Instant Notes: Analytical Chemistry

order of increasing polarity. This mode is not used extensively, but is suitable

for mixtures of structural isomers and solutes with differing functional

groups. Members of a homologous series can not be separated by adsorption

chromatography because the nonpolar parts of a solute do not interact with

the polar adsorbent surface.

● Modified partition orbonded-phase chromatography (BPC). Most HPLC

stationary phases are chemically-modified silicas, or bonded phases, by far

the most widely used being those modified with nonpolar hydrocarbons. The

solute sorption mechanism is described as modified partition, because,

although the bonded hydrocarbons are not true liquids, organic solvent

molecules from the mobile phase form a liquid layer on the surface.

The most popular phase is octadecyl(C18or ODS), and most separations

are reversed-phase, the mobile phase being a blend of methanol or aceto-

nitrile with water or an aqueous buffer. For weakly acidic or basic solutes,

the role of pH is crucial because the ionized or protonated forms have a

much lower affinity for the ODSthan the corresponding neutral species, and

are therefore eluted more quickly. The dissociation of weak acids and the

protonation of weak bases are shown by the following equations

RCOOH ÆRCOO-+H+

RNH 2 +H+ÆRNH 3 +

Thus, at low pH, bases are eluted more quickly than at high pH, whilst

the opposite holds for weak acids (Fig. 1).

D7 – HPLC: modes, procedures and applications 167

16

14

12

10

8

6

4

2

0

0246

pH

8 101214

pH range for polymeric supports

pH range for silica supports

Weak

acid Ampholyte

Weak base

k

Fig. 1. Relation between retention factor, k, and the pH of the mobile phase for weak acids,

bases and ampholytes in reversed-phase separations.