Type of Analyte Ion and Ion Pair Reagent

In a reversed phase chromatographic system, the ionized form of a molecule has a much lower retention factor than the corresponding non-ionized form. The main reason is that charged species have a lower energy in a medium of high dielectric constant (i.e. the mobile phase) than in a medium of low dielectric constant (i.e. the hydrophobic surface layer). Within a series of analyte ions of identical charge but differ ent hydrophobicity, the retention order follows the same rules as for non-ionized analytes, i.e. the more hydrophobic analyte is more retained. This property is reflected in eqns [3] and [6] by different k0B values for different fully ionized analytes.

Figure 4 Retention factor data for mono-charged (A, 1-naph-thylamine-4-sulfonic acid; *, 6-naphthol-2-sulfonic acid) and double-charged sulfonic acids (□, 2-naphthol-6,8-disulfonic acid; O, naphthalene-2,7-disulfonic acid) as a function of eluent concentration (cA) of the double-charged pairing ion, hexamethonium bromide (zA =+ 2). (Reproduced with permission from Bartha A and Stahlberg J (1994) Journalof ChromatographyA 668: 255.)

Figure 4 Retention factor data for mono-charged (A, 1-naph-thylamine-4-sulfonic acid; *, 6-naphthol-2-sulfonic acid) and double-charged sulfonic acids (□, 2-naphthol-6,8-disulfonic acid; O, naphthalene-2,7-disulfonic acid) as a function of eluent concentration (cA) of the double-charged pairing ion, hexamethonium bromide (zA =+ 2). (Reproduced with permission from Bartha A and Stahlberg J (1994) Journalof ChromatographyA 668: 255.)

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Figure 5 Retention factor of adrenaline as a function of the stationary phase surface concentration (nA) of sodium butyl- (A), hexyl- (*) and octyl- (□) sulfonate pairing ions measured at constant ionic strength (175 mM Na #) of the phosphate buffer (pH 2.1) on an ODS-Hypersil column. (Reproduced with permission from Bartha A and Stahlberg J (1994) Journal of Chromatography A 668: 255.)

0 100 200 300 400

Figure 5 Retention factor of adrenaline as a function of the stationary phase surface concentration (nA) of sodium butyl- (A), hexyl- (*) and octyl- (□) sulfonate pairing ions measured at constant ionic strength (175 mM Na #) of the phosphate buffer (pH 2.1) on an ODS-Hypersil column. (Reproduced with permission from Bartha A and Stahlberg J (1994) Journal of Chromatography A 668: 255.)

Under the condition of identical mobile phase concentration of two ion pair reagent ions of different hydrophobicity, the more hydrophobic reagent ion gives a higher retention for an oppositely charged analyte. This is attributed to higher adsorption to the stationary phase surface of the more hydrophobic reagent and a correspondingly higher induced electrostatic surface potential. Since a relative change in retention factor is primarily caused by a change in electrostatic surface potential, it is the surface concentration of the ion pair reagent, and not its detailed molecular structure, which is of importance for retention changes. This important fact is illustrated in Figure 5 where the retention factor of adrenaline is solely a function of the surface concentration of the three different alkylsulfonates and is independent of the type of ion pair reagent.

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