Normal-phase chromatography example

Normal-Phase Chromatography Example. Vitamin E, an antioxidant, is a complex made up of tocopherols and tocotrienols (Figure 8-62), which are sometimes used to stabilize formulations. Tocopherols are a series of related benzopyranols with a C16 saturated side chain. Tocotrienols contain three double bonds on the C16 side chain [32]. 

Adsorption chromatography on bare silica is an example of normal-phase chromatography, in which we use a polar stationary phase and a less polar solvent. A more polar solvent has a higher eluent strength. Reversed-phase chromatography is the more common... 

A less obvious example of normal-phase chromatography is the separation of saccharides and oligosaccharides in foods910 and in biological mixtures,1112 using a mobile phase consisting of acetonitrile/water or acetonitrile/dilute phosphate buffer. Although the separation mode has occasionally been misidentified as reversed phase, it is normal phase by virtue of the fact that increased aqueous levels of the mobile phase reduce carbohydrate retention, and elution order follows carbohydrate polarity.1... 

Any sample type that has been separated by normal-phase TLC is appropriate for a liquid-solid chromatographic (LSC) separation. An example of this is shown in Figure 5-45. In this separation, both the TLC plate and HPLC separations were done using the same mobile phase. Several good references on the early work in TLC (22,23) and on adsorption chromatography (24-26) should be consulted by those interested in a historical perspective of the use of normal-phase chromatography. 

High performance preparative liquid chromatography can often be employed to rapidly fractionate the sample. Maximum separation efficiency can be obtained 1f different chromatographic systems are used 1n the preparative and analytical separations. For example, normal phase chromatography could be used for the Initial fract1on1zat1on and reverse phase chromatography used for the anlaytlcal separation. 

NPC is ideally suited for the analysis of compounds prone to hydrolysis because it employs nonaqueous solvents for the modulation of retention. An example of the use of NPC in the analysis of a hydrolysable analyte was demonstrated by Chevalier et al. [28] for quality control of the production of benorylate, an ester of aspirin. A major issue in benorylate production is the potential formation of impurities suspected of causing allergic side effects therefore monitoring of this step is critical to quality control. The presence of acetylsalicylic anhydride prohibited the use of RPLC since it can be easily hydrolyzed in the water-containing mobile phase. However, an analytical method based on the use of normal-phase chromatography with alkylnitrile-bonded silica as the stationary phase provided an ideal solution to the analysis. Optimal selectivity was achieved with a ternary solvent system hexane-dichloromethane-methanol, containing 0.2 v/v% of acetic acid to prevent the ionization of acidic function and to deactivate the residual silanols. The method was validated and determined to be reproducible based on precision, selectivity, and repeatability. 

The retention factor, Eq. (7.2), for each species / is calculated knowing the dead time, t(), and the retention time of species i at infinite dilution, /r,./- There are known methods in the literature for calculating the dead time or retention time for a non-retained peak in normal-phase, reversed-phase and ion-exchange chromatography [67]. For example, in normal-phase chromatography, pentane in 95 5 hexane-acetone is unretained. In reversed-phase chromatography, a common measure of void volume is from the refractive index response obtained when the sample solvent composition is different from the mobile-phase composition. 

Normal-phase chromatography is a close parallel to adsorption chromatography. Briefly, adsorption chromatography most often uses polar silica gel as the stationary phase and a mobile phase that is predominately nonpolar, possibly with some polar modifier. An example of such a mobile phase would be hexane with 2% ethanol. When increasing the percentage of the polar modifier, the elution times decrease. 

In normal-phase chromatography the native, non-modified adsorbent is employed with organic solvent mixtures as eluents. Normal phase chromatography was the classical chromatography mode performed with native silica or alumina, i.e. the adsorbent s surface is hydrophilic and the interaction with the solutes takes place via the hydroxy] groups on the surface. As an example the surface of silica consists of... 

Normal-phase sorbents such as silica and Florisil are used to isolate low to moderate polarity species from nonaqueous solutions. Examples of applications include lipid classification, plant pigment separations, and separations of fat-soluble vitamins from lipid extracts, as well as the clean-up of organic solvent concentrates obtained from a previous SPE method or liquid-liquid extraction. Alumina is used to remove polar species from nonaqueous solutions. Examples include vitamins in feeds and food and antibiotics and other additives from feed. Normal-phase chromatography has been used for a number of years, and most applications for normal-phase column chromatography may be easily transferred over to normal-phase SPE. 

The extraction of aromatic hydrocarbons (Fig. 5.5) from crude oil uses two sorbents in series, first a cyanopropyl column attached to a second silica column. In this procedure, aromatic hydrocarbons are sorbed on both the cyanopropyl sorbent and on the silica sorbent. The heteroatom hydrocarbons (containing nitrogen, oxygen, and sulfur) are trapped on the cyanopropyl sorbent and eluted as a separate fraction from the silica column. Because the major interaction of aromatic heterocyclic hydrocarbons is through hydrogen bonding to the surface of the sorbent, the cyanopropyl sorbent is easier to elute than a silica sorbent alone. For this reason, the cyanopropyl column is used before the silica column. The separation of hydrocarbons from crude oil is an example of normal-phase chromatography that has been performed for many years on silica gel prior to the introduction of SPE. 

There are a number of modes of HPLC available to the analyst. The main difference between them is the type of column used. Two have been mentioned already - reversed-phase and normal phase chromatographies. Five are be discussed briefly here, each having their own applications and suitabilities for certain types of compound. An example of an LC instrument is shown in Figure 3.15. 

Normal phase/reverse phase If the stationary phase is more polar than the mobile phase, this is normal phase chromatography. An example of this is a silica column with its polar silanol groups and a mobile phase of an organic solvent. When the stationary phase is less polar than the mobile phase, this is reverse phase chromatography, exemplified by the hydrocarbons bound to the silica support and a water/acetonitrile mobile phase. Reverse phase chromatography is very widely used as a form of HPLC (see Chapter 6) and most natural products have a region of hydrophobicity that leads to their retention to some extent on a reverse phase column. 

An example of the use of normal phase chromatography to resolve a mixture of androgens is shown in Fig. 11.6.3. Resolution of the epimers of the monohydroxy metabolites of testosterone and androstanedione has been achieved using a mobile phase of hexane-tetrahydrofuran-2-propanol (80 15 5) with a silica stationary phase (Kawalek et al., 1981). 

The most popular chromatographic modes for the resolution of the flavins are reversed phase and normal phase chromatography. For example, flavins can be determined in meat extracts by using a stationary phase of silica gel with a mobile phase of chloroform-methanol (90 10) (Ang and Moseley, 1980). Identification of the by-products of FMN formed during chemical synthesis has recently been improved by the development of a reversed phase system using a C g stationary phase and a mobile phase of methanol-0.1 M ammonium formate, pH 3.7 (17 83). Interestingly, alteration of the mobile phase to methanol-5 mM tetrabutylam-monium formate, pH 3.5 (27 73) results in the elution order of the mono- and diphosphate forms being reversed (Nielsen et al., 1983). 

The properties of solvents has been studied extensively by Snyder (5), who created a dassification of the solvent properties of common solvents. It has been found (7) that (excluding proton donors such as alcohols) the maximum difference in mobile-phase selectivity is obtained if the polar solvents have a large difference in basidty. Thus, for maximum selectivity differences, one solvent should have a low basidty. Solvents of this type are acetonitrile, ethyl acetate or other esters, and acetone or other ketones. The other solvent should have a high basidty examples are ethers such as tert-butyl methyl ether, diethyl ether or tetrahydrofuran, or amines such as triethylamine. Between these groups and alcohols, large differences in chromatographic selectivity can be obtained in normal-phase chromatography (10). 

Retention can also be influenced by other components of the mobile phase present in small concentrations. The user may not be aware of the presence of these components. In such a case, troubleshooting can be quite difiicult. A common example of this problem is water in normal-phase chromatography. Water is always present in all solvents (see Table 9.3) and can shift retention substantially. However, this is not the only example. For instance, a contamination of methanol with amines that influences the retention of basic analytes in reversed-phase chromatography has been observed. To avoid this situation, the use of HPLC-grade solvents is generally recommended for HPLC applications. 

The most popular bonded phases for normal-phase chromatography are aminopropyl, cyanopropyl, and diol. Other polar bonded phases, including bonded ion exchangers, can be used as well. Another, less commonly used polar bonded phase is the nitrophenyl bonded phase. The retentivity of the aminopropyl and the diol phase is comparable to that of the oxides. Cyanopropyl and nitrophenyl phases have a smaller retentivity. Although differences in selectivity can be found for the different bonded phases, they are typically small. Thus the elution order of most members in a family of related compounds is largely preserved from packing to packing. For example, in a study of the elution behavior of steroids on silica, and aminopropyl and cyanopropyl bonded phases, an inversion in elution order was ol rved only for two to four pairs out of IS analytes (16). 

There are a few techniques related to normal phase chromatography that are worth mentioning. Silica can be coated with the salts of heavy metals. This results in unique selectivities for analytes that form complexes with the metal ions. An example of this is argentation chromatography (20). The silica is coated with silver nitrate, which gives it a spedal sel ivity for compounds with al atic double bonds. The technique has acquired a broad range of applications. However, while Ag -coated TLC plates are commercially available, HPLC columns have to be prepared by the user. Other metal salts can be used in a similar manner for different applications. 

In order for the separation to take place, a more polar solvent (than the original sample matrix) will be used to effect the desorption of the analyte molecules from the packing material. Examples of packing material include silica bonded with cyano, amine, and diol groups, as with the stationary phase of columns in normal phase chromatography (see Chapter 4 for further explanation). 

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