Reversed Phase IPC

1 Silica-Based Stationary Phases and Silanophilic Interactions 

Even if chain length is the key parameter, the ligand bonding density (usually above 2.5 umol/m ) may be very influential in determining overall stationary phase hydrophobicity. When the monolayer capacity, theoretically estimated by [L]j, increases as a result of increased bonding density, adsorption competitions are less operative and enhanced retention is expected. It should be noted that ligand bonding density can be calculated on the basis of the column carbon load and the total surface area of the column. 

The former represent the weight percent of carbon atoms on the adsorbent sample measured by elemental analysis. The bonding density of a given carbon load decreases with increasing surface area of the silica support porosity multiplies the available particle surface. The specific surface area (surface area in 1 g adsorbent) is said to be inversely proportional to pore diameter (at constant specific pore volume) and obviously increases with increasing specific pore volume. 

The estimate of the surface area of chromatographic silica support is a complicated issue. It is usually performed via the BET method using low temperature nitrogen adsorption (N2.- sorptometry). The total surface area of the adsorbent is the product of the number of adsorbed molecules and the surface area per molecule. However, if the pore size distribution is not very narrow, an estimate of bonding density on the basis of carbon load and surface area may yield a large error because the smallest pores are not available for derivatization and the calculated bonding density is lower than the actual one. 

Pore diameter sanctions the ability of analyte molecules to penetrate inside the particle and interact with its inner surface. Small pores (less than 10 50 A) should 

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Reversed-phase IPC has quickly gained widespread acceptance- as a versatile and efficient method for the separation of ionized and easily ionizable analytes [342,355,360-364]. It is complementary to ion-exchange (section 4.5.8) and ion... 

Figure 4.18 A, separation of antihistanine and decongestant drugs by reversed-phase IPC. Mobile phase nethanol-water (1 1) containing 5 aM hexanesulfonate and 1 % acetic acid at a flow rate of 3 al/nin. B, separation and indirect OV detection of carboxylic acids by reversed-phase IPC. Coaponents 1 acetic acid, 2 = propionic acid, 3 butyric acid, 4 = valeric acid, 5 caproic acid, and S -. systea peak. Mobile phase 0.3 aM l-phenethyl-2-picoliniua in acetate buffer at pH 4.6.

Several theoretical models, such as the ion-pair model [342,360,361,363,380], the dyneuaic ion-exchange model [342,362,363,375] and the electrostatic model [342,369,381-386] have been proposed to describe retention in reversed-phase IPC. The electrostatic model is the most versatile and enjoys the most support but is mathematically complex euid not very intuitive. The ion-pair model emd dynamic ion-exchange model are easier to manipulate and more instructive but are restricted to a narrow range of experimental conditions for trtilch they might reasonably be applied. The ion-pair model assumes that an ion pair is formed in the mobile phase prior to the sorption of the ion-pair complex into the stationary phase. The solute capacity factor is governed by the equilibrium constants for ion-pair formation in the mobile phase, extraction of the ion-pair complex into the stationary phase, and the dissociation of th p ion-pair complex in the... 

While a hydrophobic ion-pair is retained on hydrophobic stationary phases better than an ionized analyte, the retention of the duplex on normal phases is easily predicted to be lower than that of the ionized analyte because polar interactions are reduced. Actually the trend of k versus IPR concentration under normal phase IPC is the opposite of reversed phase IPC [34]. An aminopropyl, a cyanoethyl, and a silica stationary phase were compared for the analysis of alcohol denaturants. The cyanoethyl phase was selected and anionic IPRs were used to reduce retention of cationic analyte, suppressing their interactions with negatively charged silanols... 

Reversed-phase IPC, where the liquid stationary phase is organic and the pairing ion is introduced in the aqueous mobile phase Reversed-phase IPC, using a chemically bonded stationary phase and a hydrophobic pairing ion in the aqueous mobile phase... 

This is, by far, the most commonly used form of reversed-phase IPC. This technique has been also called soap chromatography although, in soap chromatography, the use of detergents as counterions is introduced. 

In reversed-phase IPC, maximum k values are obtained at intermediate values of pH, where the sample compounds are completely ionized and ion-pair formation is at a maximum. As the pH of the mobile phase is lowered, sample anions begin to form the un-ionized acids HA, leading to a smaller number of sample ion pairs in the stationary phase. Adds are usually separated at a pH of 7-9, whereas bases are separated at a pH of 1-6. 

Reversed-phase IPC, where the liquid stationary phase is organic and the pairing ion is introduced in the aqueous mobile phase. 

An increase in the alkyl chain length of the counter-ion increases retention in reversed-phase IPC by up to 2.5 times per added -CH2- group in the counterion. 

Figure 4.27 Flow chart for coluwi selection based on sample type (m - molecular weight). PLC precipitation-liquid chromatography SEC = size-exclusion chromatography lEC - ion-exchange chromatography HIC hydrophobic interaction chromatography LSC liquid-solid chromatography RPC - reversed-phase liquid chromatography BPC (polar) bonded-phase chromatography and IPC - ion-pair chromatography.

The application area of IPC and IC is overlapping, whereas IPC fills the gap between IC and reversed phase chromatography [ 3]. As one borderline case IPC can be described as IC with dynamically coated exchangers. The ion pairing reagent is simply... 

Reversed phase ion-pair systems (RP-IPC) could be of the LLC type, but the use of chemically bonded (alkyl) phases has become increasingly popular, because of the increased stability and flexibility of the system. Even if an LLC system is used for RP-IPC, then a chemical modification of the surface is still required to coat an organic liquid on the particles of (for example) silica. 

The most common way to create an RP-IPC system is to use a genuine chemically bonded reversed phase column (e.g. C18 see section 3.2.2.1) and to use large pairing ions with a hydrophobic alkyl chain dissolved in the mobile phase. This technique was introduced by Knox and Laird, who named it soap chromatography [380]. Because of the usually long alkyl chains of the pairing ions, the use of Cl 8 phases is to be recommended in order to avoid effects that are related to the critical chain length (see section 3.2.2.1). 

As IPC is currently practiced, a bonded reverse phase separation is usually the first system tried. If some ions cannot be retained, an ion pair reagent is added. A typical separation is shown in Figure 9.16. 

Ion Pair Chromatography (IPC). This discussion is limited to the most common type of IPC—reverse phase mode using bonded alkyl ligates (like C)8 and Cg) as the stationary phase. Its main advantage over regular reverse phase LC is that it facilitates the analysis of samples that contain both ions and molecular species. 

TABLE VIII. Comparison of the General Effect of Variables on Retention in Reversed-Phase Ion-Pair (RP-IPC) and Micellar Liquid Chromatography (MLC)... 

Since the discovery of IPC, this separation strategy has been devoted to increasing the mediocre retention of ionized samples on reversed phase stationary phases via ion-pair formation with a suitable IPR to increase the analyte hydrophobicity and, in turn, its retention. It is therefore clear that most IPC separations are performed under reversed phase conditions. Even if normal phase chromatography exploits polar... 

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