Normal phase SPE

In normal phase soUd phase extraction, the stationary phase is composed of a modified silica polar material. Ihe analytes of interest will be polar, in order to have an affinity for the packing material, and the matrix should be nonpolar, in order for the extraction to take place. 

In comparison to reverse phase SPE, the separations in normal phase SPE occur due to the polar functional groups of the packing material and the polar functional groups of the analytes of interest interacting. The interactions that occur are based on more than one intermolecular force and include hydrogen bonding, dipole-dipole interactions, and tt-tt interactions, as well as others. 

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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For normal-phase SPE, cyanopropyl (CN), aminopropyl (NH2), and diol functional groups are chemically bonded to the silica gel. The loading on the cyano, amino, and diol colunms are sufficiently large ( 6-10% as carbon) that they may sometimes be used for reversed-phase applications, especially for the removal of hydrophobic solutes from water or other polar solvents. These hydrophobic solutes would otherwise sorb too strongly to a more hydrophobic C-8 or C-18 sorbent and would be difficult to elute. Straight silica gel also is... 

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. 

As early as the 1930s, silica, alumina, Florisil, and kieselguhr (diatomaceous earth) were used as solid sorbents for normal-phase sample concentration, for what would now be called normal-phase SPE. The next major development occurred in 1941 when Martin and Synge (Nobel Prize for work in chromatography) published their work on partition chromatography. They... 

However, the R group can contain any of the functional groups shown in Table 1.1. This includes reversed phases, such as C-2, C-4, C-8, C-18, cyclohexyl, phenyl, and even a single C-1 is available. The cyclohexyl and phenyl groups typically are bonded with a three-carbon alkyl chain connecting them to the surface of the silica gel. Likewise, the normal-phase sorbent and ion-exchange sorbents typically will contain a three-carbon linking chain to the silica-gel surface. Structures are shown in later chapters on reversed-phase and normal-phase SPE. 

Normal-phase SPE refers to the sorption of an analyte by a polar surface. It is ... 

The types of nonbonded phases used for normal-phase SPE are silica, alumina, and magnesium silicate (Florisil). The most popular phase is silica. Several bonded phases may also be used for normal-phase SPE, including aminopropyl, cyanopropyl, and propyldiol (Table 1.1, Fig. 2.7). Water is not used in the mobile phase in normal-phase SPE because it will sorb to the active sites of the sorbent and reduce the interaction between analyte and sorbent. Typically, normal-phase SPE is used as a clean-up procedure for organic extracts of water, soil, food, or other materials. Normal-phase SPE is also used for the isolation of analytes from organic liquids, such as oils. 

The elution of the solute from a normal-phase sorbent is typically a function of the eluotropic strength and polarity of the eluting solvent. Table 2.1 shows the solvent eluotropic strength and polarity for a range of organic solvents typically used in normal-phase SPE. Silica will adsorb moderately polar... 

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... 

For these reasons, normal-phase SPE does not use polar solvents, such as water or alcohols, but works extremely well with nonpolar solvents such as hydrocarbons (hexane), chlorinated solvents (methylene chloride), or ether solvents (petroleum ether). Thus, normal-phase SPE is a popular method for applications that require the removal of organic analytes from nonpolar... 

Alumina may also be used as a sorbent in normal-phase SPE. It may be acidic, neutral, or basic depending on the pH of the wash solution used on the alumina. It too may bind solutes by hydrogen bonding, weak ion exchange, and other polar reactions. The type of alumina generally used will depend on the analytes, with basic solutes sorbed to acidic alumina and acidic analytes sorbed to basic alumina. The magnesium silicate, Florisil, may also be used in a similar fashion to silica. 

Table 5.1 shows the various grades of these materials that are available for normal-phase SPE. The sorbents come in a variety of diameters from 25 to 200 tm, with sizes typically between 60 and 100 Ltm. Because of the long history of silica use, it has the most applications in the literature for normal-phase isolation and separation. 

Although there have been many important uses for silica in normal-phase SPE, there is an important shift to newer silica-based bonded-phase sorbents for methods development. The reason for this shift is because of the occurrence of strong, often irreversible sorption sites on silica gel caused by free... 

Normal-phase SPE methods development is a straightforward process, and for this reason, many methods have been developed on silica sorbents, especially with the broad and comprehensive literature available in thin layer chromatography (TLC). The development of the silica bonded phases has introduced a new and important aspect to methods development with normal-phase SPE. 

Fats, oils, and lipids are common components of meats, nuts, and dairy products and manufactured goods, such as potato chips, cookies, and chocolate. They are soluble in nonpolar solvents, such as hexane and methylene chloride. The analyte, of course, should also be soluble in the extraction solvent. Typically normal-phase SPE would be used to retain a compound from this extraction solvent. A solid fat may be homogenized in a blender with hexane, filtered or centrifuged, then the solvent would be passed through a normal-phase column for retention of the solute. Another approach is the use of matrix solid-phase dispersion, where the solid would be ground into the silica and C-18 directly, then the analyte eluted directly from the ground mixture with either hexane or methylene chloride. The hexane or methylene chloride extract could then be applied directly to a normal-phase sorbent for separation. Liquid oils may be directly diluted with hexane or methylene chloride and applied to the normal-phase sorbent. Other lipid substances may be handled either as solids or liquids depending on their form. 

Other sorbents include mixed mode sorbents, normal-phase SPE sorbents or restricted access matrix sorbents. 

Concentration using aCi8 SPE Continuous-flow (methanol), high-temperature (65°C), sonicated extraction system to isolate APEO metabolites from sediment samples (low-power ultrasonic energy) sediment extraction was complete after 7 min with a total solvent consumption of 3.5 ml/sample two-step cleanup, normal-phase SPE, reversed-phase... 

Normal-phase solid-phase extraction refers to the mechanism by which the analyte is adsorbed onto the polar surface of sorbent from a nonpolar solvent. The mechanism of interaction between the analyte and sorbent is a polar interaction, such as hydrogen bonding, dipole-dipole interaction, n-K interaction, and induced dipole-dipole interaction. The sorbents widely used in normal-phase SPE are silica, alumina, and magnesium silicate (Florisil), and the siUca chemically modified with polar groups like amino, cyano, or dioLThe samples for normal-phase SPE are typically dissolved in hexane or isooctane. Step elution with solvents of increasing polarity allows the separation into fractions on the basis of difference in polarity. 

Normal-phase SPE has been in the purification of a library of N-alkylated L-amino acids. In the synthesis of this library, the final N-alkylated L-amino acid products were usually contaminated with small amount of alcohols. These alcohols are less polar than V-alkylated L-amino acids and could be removed by SPE using silica cartridge, washing with 9 1... 

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