When you are doing daily testing, you will find that some items, such as sodium heptane sulfonate, tetrabutylammonium hydroxide, tetrabutylammonium bromide and other reagents are added to the mobile phase used in the test standard. We call these reagents ion pairing Reagents, which can be used to improve separation and peak shape, narrow the retention range of samples, etc. Ion pairing reagents can be seen as a manifestation of the introduction of ion chromatography in high performance liquid chromatography. Today I will talk to you about the fundamentals of retention and some special issues in ion-pair chromatography.
Ion-pair chromatography (IPC) can be viewed as a modified form of reversed-phase chromatography with the goal of separating ionic samples. The only difference between IPC and RPC is that IPC adds ion-pairing reagents R+ or R- to the mobile phase, which can interact with A- for acidic compounds or BH+ for basic compounds during equilibrium:
ionized solute ion pair
(acid) A-+R+ ⇔ A-R+
(base) BH++R- ⇔ BH+R-
Hydrophilic solute Hydrophobic ion pair
(less reserved in RPC) (more reserved in RPC)
Using IPC can produce a change in sample retention behavior similar to changing the pH of the mobile phase, but IPC can better control the retention behavior of acidic or basic solutes without the need to use extreme pH (such as pH<2.5 or pH>8 ). Typical ion-pairing reagents include alkyl sulfonates R-SO3-(R-) and tetraalkylammonium salts R4N+(R+), and strong (usually ionized) carboxylic acids (tetrafluoroacetic acid, TFA; heptafluoroacetic acid) Butyric anhydride, HFBA (R-), and so-called chaotropes (BF4-, ClO4-, PF6-).
There are currently two theories about the retention mechanism of IPC.
One argument is that ion pairs are formed in solution and then retained on the column, and the solute retention equilibrium process is as follows (taking the ion pair formed by the ionized acidic solute A- and the tetraalkylammonium salt R+ as an example):
A-R+ (mobile phase) ⇔ A-R+ (stationary phase)
According to this statement, solute retention is determined by:
① The ionized part of the 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 way of saying that the IPC reagent is first retained by the stationary phase, and then the solute retention is a process of ion exchange, 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-pairing reagent R+X- is adsorbed on the stationary phase first, and then the sample ion A- replaces the counter ion X- on the stationary phase. Both of these IPC retention processes may prevail in any given separation, but which mechanism plays a more important role is neither easy to determine nor practically important.
In IPC, separation conditions that can be used to control selectivity include:
➩ Types of IPC reagents (sulfonates, quaternary ammonium salts, chaotropic agents)
➩ Concentration of IPC reagent
➩ Solvent Strength (B%)
➩ Solvent type (methanol, acetonitrile, etc.)
➩ Column Type
➩ Type and concentration of buffer solution
Inorganic reagents (or “chaotropic agents”) such as ClO4-, BF4- and PF6- can be used in place of commonly used alkyl sulfonates as IPC reagents. Inorganic reagents have 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 chaotropes are better for gradient elution (less baseline noise and drift) and dissolve better in the mobile phase when the B% is higher.
However, the use of ion-pairing reagents has some special problems, in some cases tight control of mobile phase pH is required; reproducibility of temperature control must be high (more required than RPC), in addition, some problems in IPC do not occur in RPC Appears in separation or differs from other RPCs. There are also spurious peaks, slow column equilibration changing the mobile phase, and poor chromatographic peak shapes for unknown reasons.
The first is spurious peaks. When injecting the sample solvent (without sample) into the IPC (i.e. blank experiment), we sometimes observe positive and negative peaks at the same time. Artifact peaks are usually caused by differences in the composition of the mobile phase and the sample solvent. The use of impure IPC reagents, buffers, or other mobile phase additives can exacerbate the problem of spurious peaks.
This is followed by slow column equilibration. When using a new mobile phase, the column must be flushed with a sufficient volume of mobile phase to equilibrate the column. In IPC, the adsorption and desorption of IPC reagents on the column is in some cases very slow, which can cause the column not to be fully equilibrated with the new mobile phase. So, whether the old mobile phase or the new mobile phase contains IPC reagents, we must be sure that the retention of the sample after changing the mobile phase is reproducible (requiring several hours of flushing the column with the new mobile phase to achieve full equilibration ). When replacing the IPC reagent, first use a special eluent to elute the IPC reagent previously adsorbed on the chromatographic column, and then equilibrate with a new mobile phase chromatographic column.
Anionic reagents (such as alkyl sulfonates) can be eluted with eluents composed of 50% to 80% methanol-water; quaternary ammonium salts require a 50% methanol-buffer (eg, pH 4 to 5). 100mmol/L dipotassium hydrogen phosphate solution, adding dipotassium hydrogen phosphate is to reduce the interaction between the quaternary ammonium group and the ionized silanol group on the stationary phase). In either case, the column should first be flushed with at least 20 column volumes of eluent before equilibrating the column with new mobile phase. In addition, weaker ion-pairing buffers such as trifluoroacetic acid (TFA) and chaotropic agents do not slow down the column equilibration process, and flushing the column with 10-20 times the mobile phase containing TFA or chaotropic agent is usually sufficient to achieve Column balance.
Initial equilibration of the column with a mobile phase containing IPC reagents can be very slow. To avoid 12h equilibration before each new series of routine experiments, we recommend that the column be soaked in mobile phase (with IPC reagents) for storage after each series of experiments. This expedient method allows for faster column equilibration with IPC assays; we also recommend this method if daily or two-day repetitions are required, however, its useful life may be limited when stored in this manner will shorten.
Due to the slow equilibration process of the IPC reagent with the column, it is impossible to completely elute the IPC reagent from the column even with a more vigorous elution procedure. For this reason, we recommend that columns that have been separated by IPC should not be used for RPC separations without IPC reagents (with the exception of TFA and chaotropes).
If poor peak shape and/or low N values for column plate number are observed in IPC, consider changing the column temperature.
The above is the retention principle of ion-pair chromatography and the solutions to some special problems. I hope it will be helpful for everyone to use ion-pair chromatography in the future.