When doing daily testing, we will find some items. Reagents such as sodium heptanesulfonate, tetrabutylammonium hydroxide and tetrabutylammonium bromide are added to the mobile phase used in the test standard. These reagents are called ion pair reagents, which can be used to improve the separation and peak shape and narrow the retention range of samples. Ion pair reagent can be regarded as a manifestation of the introduction of ion chromatography into high performance liquid chromatography. Today, we will talk about some basic principle and some special problems of ion pair chromatography.
Ion pair chromatography (IPC) can be regarded as an improved form of reversed-phase chromatography aimed at separating ion samples. The only difference between IPC and RPC is that IPC adds ion pair reagent R + or R – to the mobile phase, which can interact with a – of acidic compounds or BH + of basic compounds in the process of equilibrium:
Ionized solute ion pair
(acid) A–+R+ ⇔ A–R+
(alkali) BH++R– ⇔ BH+R–
Hydrophilic solute Hydrophobic ion pair
(less reserved in RPC) (more reserved in RPC)
Using IPC can make the retention behavior of the sample change similar to changing the pH of the mobile phase, but IPC can better control the retention behavior of acidic or alkaline solutes without using extreme pH (such as pH < 2.5 or pH > 8). Typical ion pair reagents include alkyl sulfonate R-SO3–(R–) and tetraalkyl ammonium salt R4N+(R+), strong carboxylic acid (usually ionized) (tetrafluoroacetic acid, TFA; heptafluorobutyric anhydride, HFBA（R–））, and so-called liquid separation agents (BF4–、ClO4–、PF6–).
There are two theories about the retention mechanism of IPC.
One theory is that the ion pair is formed in the solution and then retained on the chromatographic column. The solute retention equilibrium process is as follows (take the ion pair formed by ionized acidic solute A– and tetraalkyl ammonium salt R+ as an example):
AA–R+ (mobile phase) ⇔ A–R+ (stationary phase)
According to this statement, solute retention is determined by the following factors:
① The ionized part of solute molecule a in the mobile phase (depending on the pH of the mobile phase and the pKa of the solute);
② The concentration of IPC reagent and its tendency to form ion pairs;
③ K value of ion pair complex A–R+.
Another theory holds that IPC reagent is retained by the stationary phase first, and then the retention of solute is an ion exchange process. For example, ionized acidic mobile phase a – and IPC reagent R+X–:
A– (mobile phase) + R+X–(stationary phase)
A–R+(stationary phase) + X–(mobile phase)
That is, the ion pair reagent R+X– is first adsorbed on the stationary phase, and then the sample ion A– replaces the counter ion X– on the stationary phase. The retention processes of these two IPC may be dominant in any given separation, but which mechanism plays a more important role is neither easy to determine nor important for practical operation.
In IPC, separation conditions that can be used to control selectivity include:
➩ Type of IPC reagent (sulfonate, quaternary ammonium salt, liquid separation agent);
➩ Concentration of IPC reagent;
➩ Solvent strength (B%);
➩ Solvent type (methanol, acetonitrile, etc.);
➩ Column type;
➩ Type and concentration of buffer solution.
Inorganic reagents (or “liquid separation agents”) such as ClO4 -, BF4 – and PF6 – can be used to replace commonly used alkyl sulfonates as IPC reagents. Inorganic reagent has less retention on the stationary phase, and its retention mechanism is closer to the first statement above, forming ion pairs in the mobile phase. The eluent can be better used for gradient elution (with less baseline noise and drift), and can be better dissolved in the mobile phase when B% is high.
However, the use of ion pair reagents also has some special problems. In some cases, it is necessary to strictly control the pH of the mobile phase; The reproducibility of temperature control must be high (more needed than RPC), in addition, some problems in IPC will not appear in RPC separation or different from other RPCs. There are also pseudo peaks, slow changes in the balance of the mobile phase and poor chromatographic peak patterns caused by unknown reasons.
The first is the pseudo peak. When the sample solvent (without sample) is injected into IPC (i.e. blank experiment), we sometimes observe the simultaneous occurrence of positive and negative peaks. The cause of pseudo peak is usually caused by the difference between the composition of mobile phase and sample solvent. The use of impure IPC reagent, buffer or other mobile phase additives will make the problem of pseudo peak more serious.
The second is slow column balance. When a new mobile phase is used, the chromatographic column must be washed with a sufficient volume of mobile phase to make the chromatographic column reach equilibrium. In IPC, the adsorption and desorption of IPC reagent on the chromatographic column are very slow in some cases, which will cause the chromatographic column to not be completely balanced by the new mobile phase. Therefore, whether the old mobile phase or the new mobile phase contains IPC reagent, we must make sure that the retention of the sample after changing the mobile phase is reproducible (the chromatographic column needs to be washed with the new mobile phase for several hours to achieve complete equilibrium). When replacing IPC reagent, first elute the IPC reagent previously adsorbed on the chromatographic column with a special eluent, and then balance it with a new flow relative to the chromatographic column.
Anionic reagent (such as alkyl sulfonate) can be eluted with eluent composed of 50% ~ 80% methanol water; 50% methanol buffer solution is required for quaternary ammonium salt (for example, 100mmol / L potassium hydrogen phosphate solution with pH of 4 ~ 5 is added to reduce the interaction between quaternary ammonium group and ionized silanol group on the stationary phase). In either case, wash the column with an eluent equal to at least 20 times the column volume, and then balance the column with a new mobile phase. In addition, the weak ion pair buffer trifluoroacetic acid (TFA) and liquid separation agent will not slow down the process of column equilibrium. Generally, washing the chromatographic column with 10 ~ 20 times of mobile phase containing TFA or liquid separation agent is enough to achieve column equilibrium.
If the mobile phase containing IPC reagent is used for the initial equilibrium of the chromatographic column, the equilibrium process may be very slow. In order to avoid 12h equilibrium before each new series of routine experiments, we recommend that the chromatographic column be soaked in the mobile phase (including IPC reagent) for storage after completing each series of experiments. This expedient method makes it possible to achieve column equilibrium faster when using IPC for content determination; If it needs to be repeated every day or every two days, we also recommend using this method. However, when stored in this way, its service life may be shortened.
Due to the slow equilibrium process between IPC reagent and chromatographic column, it is impossible to completely elute IPC reagent from the chromatographic column even if a more intense elution procedure is used. For this reason, we suggest that the chromatographic column that has been separated with IPC should not be used for RPC separation without IPC reagent (with the exception of TFA and liquid separation agent).
If a bad peak pattern is observed in IPC and / or the n value of the number of column trays is low, it can be considered to change the column temperature.
The above is the retention principle of ion pair chromatography and the solutions to some special problems. We hope it will be helpful for our viewers.
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