Ion-pairing reagent, a kind of reagent used in lab, can strengthen the retention of ion samples if added in mobile phase of reverse-phase chromatography. Ion-pairing reagents can improve the ion exchange ability on the surface of the hydrophobic bonded phase and can better control the retention of acidic or alkaline samples under mild mobile phase pH conditions.
Ion-pairing reagents absorb on the column bed through hydrophobic part of reagent( Heptane group, dodecyl group,etc.) to interact with stationary phase of column, which enables stationary phase to have ion-exchange ability, thus strengthening the retention of analytes with opposite charge.
eg. Sodium octanesulfonate
Although the ion-pairing reagent is added in the mobile phase, it works in the stationary phase of column. Take sodium octane sulfonate as an example, the ion-pairing reagent added in the mobile phase enters the column with the mobile phase, and then the ion-pairing reagent achieves the balance between the stationary phase and the mobile phase of column.
According to the principle of same-sex phase absorption, the carbon chains of eight carbon atoms in sodium octane sulfonate will be adsorbed by the stationary phase of the column with weak polarity, while the sulfonic acid ions with strong polarity are exposed to the mobile phase outside the stationary phase. The stationary phase of the column adsorbed octane sulfonate ions is negatively charged, ensuring to adsorb and retain the positively charged compounds.
Beside from improving the retention ability, ion-pairing reagent can modify peak shape.
The matrix of the stationary phase of the reversed phase column is mainly silica gel which has a large number of hydroxyl groups on the surface of silica gel. During the preparation of column, the grafted stationary phase and the later end-capping process will consume some hydroxyl groups, but there are still a large number of hydroxyl groups remaining on the surface of silica gel. The interaction of silica hydroxyl groups and compounds on the surface of silica gel matrix will lead to tailing peak.
The ion-pairing reagent will be filled into the gap of the stationary phase, which can shield the residual hydroxyl groups on the surface of silica gel, prevent the hydroxyl interaction between the target compound and the silica gel surface and improve the peak shape.
Common Ion-Pairing Reagent
Interact with the acid compounds:
Tetrabutyl ammonium hydroxide, tetrabutyl ammonium chloride (tetrabutylammonium bromide), dodecyl trimethyl ammonium chloride.
Interact with the alkaline compounds:
Various alkyl sulfonate groups
PFC-substituted straight-chain organic acids, such ad trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, etc. (they are special for that they are both ion-pairing reagents and strong acids to adjust pH).
But the use of ion-pairing reagent will inevitably cause some problems.
1. Equilibrium Requires Longer Time
Compared with the reverse-phase chromatography method, ion-pairing chromatography involves very complex equilibrium process, so equilibrium time of the column that uses ion-pairing reagent is relatively long. Since the concentration of ion-pairing reagents is 2-5 mmol/L or lower, if they are adsorbed on the surface of packing materials, the surface concentration is in the range of 0.5-2μmol/m2. A column of 4.6×250 mm has about 3g packing materials and requires 2 mmol of ion-pairing reagents for complete column equilibrium. If the mobile phase concentration is 2 mmol, 1 L of mobile phase is required to equilibrate. Although this is of course an extreme condition, it is normal to equilibrate the column with hundreds of ml of mobile phase.
Because of the long equilibrium time, the choice of gradient elution method when using ion-pairing reagent may lead to a series of problems, such as poor reproducibility and retention, unstable baseline and other separation problems. Therefore, gradient method is not recommended in ion-pairing method. When using small molecular ion-pairing reagents such as trifluoroacetic acid (TFA) and triethylamine (TEA), the column equilibrium is fast, and using gradient elution is viable.
If the column needs to be cleaned, it is recommended to use 50% methanol-aqueous solution to wash and maintain.
2. Peak Shape Problem
Using ion-pairing reagent can ameliorate the peak shape problem caused by the residual silanol groups on the column. If there is a peak shape problem in the ion-pairing method, such as leading area, it can be solved by changing the temperature. Therefore, when the peak shape is not good or the number of theoretical plates decreases, the effect of temperature change on the peak shape can be studied.
3. Blank Solvent Peak
In the ion-pairing method, when it comes to blank experiment (that is, when injecting sample solvent), the positive and inverted peaks sometimes appear, and these abnormal peaks are also called the blank solvent peaks which will interfere with the establishment or daily application of liquid phase methods, so blank experiments should be applied before establishing ion-pairing methods and after separation.
The blank solvent peak problem is usually caused by the difference between the mobile phase and the sample solvent, whose impact will be amplified by the insufficient purity of the buffer salt, the addition of ions to the reagent and other mobile phase additives.
Influence of Variables in Method Development of Ion Pair
Compared with the ordinary reverse-phase method, the ion-pairing method can meet the requirements of resolution by adjusting the parameters, including pH, the type and concentration of ion-pairing reagent, temperature and so on.
1. The pH of mobile phase: it not only affects the degree of ionization of the analyte, but the ion-pairing interaction with the analyte by changing ionization progress of the reagent. Therefore, the effect of pH change on the retention of analyte is more complex.
2. Temperature: the change of temperature in ion-pairing method will cause significant change of relative retention. Temperature will change the amount of ion-pairing reagents adsorbed on the column, so controlling temperature is particularly important for separation.
3. The type and concentration of ion-pairing reagent: in most cases, the concentration of ion-pairing reagent in stationary phase will affect the retention behavior of ion exchange of solute, while the type and concentration of ion-pairing reagent will directly affect its concentration in stationary phase.
When one wants to obtain a certain retention, lower concentration of ion-pairing reagent with stronger hydrophobic ability or higher concentration of ion-pairing reagent with weaker hydrophobic ability can be used.
When the ion-pairing reagent in the stationary phase reaches saturation, keeping increasing the concentration of ion-pairing reagent will reduce the retention of the analyte, because the increase of concentration of ion-pairing reagent will strengthen the ion exchange competition with the analyte.
If the pH of mobile phase and the concentration of ion-pairing reagent change simultaneously, the retention range and relative retention of ion sample can be controlled to a great extent.
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