Ion pair reagents are commonly used in the laboratory and their addition to reversed-phase chromatography mobile phase can enhance ion sample retention. The use of ion-pair reagents can improve the ion-exchange capacity of the hydrophobic bonding phase surface, allowing for better control of acidic or alkaline sample retention under mild mobile phase pH conditions.
The ion-pair reagent adsorbed on the column bed through the interaction between the hydrophobic part of the reagent (commonly heptyl and dodecyl) and the stationary phase of the column, so that the stationary phase has a certain ion exchange ability, thus enhancing the retention of analytes with opposite charges.
For example, the common octane sulfonate
Ion-pair reagents are added to the mobile phase, but their place of action is in the stationary phase of the column. Let’s take sodium octane sulfonate as an example. The ion pair added to the mobile phase enters the column with the mobile phase and is then balanced between the stationary and mobile phases of the column.
According to the principle of isotropic phase absorption, the carbon chain of 8 carbon atoms in sodium octane sulfonate will be adsorbed by the stationary phase of the weak polarity column, while the large polarity sulfonate ions are exposed in the mobile phase outside the stationary phase of the column. The stationary phase of the column, adsorbed with octane sulfonate ions, is negatively charged and thus adsorbed and retained positively charged compounds.
In addition to improving the retention of the chromatographic column on the target compound, the ionic reagent has an effect of improving peak type.
The matrix of the fixed phase of the inverter chromatography column is mainly silica gel, and there is a large amount of hydroxyl group on the surface of the silica gel. The fixed phase grafted during the preparation of columns and the later chastic process consumes partial hydroxyl groups, but there is still a large amount of hydroxyl residue on the surface of the silicone matrix. Silicon hydroxy groups and compounds of the silica gel matrix can cause drag-rear peaks.
As mentioned earlier, the ion pair will fill the gaps in the stationary phase, and this part of the ion pair can shield the residual hydroxyl group on the silica gel surface. Thus, the hydroxyl interaction between the target compound and silica gel surface was prevented and the trailing peak was improved.
Common ion pair reagents
Acting on acidic compounds:
Tetrabutylammonium hydroxide, tetrabutylammonium chloride (tetrabutylammonium bromide), dodecyltrimethyl ammonium chloride.
Acting on basic compounds:
Octane sodium sulfonate and other alkyl sulfonates
Trifluoroacetic acid, pentafluoropropionic acid, sevofluobutyric acid and other perfluorosubstituted straight chain organic acids. (Perfluorinated linear organic acids are special, both ionic pair reagents and strong ph-regulating acids).
However, the use of ion pairs of reagents will inevitably cause some problems.
Common Problems and solutions
Column balance time long
The ionic chromatography is relatively long because of a plurality of complex balanced processes, and thus generally use the chromatography column equilibrium time using ionic columns. Since the concentration of the ion pair reagent is at 2-5 mmol / L or even lower concentration, they should be adsorbed on the surface of the reverse chromatography column filler, the surface concentration is from 0.5 to 2 μmol / m2.1 to the column of 4.6 × 250 mm, about 3 g. The filler requires a 2 mmol ion to the reagent to completely balance. When the flow phase concentration is 2 mmol, a liter flow phase is needed to balance. This is of course extreme condition, but it is normal to use hundreds of ml of mobile to equilibrium columns.
Due to the long balance time, when the gradient elution method is selected when using ion on the reagent, a series of problems may result in a series of problems, and the retention value is poor, the baseline is unstable and other separation problems, and therefore generally does not recommend ion-pair method. Use a gradient method. However, for the use of small molecular ions, the reagents such as trifluoroacetic acid (TFA) and triethylamine (TEA) are rapid, and the gradient elution is generally no problem.
If you need to clean the column, it is recommended to use 50% methanol-aqueous solution rinsing and maintenance.
Peak problem
Typically add ion pairs of reagents improve the peak problem of residual silicol groups on the silica gel matrix column. If a peak problem occurs in ions, such as peak advances, etc., can be solved by changing the temperature. Therefore, when the peak shape is not good or the theoretical tower number is lowered, it can be investigated the impact of temperature changes on the peak shape.
Blank solvent peak
In the ion-pair method, when blank experiments (ie, injecting the sample solvent), the peaks and falling peaks sometimes appear, and these abnormal peaks are also referred to as blank solvent peaks. These blank solvent peaks interfere with the establishment or daily application of the liquid phase method, so blank experiments should be performed before the ions are established and have a desired separation.
The blank solvent peak problem is usually caused by the difference between the flow phase and the sample solvent, and the buffer salt used is insufficient, and the ionic reagent or other mobile phase additive is added to enlarge this effect.
Ion impact of variables in method development
Ions can satisfy the separation by adjusting parameters (including pH, ion pair reagents, including the type and concentration, temperature) of the parameters (including the type and concentration, temperature) of the reagent.
- PH of the mobile phase: pH in the ion pair method not only affects the degree of ionization of the analyte, but also affects the degree of ionization of the reagent to the ionization of the reagent, so the change to the analyte The impact of reservations is more complicated.
- Temperature: The change in temperature in the method can cause a significant change in relative retention value. Temperature changes the amount of ion-to-reagent adsorbed on the column, so the control temperature is particularly important.
- Ion Type and Concentration of Ion Reagents: In most cases, the concentration of the immobilized phase neutralizing the reagent affects the retention behavior of the ion exchange of the solute, and the type and concentration of the ion on the reagent directly affect its immobilization phase. concentration.
When you want to get certain reservations, a lower concentration, a more hydrophobic ion pair reagent or a more hydrophobic, a more hydrophobic ion pair reagent.
When the ion pair of the fixed phase reaches the reagent, the concentration of the ion to reducing the reagent will reducing the retention of the analyte, since the ionic concentration of the reagent is increased, and the ion exchange compete with the analyte is increased.
When the pH of the mobile phase is changed while the concentration of the ion on the reagent, the retention range and the relative retention value of the ion sample can be greatly controlled to a large extent.
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