Principles of HPLC (3) Separation modes

July 9, 2020

Separation principle

In HPLC, individual components are separated using a column, based on the difference in the degree of interaction between the sample components and the column. Components with a low degree of interaction with the column are eluted first. These interactions include adsorption, hydrophilic interactions, hydrophobic interactions, electroaffinity, penetration and exclusion (Fig. 6).

Fig. 6 Separation method

Column types and separation modes

As shown in Table 2, there are various types of columns and separation modes that can be used, and the optimum choice depends on the nature of the sample and the analysis that is required. When an organic solvent is used as the mobile phase, a normal-phase column (mainly silica gel) can separate and analyze samples composed of fat-soluble components based on adsorption. When a water/methanol solvent is used as the mobile phase, separation can be achieved based on hydrophobic interactions in reverse-phase mode. GPC columns separate sample components based on their molecular size using pores. Ion exchange columns separate ion components based on electrical affinity.

Table 2 Column types and separation modes

Mode Stationary phase Mobile phase Interaction Features
Normal phase Silica gel Organic solvent Adsorption Separation of fat-soluble components
Reversed phase Silica C18 (ODS) Water / MeOH Hydrophobic The most commonly used method
GPC (non-aqueous) Polymer Organic solvent Gel permeation Molecular weight distribution measurement
GFC (aqueous) Hydrophilic polymer Buffer Gel permeation Biopolymer separation
Ion exchange Ion exchanger Buffer Electric affinity Separation of ionic components

Normal phase vs. reverse phase

Normal-phase chromatography and reverse-phase chromatography are completely different methods. In normal-phase chromatography, a low-polarity solvent is passed through a high-polarity column and the low-polarity components are eluted first. In reverse-phase chromatography, which is the most commonly employed technique, short-carbon-chain compounds are eluted first.

Isocratic elution vs. gradient elution

In reverse-phase chromatography and ion-exchange chromatography, gradient elution may be used to improve the separation and reduce the measurement time. As an example, chlorogenic acid and rutin can be separated using an ODS column and a methanol/1% acetic acid solution.
First, let’s look at an analysis example that was performed without changing the composition ratio of the solvent. In the case of a methanol/1% acetic acid ratio of 40/60, component A could not be separated well (Fig. 7, upper left). In the case of a methanol/1% acetic acid ratio of 30/70, separation was completed but it took a long time (Fig. 7, lower left). However, by applying a gradient from a methanol/1% acetic acid ratio of =30/70 to 45/55 (changing the concentration), the retention of the column was strengthened, and component A was reliably separated and eluted (Fig. 7, right).

Fig. 7 Isocratic elution (left) vs. gradient elution (right)