the internal components of the HPLC system.

Buffers will function correctly unless they are of insufficient concentration or employed at a pH sufficiently away for their pKa – outside their operating range. In the top example (Figure 1), the buffer concentration was inadequate.  Notice that the peak shapes are broad and retention times are unstable. In the example below, the peak shape has improved when using an appropriate buffer concentration and within its operating range.  Typical buffer concentrations range from 25 to 100 mM.

Figure 1: Effect of Buffer Concentration on the Analysis of Basic Analytes in Reversed Phase HPLC.

 

Buffer type, concentration, and temperature can all affect the column lifetime in HPLC.  Ensure all buffers are flushed from the column after use.  Citrate may seem to be a more attractive buffer, however, it will attack and cause corrosion of the stainless steel components of the HPLC system.

Figure 2: Effect of buffer on column lifetime and efficiency.

 

Reversed phase separation of ionizable analytes is particularly susceptible to changes in pH which is why buffers are used to carefully control and stabilize the pH of 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.

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Figure 3: The effect of mobile phase buffer concentration on Selectivity and Peak Shape in Reversed Phase HPLC.

Key Learning Points

  • Some analytes are susceptible to changes in ionic strength which usually presents as a change in retention time
  • Higher buffer concentrations lead to improved peak shape (better efficiency and asymmetry)
  • Below 10 mM the buffer concentration has little effect on the chromatography
  • Ionic strength can critically affect the selectivity of a separation and, therefore, should be optimized and care taken to obtain correct ionic strength when making up buffered mobile phases

 

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 (Equation 1).

Where:
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 factors

  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 (Equation 2).10

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

Figure 4: Buffer capacity for a buffer pKa = 7 as percentage of the maximum buffering capacity.
 

 

 
 
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