Normal-phase

Silica has often been modified with silver for argentation chromatography because of the additional selectivity conferred by the interactions between silver and Jt-bonds of unsaturated hydrocarbons. In a recent example, methyl linoleate was separated from methyl linolenate on silver-modified silica in a dioxane-hexane mixture.23 Bonded phases using amino or cyano groups have proved to be of great utility. In a recent application on a 250 x 1-mm Deltabond (Keystone Scientific Belief onte, PA) Cyano cyanopropyl column, carbon dioxide was dissolved under pressure into the hexane mobile phase, serving to reduce the viscosity from 6.2 to 1 MPa and improve efficiency and peak symmetry.24 It was proposed that the carbon dioxide served to suppress the effect of residual surface silanols on retention. 

Typical functional groups that can interact with the sorbent are hydroxyl, amino, carbonyl, aromatics, double bonds, and groups containing heteroatoms such as oxygen, nitrogen, sulfur, and phosphor. 

Typical sorbents for normal-phase SPE are silica, cyano, did, NHz (all silica based), alumina (AI2O3 based), and Florisil (MgSi03 based) (Table 9.3). 

Since normal-phase SPE is usually based on polar interactions, it is of importance that both sample matrix, conditioning, equilibration, and wash solvent, are nonpolar organics. This is to ensure that there is no elution of the analyte (and thereby analyte loss) during sample application and sorbent wash. The analytes are eluted by 

Many of the interactions discussed in this chapter are also discussed in Section 3.5. 

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Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

Nonpolar organic mobile phases, such as hexane with ethanol or 2-propanol as typical polar modifiers, are most commonly used with these types of phases. Under these conditions, retention seems to foUow normal phase-type behavior (eg, increased mobile phase polarity produces decreased retention). The normal mobile-phase components only weakly interact with the stationary phase and are easily displaced by the chiral analytes thereby promoting enantiospecific interactions. Some of the Pirkle-types of phases have also been used, to a lesser extent, in the reversed phase mode. 

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). 

Also for analysis of some pharmaceutical substances a normal-phase mode of HPLC and a lot of organic solvents are needed, especially if it is used in routine analysis. 

We proposed using MLC for assay of azithromycin in tablets and capsules. As alternative conventional reversed-phase HPLC method MLC was used for analysis of Biseptol (sulfamethoxazole and trimethoprim) tablets and injection. The MLC was proposed to assay of triprolydine hydrochloride and pseudoephedrine hydrochloride in tablets as alternative normal-phase HPLC method described in USP phamiacopoeia. 

The results confirmed that the chloroheptane/n-heptane mixture behaves in an identical manner to carbon tetrachloride and all the points were on the same straight line as that produced using a mixture of carbon tetrachloride and toluene. These experiments are similar to normal phase chromatography using pure water instead of... 

The column was operated in the normal phase mode using mixtures of n-hexane and ethanol as the mobile phase. Equation (13) is validated by the curves relating the corrected retention volume to the reciprocal of the volume fraction of ethanol in Figure 19. It is seen that an excellent linear relationship is obtained between the corrected retention volume and the reciprocal of the volume fraction of ethanol. 

While partially concurrent eluent evaporation is easier to use, and is preferred for the transfer of normal phase solvents, concurrent eluent evaporation with co-solvent trapping is the technique of choice for transfer of water-containing solvents, because wettability is not required. 

Coupled liquid chromatography-gas chromatography is an excellent on-line method for sample enrichment and sample clean-up. Recently, many authors have reviewed in some detail the various LC-GC transfer methods that are now available (1, 43-52). For the analysis of normal phase eluents, the main transfer technique used is, without doubt, concurrent eluent evaporation employing a loop-type interface. The main disadvantage of this technique is co-evaporation of the solute with the solvent. 

Normal-phase LC tends to separate according to solute polarity since the stationary phase is polar and retention is often dominated by hydrogen bonding. Thus, normal-phase LC is useful in sorting out classes of materials according to the polarity of the solutes. Fatty acids are easily separated from monoglycerides, but the separation of individual saturated fatty acids from each other on the basis of their carbon... 

In the first version with a mobile phase of constant composition and with single developments of the bilayer in both dimensions, a 2-D TLC separation might be achieved which is the opposite of classical 2-D TLC on the same monolayer stationary phase with two mobile phases of different composition. Unfortunately, the use of RP-18 and silica as the bilayer is rather complicated, because the solvent used in the first development modifies the stationary phase, and unless it can be easily and quantitatively removed during the intermediate drying step or, alternatively, the modification can be performed reproducibly, this can result in inadequate reproducibility of the separation system from sample to sample. It is therefore suggested instead that two single plates be used. After the reversed-phase (RP) separation and drying of the plate, the second, normal-phase, plate can be coupled to the first (see Section 8.10 below). 

On the basis of the principle of grafted TLC, reversed-phase (RP) and normal-phase (NP) stationary phases can also be coupled. The sample to be separated must be applied to the first (2.5 cm X 20 cm) reversed-phase plate (Figure 8.16(a)). After development with the appropriate (5ti 5yi) mobile phase (Figure 8.16(b)), the first plate must be dried. The second (20 cm X 20 cm) (silica gel) plate (Figure 8.16(c)) must be clamped to the first (reversed-phase) plate in such a way that by use of a strong solvent system (Sj/, SyJ the separated compounds can be transferred to the second plate (Figure 8.16(d)). Figure 8.16(e) illustrates the applied, re-concentrated... 

J. W. Hofsti aat, S. Griffioen, R. J. van de Nesse and U. A. Th Brinkman, Coupling of naiTOw-bore column liquid cliromatography and thin-layer cliromatography. Interface optimization and characteristics for normal-phase liquid cliromatography , J. Planar Chromatogr. 1 220-226 (1988). 

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

Figure 10.9 shows the ehromatograms of fortified eoeonut oil obtained by using (a) normal-phase HPLC and (b) GPC/normal-phase HPLC. As ean be seen from these figures, ehemieal interferenees due to lipid material in the oil were eliminated by using the MD system that was used for quantitative analysis of all of the eom-pounds, exeept DL-a-toeopheryl aeetate, where the latter was eo-eluted with a triglieeride eompound and needed further separation. 

Figure 10.9 Cliromatogi ams of foitified coconut oil obtained by using (a) normal-phase HPLC and (b) GPC/noimal-phase HPLC. Peak identification is as follows 1 (a,b), DL-a-toco-pheryl acetate, 2 (b), 2,6-di-tert-butyl-4-methylphenol 2 (a) and 3 (b), retinyl acetate 3 (a) and 4 (b), tocol 4 (a) and 5 (b), ergocalciferol. Reprinted from Analytical Chemistry, 60, J. M. Brown-Thomas et al., Determination of fat-soluble vitamins in oil matrices by multidimensional liigh-peiformance liquid cliromatography , pp. 1929-1933, copyright 1988, with permission from the American Chemical Society.

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