Normal phase mechanisms

HILIC is a variant of normal-phase chromatography that employs polar stationary phases and RPLC-type mobile phases. Because HILIC separations occur by a normal-phase mechanism, the organic component of the mobile phase... 

Scalia and Games developed a packed column SFC method for the analysis of free bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) [32]. The baseline separation of all five bile acids was achieved on a packed phenyl column with a methanol-modified carbon dioxide in less than 4 min. The elution order showed a normal-phase mechanism because the solutes eluted in the order of increasing polarity following the number of hydroxyl groups on the steroid nucleus. The method was also applied to the assay of UDCA and CDCA in capsule and tablet formulations. The method was found to be linear in the range 1.5-7.5 ng/ml (r > 0.99, n = 6). The average recoveries (n= 10) for UDCA and CDCA were 100.2% with a RSD of 1.7% and 101.5% with a RSD of 2.2%, respectively. The reproducibility of the method was less than 1.5% (n = 10) for both UDCA and CDCA. 

Wei et al. studied the separation of amines (aniline, ephedrine, codeine, cocaine, thebaine) and quaternary ammonium compounds (berberine, jatrorrhi-zine) on a bare silica stationary phase [44], A thorough study of the separation mechanism revealed a complex multifunctional mechanism. Contributions from differential electrophoretic migration were superimposed on hydrophobic, cation-exchange, and normal-phase interactions. Retention was highly dependent on the pH (optimal pH 8.3), ionic strength, and the amount of organic modifier. As the content of acetonitrile exceeded 80%, retention was consistent with a normal-phase mechanism. 

Figure 2.11. Normal-phase mechanism in SPE for the sorption of nitrobenzene. [Reproduced from Zief and Kiser (1987) and published with permission.]...

Figure 5.1. Hydrogen bonding normal-phase mechanism in SPE using a cyanopropyl sorbent.

An example is endrin in soil (Fig. 7.7). This highly chlorinated insecticide is extracted by Soxhlet extraction with hexane from a soil that has been mixed one to one with sodium sulfate to remove water. The hexane extract is further dried over sodium sulfate and the hexane is added to a hexane-loaded silica or CN SPE sorbent that has been prepared with hexane. The endrin is sorbed to the silica or CN by normal-phase mechanisms and may be eluted with a solvent that will overcome the interaction, such as methylene chloride or ethyl acetate. The more polar interferences are sorbed to the silica or CN sorbent and are not eluted with endrin. Thus, the SPE column performs both trace enrichment and SPE clean-up. See Chapter 5 for more examples. 

Rarely, the separation of anionics is made by a normal phase mechanism on a silica gel column with a mobile phase of chloroform/ethanol containing a counter ion (10,11). Normal phase conditions may be chosen such that some separation of isomers and oligo-... 

Below about 0.5 K, the interactions between He and He in the superfluid Hquid phase becomes very small, and in many ways the He component behaves as a mechanical vacuum to the diffusional motion of He atoms. If He is added to the normal phase or removed from the superfluid phase, equiHbrium is restored by the transfer of He from a concentrated phase to a dilute phase. The effective He density is thereby decreased producing a heat-absorbing expansion analogous to the evaporation of He. The He density in the superfluid phase, and hence its mass-transfer rate, is much greater than that in He vapor at these low temperatures. Thus, the pseudoevaporative cooling effect can be sustained at practical rates down to very low temperatures in heHum-dilution refrigerators (72). 

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

Although SPE can be done in a batch equilibration similar to that used in LLE, it is much more common to use a small tube (minicolumn) or cartridge packed with the solid particles. SPE is often referred to as LSE, bonded phase or sorbent extraction SPE is a refinement of open-column chromatography. The mechanisms of retention include reversed phase, normal phase, and ion exchange. 

The TLC process is an off-line process. A number of samples are chromatographed simultaneously, side-by-side. HPTLC is fast (5 min), allows simultaneous separation and can be carried out with the same carrier materials as HPLC. Silica gel and chemically bonded silica gel sorbents are used predominantly in HPTLC other stationary phases are cellulose-based [393]. Separation mechanisms are either NPC (normal-phase chromatography), RPC (reversed-phase chromatography) or IEC (ion-exchange chromatography). RPC on hydrophobic layers is not as widely used in TLC as it is in column chromatography. The resolution capabilities of TLC using silica gel absorbent as compared to C S reversed-phase absorbent have been compared for 18 commercially available plasticisers, and 52 amine and 36 phenolic AOs [394]. 

In lc there are other sorption mechanisms that can cause separation, depending on whether we choose to use a liquid or a solid as the stationary phase, or what kind of solid we use. Liquid-liquid chromatography (11c) uses a liquid stationary phase coated onto a finely divided inert solid support. Separation here is due to differences in the partition coefficients of solutes between the stationary liquid and the liquid mobile phase. In normal phase 11c the stationary phase is relatively polar and the mobile phase relatively non-polar, whilst... 

In another study, the authors reported a comparative study of the enantiomeric resolution of miconazole and the other two chiral drugs by high performance liquid chromatography on various cellulose chiral columns in the normal phase mode [79], The chiral resolution of the three drugs on the columns containing different cellulose derivatives namely Chiralcel OD, OJ, OB, OK, OC, and OE in normal phase mode was described. The mobile phase used was hexane-isopropanol-diethylamine (425 74 1). The flow rates of the mobile phase used were 0.5, 1, and 1.5 mL/min. The values of the separation factor (a) of the resolved enantiomers of econazole, miconazole, and sulconazole on chiral phases were ranged from 1.07 to 2.5 while the values of resolution factors (Rs) varied from 0.17 to 3.9. The chiral recognition mechanisms between the analytes and the chiral selectors are discussed. 

A normal-phase HPLC method was employed for the separation of 11 -cis- and all-frany-retinals. Separation was performed in a silica column (150 X 6.0 mm i.d. particle size 3 jum) with an isocratic mobile phase (n-hexane with 15 per cent ethyl acetate and 0.15 per cent ethanol). The flow rate was 1 ml/min and retinols were detected at 360 nm. The method separated well the 11 -cis- and all-/ran.v-retinals as demonstrated in Fig. 2.162. The results emphasize again the decisive role of chromatographic methods in the elucidation of the mechanism of various biochemical processes [334],... 

Two types of system are used for ion-pair liquid chromatography. When polar stationary phase materials, such as silica gel, are used an ion-pair partition mechanism is applied. When non-polar stationary phase materials, such as octadecyl-bonded silica gel and polystyrene gel, are employed a paired-ion adsorption mechanism is involved. The former is called normal-phase ion-pair partition liquid chromatography, and the latter is called reversed-phase ion-pair liquid chromatography. 

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