HPLCWOOD: MOBILE PHASE DISCRIPTION AND PROPERTIES

MOBILE PHASE DISCRIPTION AND PROPERTIES

Mobile phase has been rightly termed as the lifeline of the HPLC system. It plays the important role of transport of the sample through the separation column and subsequently to the detector for identification of the separated components.

Mobile phase is seldom a single solvent. It consists of combination of water with organic solvents, aqueous buffers with polar solvents or mixtures of organic solvents in desired proportions. The two operational modes commonly used are:

Isocratic mode – in the Isocratic mode the composition of the mobile phase remains constant throughout the analytical run ,i.e, the proportion of solvents in the mixture is pre-decided and remains unchanged during analysis

Gradient Elution – the composition is varied by the software through the analytical run in a predetermined mode and at the end of the run the proportion of solvents is different from the initial proportion.

The objective of using different solvent mixtures is to achieve the desired polarity of mobile phase for complete miscibility of the sample and control interaction of sample components with the stationary phase to achieve the desired degree of resolution of separated component peaks in the shortest possible time.

In order to meet the required objectives the mobile phase should have the following essential features:

The sample should be fully soluble in the mobile phase. Any insolubility will result in flow restrictions. Always check sample solubility in mobile phase before injection into the system.
Mobile phase components should be non-hazardous and non-toxic. They should not pose any health hazard to the operator.
Mobile phase shouId be inert towards sample constituents and the stationary phase. Any reactions can lead to formation of insoluble suspensions which can result in column blockages.
The mobile phase should not give its own response on passing through the detector. In other words the detector signals should reflect only the response of the sample constituents. This, however, is not applicable in bulk property detectors such as refractive index detector which respond to overall changes in refractive index of the mobile phase containing the eluting compound.
The mobile phase should be affordable and the proportion of solvents used should make the analysis economically viable.

It is important to mix the solvents of specified purity and from same source to get consistency of results and also to adopt the same off-line mixing technique to avoid errors due to heat of mixing errors. In case buffers are to be used always adjust the pH of the aqueous phase prior to making up the final volume with the organic phase.

HPLC technique holds great promise for applications in diverse fields. Control of operational conditions including consistency of mobile phase will ensure repeatability of results.

The Role of pH IN MOBILE PHASE -

The pH of the mobile phase (eluent) is adjusted to improve component separation and to extend the column life. This pH adjustment should involve not simply dripping in an acid or alkali but using buffer solutions, as much as possible. Good separation reproducibility (stability) may not be achieved if buffer solutions are not used.

A buffer solution is prepared as a combination of weak acids and their salts (sodium salts, etc.) or of weak alkalis and their salts. Common preparation methods include: 1) dripping an acid (or alkali) into an aqueous solution of a salt while measuring the pH with a pH meter and 2) making an aqueous solution of acid with the same concentration as the salt and mixing while measuring the pH with a pH meter. However, if the buffer solution is used as an HPLC mobile phase, even small errors in pH can lead to problems with separation reproducibility. Therefore, it is important to diligently inspect and calibrate any pH meter that is used. This page introduces a method that does not rely on a pH meter. The method involves weighing theoretically calculated fixed quantities of a salt and acid (or alkali) as shown in the table below. Consider the important points below.

Denoting Buffer Solutions

A buffer solution denoted, "100 mM phosphoric acid (sodium) buffer solution pH = 2.1," for example, contains phosphoric acid as the acid, sodium as the counterion, 100 mM total concentration of the phosphoric acid group, and a guaranteed buffer solution pH of 2.1.

Maximum Buffer Action Close to the Acid (or Alkali) pKa

When an acetic acid (sodium) buffer solution is prepared from 1:1 acetic acid and sodium acetate, for example, the buffer solution pH is approximately 4.7 (near the acetic acid pKa), and this is where the maximum buffer action can be obtained.

Buffer Capacity Increases as Concentration Increases

The buffer capacity of an acetic acid (sodium) buffer solution is larger at 100 mM concentration than at 10 mM, for example. However, precipitation occurs more readily at higher concentrations.

Beware of Salt Solubility and Precipitation

The salt solubility depends on the type of salt, such as potassium salt or sodium salt. Salts precipitate out more readily when an organic solvent is mixed in.

In addition, avoid using buffer solutions based on organic acids (carboxylic acid) as much as possible for highly sensitive analysis at short UV wavelengths. Consider the various analytical conditions and use an appropriate buffer solution, such as an organic acid with a hydroxyl group at the α position to restrict the effects of metal impurity ions

Differences in Preparation Method May Result in Different Chromatograms -

A water-based solvent, organic solvent, or a mixture of the two is mainly used as the mobile phase for HPLC. A buffer solution is often used as the aqueous solvent. describes the actual preparation methods for typical buffer solutions used with HPLC. However, it seems that many people have a vague understanding of buffer solutions.

If the preparation method is not the same as the method described in the material for the analysis method, differences can occur in the mobile phase that affect the chromatograms and analysis results. Apart from the buffer solution, many other unforeseen factors affect the mobile phase preparation, such as the method of mixing the solvent.

Here, we use actual examples to consider the effects of the mobile phase preparation method on the analysis results.

Preparing Buffer Solutions

In practice, how do we go about preparing a buffer solution that is denoted as "20 mM phosphoric acid buffer solution (pH=2.5)"? Let's examine a few possible issues.
First, we know that it is a phosphoric acid buffer solution, but what is the counterion? If sodium ions are unambiguously the counterion, does "20 mM" refer to the phosphoric acid concentration or the sodium phosphate concentration?

For a 20 mM phosphoric acid (sodium) buffer solution, "20 mM" could be considered to be the phosphoric acid concentration. On the other hand, considering "20 mM" to be the sodium concentration, the buffer solution could be a buffer solution prepared by adjusting the pH using a 20 mM aqueous solution of sodium dihydrogen phosphate. (However, a 20 mM aqueous solution of sodium dihydrogen phosphate has a pH just under 5, so that pH adjustment with some type of acid is required to achieve pH 2.5.)
However, an ion-pair effect due to the acid used for pH adjustment could affect the analysis results. This is potentially dangerous, as it leads to several possible interpretations of a denoted buffer solution.

The example above leads to three potential interpretations. Fig. 1 shows how this affects the analysis results.
These differences can significantly affect the retention time, as shown for dihydrocodeine in the example, and can lead to problems with the robustness of the analysis method. Clearly identifying the buffer solution when specifying the preparation method can prevent problems due to differences in interpretation.