In the previous lesson we focussed on LC techniques for separating (low-molecular-weight) molecules. Now, we focus on the separation of ions. The main techniques discussed are ion-pair chromatography (IPC) and ion-exchange chromatography (IEC).
Learning Goals #
After this course you will
- Explain the working principles and applications of ion-pair chromatography and ion-exchange chromatography for the separation of ionic analytes.
- Describe how pH and ionization influence retention behavior of weak acids and bases in LC, and how this can be manipulated for method development.
- Evaluate the roles of ion-pair reagents and counter ions in modulating selectivity and retention in IPC and IEC, respectively.
- Interpret retention models and elution behavior based on analyte charge, counter-ion concentration, and the nature of the stationary phase.
1. Introduction #
In Lesson 6 we have seen that reversed-phase LC (RPLC) is a strong favourite among LC techniques. However, we have also learned that (small) ions exhibit little or no retention and in RPLC. The factor k/(1+k) in the resolution equation implies that ions are hard to separate by RPLC.
There are a number of methods to separate ions, as illustrated in the figure below. One way is to suppress the ionization by varying the pH or by adding an ion-pair reagent to the mobile phase. In this way we obtain neutral molecules or complexes, which can be separated using the LC modes described in the previous class (almost always RPLC). Alternatively, we may separate ions using ion-exchange chromatography (IEC) or capillary electrophoresis (CE). The LC methods are discussed in this class. CE is discussed in three other classes.
Figure 1. Overview of methods for the separation of ions.
READ SECTION 3.5.4
Mobile Phase pH
2. Weak acids and bases in RPLC #
We may assume that the retention factor of an ionogenic analyte (a weak ion) is the weighted average of that of the different forms. For example, for a weak acid \text{HA} we may write
If we introduce the acid distribution coefficient
or
we find
or
This equation represents a sigmoidal curve, as illustrated in Figure 2. Under acidic conditions, where \text{pH} \ll \text{p}K_{\text{a}}, this reduces to k_{\text{A}}=k_{\text{HA}}. When \text{pH} \gg \text{p}K_{\text{a}} we find k_{\text{A}}=k_{\text{A}^-}.
Figure 2. Schematic illustration of retention behaviour of some weak acids and bases as a function of pH.
For a weak monoprotic base the corresponding equation is
where k_{\text{B}} is the retention factor of the intact acids, k_{\text{HB}^+} is the retention factor of the protonated base. It is clear from Figure 2 that the pH has a strong effect on retention and on selectivity of ionogenic solutes in RPLC.
EXERCISE 1
3,4-Methylenedioxymethamphetamine (MDMA, or “ecstasy”) has an estimated \text{p}K_{\text{a}} value of 9.9. It was found to elute nearly unretained on an ODS column using ACN-water mixtures at \text{pH} = 7 as mobile phase.
Why do you think MDMA is nearly unretained at \text{pH} = 7?
Correct! Because MDMA is fully protonated and retention of the charged form is low.
Incorrect. Check the pKₐ: MDMA is still ionized at pH 7. Hydrophobic interactions dominate in reversed-phase LC, but only for neutral species.
At which pH do you expect higher retention for MDMA?
To effectively suppress its ionization, we would need to increase the pH well above 9.9. It must be well above because (i) when the \text{pH} is equal to the \text{p}K_{\text{a}} half of the molecules are neutral, and (ii) in the vicinity of the \text{p}K_{\text{a}} peak shapes tend to be poor.
Incorrect! The general pH preference doesn’t override analyte-specific ionization. Stability isn’t the issue: consider ionization and hydrophobicity.
Why are experimental data at this higher pH not commonly available?
Silica-based ODS columns degrade under basic conditions.
This is not correct. The limitation is related to the column, not the analyte or detection.
READ SECTION 3.7.3
Ion-Pair Chromatography
3. Ion-pair chromatography #
Adjusting the pH suffices to achieve retention for weak acids and bases. For strong acids, such as sulphonic acids, or strong bases, such as MDMA this is not practical. Instead, ionization can be suppressed by forming complexes (“ion pairs”) with ions larger than H3O+ or OH–. Such pairing ions or ion-pair reagents are typically strong ions, such as alkyl sulphonic acids (anions) or alkyl-trimethyl ammonium salts. The extent of retention can be varied by adjusting the alkyl chain length, but many other parameters play a role in ion-pair chromatography. This is because various equilibria all exist simultaneously, as illustrated in Figure 4.
Figure 3. Schematic illustration of the equilibria underlying ion-pairing chromatography. All four depicted equilibria occur simultaneously and at the same location.
The figure illustrates the different equilibria in four windows (that do not represent different parts of the column).
- Depending on its structure, the IPR, denoted with the dark-grey hydrophobic groups and a light-blue anionic group, may adsorb on an ODS stationary phase. More adsorption is expected for longer alkyl chains in the IPR. This equilibrium is illustrated in the second window from the left. (Equilibrium #2)
- It will then act as a dynamic ion exchanger, analogously to the ion-exchange chromatography (IEC). Analyte ions A+ exchange with counter ions C+. This is illustrated in the left window. (Equilibrium #1)
- The formation of ion pairs is based on an exchange equilibrium of (loosely associated) counter ions C+ with analyte ions A+. (Equilibrium #3)
- The neutral ion pair IPR-A engages in equilibrium #4 between the mobile phase and the stationary phase. The larger and more hydrophobic the ion pair, the higher its retention.
The result is a complex mechanism that is much more difficult to describe mathematically than the retention of weak acids or bases discussed before.
An good example of ion-pair chromatography is the separation of oligonucleotides with various IPRs, as shown in Figure 3.
Figure 3. Separation of 15, 20 25, 30, 35, 40, 50, and 60 mer oligodeoxythymidines using selected 100 mM ion-pairing buffers. Adapted from [1].
READ SECTION 3.7.1
Ion-Exchange Chromatography
4. Ion-exchange chromatography #
We already touched upon the principle of ion exchange above. Charged groups on the stationary phase may exchange counter ions against analyte ions. In contrast to ion-pair chromatography, however, the ionic groups are (covalently) attached to the surface.
Table 1. Overview of anion and cation exchangers for strong and weak ion-exchange. Not exhaustive.
| Anion exchangers | Cation exchangers | |
|---|---|---|
|
Strong |
Quaternary ammonium –CH2–N+–(CH3)3 |
Sulphopropyl –CH2–CH2–CH2–SO3– |
|
|
|
Methyl sulphonate –CH2–SO3– |
|
Weak |
Diethyl aminoethyl (DEAE) –CH2–CH2–N–(CH2–CH3)2 |
Methyl sulphonate –CH2–COO– |
|
|
Diethyl aminopropyl (ANX) –CH2–CHOH–CH2–N–(CH2–CH3)2 |
|
A distinction can be made between anion exchangers and cation exchangers, and between weak and strong ones. The ionization of weak exchangers is affected by the pH, while that of strong exchangers is not. Examples are given in Table 1.
4.1. Mechanism #
Ion exchange follows a competitive adsorption model, similar to that described for NPLC in Lesson 6. For example, in case of cation exchange, the ion-exchange sites on the stationary surface can either be occupied by counter ions \text{C}^{z^+_{\text{c}}}, with z_{\text{c}} positive charges, or by analyte \text{A}^{z^+_{\text{a}}} with z_{\text{a}} positive charges. The exchange equilibrium is then described by
Equation 3.34: z_{\text{c}} \text{A}^{z^+_{\text{a}}}+z_{\text{a}}\text{CX} \leftrightarrow z_{\text{c}} \text{AX} + z_{\text{a}} \text{C}^{z^+_{\text{c}}}
where \text{X} denotes the ion-exchange stationary phase. The corresponding equilibrium coefficient is
Equation 3.36: K_{\text{AC,IEC}}=\frac{[\text{AX}]^{z_{\text{c}}}}{[\text{A}^{z^+_{\text{a}}}]^{z_{\text{c}}}} \cdot \frac{[\text{C}^{z^+_{\text{c}}}]^{z_{\text{a}}}}{[\text{CX}]^{z_{\text{c}}}}
The ratio {[\text{AX}]^{z_{\text{c}}}}/{[\text{A}^{z^+_{\text{a}}}]^{z_{\text{c}}}} is proportional to the retention factor k_{\text{A}} of analyte A (k_{\text{A}}=\Phi [\text{AX}]/[\text{A}^{z^{+}_{\text{a}}}], where \Phi is a phase ratio). At low concentrations of analyte ions (the usual case in chromatography) nearly all exchange sites are occupied with counter ions, so that [\text{CX}][\latex] is constant and
Equation 3.37:
\log{k_{\text{A}}}=\frac{1}{z_{\text{c}}} \log{(K_{\text{AC,IEC}} \Phi [\text{CX}]^{z_{\text{a}}})}-\frac{z_{\text{A}}}{z_{\text{c}}} \log{[[\text{C}^{z^+_{\text{c}}}]}= \log{k_{\text{A},1}} – \frac{z_{\text{A}}}{z_{\text{c}}} \log{[\text{C}^{z^+_{\text{c}}}]}where \log{k_{\text{A},1}} is a (temperature- and column-dependent) constant that is numerically equal to the logarithm of the retention factor at unit concentration of counter ion. Note that [\text{C}^{z^+_{\text{c}}}] can be expressed in one of various concentration units (e.g. mol\cdotL-1 or g\cdotL-1). The conversion factor will be reflected in the value of \log{k_{\text{A},1}}.
Equation 3.37 shows that a log-log model describes retention as a function of counter-ion concentration in IEC and that the slope of the line is proportional to the charge of the analyte ions. This is apparent from Figure 4B, that is applicable to polyphosphate anions with charges 3, 4, 6, and 8.
Figure 4. (A) Effect of change in counter-ion-concentration on retention; (B) Dependence of \log{k} on counter-ion concentration (\log{C}). Squares = Na3P3O9 (“tri”), Diamonds = Na4P4O12 (“tetra”), Triangles = Na6P6O18 (“hexa”), Circles = Na8P8O24 (“octa”). Based on data from [2].
Different counter ions may show different affinities for the stationary-phase and may thus be stronger or weaker displacers (eluents). Multivalent counter ions tend to be stronger than monovalent ones. For anions we have the following series for the elution strength (at equal concentrations)
F– < OH– < acetate– < Cl– < SCN– < Br– < NO3– < I– < oxalate2– < SO42– < citrate3–
while for cations
Li+ < H+ < Na+ < NH4+ < K+ < Rb+ < Cs+ < Ag+ < Zn2+ < Co2+ < Cu2+ < Cd2+ < Ni2+ < Ca2+ < Pb2+ < Ba2+
EXERCISE 2
You are performing a cation-exchange experiment and you observe a peak in the chromatogram at a retention time of 226 s. You double the ammonium concentration (the counter-ion) and the peak shifts to 106 s. You observe an unretained peak at 66 s in both chromatogram.
What is the charge of this unknown ion?
Use Equation 3.37
\log{k_{\text{A}}}= \log{k_{\text{A},1}} – \frac{z_{\text{A}}}{z_{\text{c}}} \log{[\text{C}^{z^+_{\text{c}}}]}where k is the retention factor, \text{A} denotes the analyte, z the charge of the indicated ion, and \text{C} the concentration of the count ion.
Note that for ammonia z_{\text{c}} = 1.
The easiest way to solve this problem is to subtract the two equations for the two chromatograms, i.e.
\log{k_{\text{A},2}}-\log{k_{\text{A},1}}=-z_{\text{A}} (\log{C_2}=\log{C_1})On the left-hand side of the equation, we get
\log{k_{\text{A},2}}-\log{k_{\text{A},1}}=\log{\frac{k_{\text{A},2}}{k_{\text{A},1}}}=\log{\frac{t_{\text{R,A},2}-t_0}{t_{\text{R,A},1}-t_0}}=\log{\frac{106-66}{266-66}}The ultimate answer can be substituted in the resulting equation from Hint 2.
On the right-hand side of the equation from Hint 2, we get:
\log{C_2} – \log{C_1} = \log{\frac{C_2}{C_1}}The actual concentrations do not matter. The only thing that matters is that it doubled, e.g. C_2 = 2 and C_1 = 1.
Fill in the charge of the unknown ion in the field below. Enter an integer number (no decimals).
Correct!
Unfortunately, this is not correct. Check the hints!
4.2. Ion chromatography #
Ion chromatography (IC) is a term coined for the separation of small ions. Often a suppressor is used that allows suppressing the conductivity of the eluent. For example, an small cation exchange column saturated with H+ ions can be used to virtually eliminate the conductivity of a sodium-bicarbonate (Na+ and HCO3–) solution by forming the neutral carbonic acid (or H2O + CO2). Alternatively, membrane suppressors can be used that are based on dialysis rather than ion exchange. Low capacity ion exchangers and low counter-ion concentrations are needed for prolonged operation of the suppressor and to minimize the contribution of the latter to extra-column band broadening.
Concluding remarks #
In this session, we explored two essential techniques for the chromatographic separation of ions: ion-pair chromatography (IPC) and ion-exchange chromatography (IEC).
IPC allows for the separation of strong acids and bases by forming neutral ion pairs with suitably chosen reagents, offering flexible control over retention and selectivity through multiple adjustable parameters. IEC, in contrast, relies on permanent ionic groups bound to the stationary phase and operates based on competitive adsorption equilibria between analyte and counter ions.
Both techniques address limitations of reversed-phase LC for ionic species and significantly expand the chromatographer’s toolbox.
References #
[1] M. Donegan, J.M. Nguyen, M. Gilar, Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides, J. Chromatogr. A, 2022, 1666, 462860, DOI: 10.1016/j.chroma.2022.462860
[2] Y. Baba, G. Kura, Computer-assisted retention prediction system for inorganic cyclic polyphosphates and its application to optimization of gradients in anion-exchange chromatography, J. Chromatogr. A, 1991, 550, 5-14, DOI: 10.1016/S0021-9673(01)88526-5
