Normal with bonded polar functional groups

Normal-phase partition chromatography also uses a polar stationary phase and an nonpolar mobile phase such as n-hexane, methylene chloride, or chloroform. In this LC mode, however, the stationary phase is a bonded siloxane with a polar functional group, which in order of increasing polarity may be the cyano (-C2H4CN), diolC-CjHfiOCHzCHOHCHzOH), amino (-C d I N11,). or dimethylamino (-CsHeNfCHsiz) group. 

Liquid-solid chromatography (LSC), sometimes referred to as normal phase or straight phase chromatography, is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase with polar functional groups and a nonaqueous mobile phase... 

Residual Silanol-Solute. Even after bonding hydrocarbon chains to the silica, some residual unreacted silanol groups remain. These polar groups can interact with polar functional groups of solutes, i.e., normal phase attraction. The precise role they play in retention and selectivity is not well understood, but the different selectivities exhibited by various packings probably result at least in part from their different amounts of unreacted silanol groups. 

Different polar functional groups bonded onto silica can also be used for normal phase. The most frequently used bonded phases are amino-, diol-, cyano-, and nitro-groups. These phases, when used in the normal mode, are similar to adsorption onto silica gel. The use of bonded-phase columns in the normal phase mode is shown in Figure 5-44. There is a different order of elution on the amino-bonded phase compared to the cyano phase and different elution than on silica (ortho < meta < para), which is not shown. This is due to interactions with the functional groups on the packing rather than solely with the hydroxyl groups on the silica gel. 

In summary, retention of analytes by normal-phase SPE is facilitated by the dissolution of the sample in nonpolar solvents that do not compete for polar sorption sites on the solid sorbent. Elution of the sample from the sorbent is facilitated with polar solvents that can disrupt the hydrogen bonding between functional groups of the analyte and the sorbent surface. Examples of the use... 

Bonded-phase packings are classitied as reversed phase when the bonded coating is nonpolar in character and as normal phase when the coating contains polar functional groups. It has been estimated that more than three quarters of all IIPLC separations are currently performed in columns with reversed-phase packings. The major advantage of reversed-phase separations is that water can be used as the mobile phase,... 

It is important to note that besides aromatic rings, vr-type interactions are also peculiar to aliphatic compounds with double bonds, as well as functional groups that expose lone electron pairs at heteroatoms of O, N, and S. Therefore, numerous compounds incorporating polar functional groups can also be successfully separated under quasi-normal phase conditions on hypercrosslinked polystyrene packings. 

Before the development of reversed-phase bonded phases, normal-phase chromatography was the most popular separation technique. It relies on the interaction of analytes with polar functional groups on the surfooe of the stationary phase, which is strongest when nonpolar solvents are used as mobile phase. Previously, it was also called adsorption chromatography. However, the technique has expanded from the exclusive application of metal oxide adsorbents such as silica and alumina as stationary phases to the use of polar bonded phases. Thus the name adsorption chromatography has become too narrow. 

In the normal-phase extraction, compounds with polar functional groups are extracted from a nonaqueous sample. Retention is based on polar interactions such as charge-based interactions, hydrogen bonding, dipole-dipole interactions, and dispersion interactions between the sorbent and the analyte. Charge-based interactions are often not required in the normal phase, since they are very strong and difficult to disrupt (Figure 9.4). 

CYANO PHASE. Silica gel chemically bonded with a CN functional group through a hydrophobic spacer. The layer has polarity between amino and di-methylsiloxane reversed-phase plates and can function with normal- or re-versed-phase mechanisms, depending on the mobile phase. 

In the most common application of this separation mode, components are separated according to the number and nature of the polar functional groups (e.g., ester bonds, phosphate, hydroxyl, and amine groups) in lipid molecules. Since the head group of an individual lipid class predominantly determines the polar interactions with stationary phase, normal-phase HPLC separates a lipid extract solution into the lipid classes rather than into molecular species. 

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