Bonding,stationary phase,effect

Ion exchange. This postulates that the lipophilic tail of the counter-ions locates onto the bonded stationary phase, effectively causing the column to behave as an ion exchanger. 

Jinno, K., Effect of the aUcyl chain length of the bonded stationary-phase on solute retention in reversed phase high performance hquid chromatography, Chromato-graphia, 15, 667, 1982. 

Jrnno, K., Nagoshi, T., Tanaka, N., Okamoto, M., Fetzer, J.C., and Biggs, W.R., Effect of column temperature on the retention of peropyrene-type polycyclic aromatic hydrocarbons on various chemically bonded stationary phases in re versed-phase liquid chromatography, J. Chromatogr., 436, 1, 1988. 

Separations were performed on either silica- or amino-bonded stationary phase columns, using carbon dioxide with various modifiers as mobile phases. Each column exhibited distinctly different selectivities to the examined sulfonamides, the amino-bonded column being much more sensitive to modifier variations. In a continuation of the sulfonamide study, packed-column SFC was further evaluated for possible application to the analysis of furazolidone, chloramphenicol, and lincomycin residues (82). Separation was effected on an amino-bonded stationary phase using carbon dioxide with methanol modifier as the mobile phase, whereas detection was accomplished by MS. 

The separation of enantiomers can be effected either by transforming them into diastereoisomers using a chiral reagent and separating them on conventional phases or by separating the enantiomers on chiral phases. The utilization of chiral phases has not yet become routine, but studies of enantiomeric dipeptides have been carried out (115,116). Pirkle et al. (117) and Hyun et al. (118) separated enantiomeric di- and tripeptides (methyl esters of /V-3-5-dinitrobenzoyl derivatives) on chiral stationary phases (CSPs) derived from (R)-a-arylalkylamines, (S)-N-(2-naphthyl) valine, or (S)-1 -(6,7-dimethyl-1 -naphthyl) isobutylamine. These workers were able to separate four peaks for each dipeptide derivative, corresponding to the two enantiomeric pairs (R,R)/(S,S) and (R,S)/(S,R). Cyclodextrin-bonded stationary phases and chiral stationary immobilized a-chymotrypsin phases were used to separate enantiomeric peptides (118a,b). 

In order to improve the separation efficiency and speed in biopolymer analysis a variety of new packing materials have been developed. These developments aim at reducing the effect of slow diffusion between mobile and stationary phase, which is important in the analysis of macromolecules due to their slow diffusion properties. Perfusion phases [13] are produced from highly cross-linked styrene-divinylbenzene copolymers with two types of pores through-pores with a diameter of 600-800 mu and diffusion pores of 80-150 nm. Both the internal and the external surface is covered with the chemically bonded stationary phase. The improved efficiency and separation speed result from the fact that the biopolymers do not have to enter the particles by diffusion only, but are transported into the through-pores by mobile-phase flow. 

Reversed-phase stationary phases are more or less hydrophobic, and the degree of this property is characterized by their hydrophobicity H. As a general rule, retention times are longer the more C atoms the bonded stationary phase contains. (The reason is that the volume taken up by the bonded nonpolar groups, i.e. that required by the actual stationary phase, is greater with long chains than it is with shorter chains retention is directly proportional to the volume ratio between the stationary and mobile phases see Section 2.3.) Figure 10.7 demonstrates this effect. 

The separations occur due to the interactions of the alkyl (-CH2-) groups on the analytes within the mixture and the functional groups on the surface of the silica. These interactions are known as Van der Waals forces (see Chapter 2 for further explanation), but the mechanisms of retention of analytes are complex and it is possible that more than one mechanism is operating at the same time. For example, hydrophobic interactions may be taking place on the surface of the bonded stationary phase, which can cause partitioning effects with solutes as well as adsorption effects on unreacted silanol (-Si-OH-) groups. 

Soon, Knox thought to use the hydrophobic effect to form ion-pairs in aqueous phases. He developed the first use of reversed-phase ion-pair chromatography in 1976 [6]. He termed it soap chromatography. He added ionic surfactants to the polar hydro-organic mobile phase. They adsorb on the alkyl-bonded stationary phase. They also associate with the hydrophobic and ionic analytes. 

The characteristics of alkyl-bonded stationary phases in RPLC with micellar and aqueous-organic mobile phases are another noteworthy difference. The extraction of organic solvent by the grafted alkyl phase depends on the composition of the aqueous-organic mobile phase, which has a profound effect on retention. With micellar eluents, however, alkyl-bonded phases may be modified with an approximately constant concentration of... 

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