Normal phase silica

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

Normal phase silica column Chloroform-methanol-ammonia solution (86.8 12.5 0.7) 254 nm Assay of primaquine and hepatic targeting neoglycoalbumin-primaquine in whole blood and liver of mouse by reversed-phase HPLC. [105]... 

There are much better parallels to HPLC TLC or column chromatography. Vary the eluents in these techniques and you get widely different results. With a normal-phase silica-based column, you can get results similar to those from silica gel TLC plates. 

Normal phase Silica(weak) 5-15 Ester, ether, diester... 

These compounds were analyzed by using normal phase silica columns operated at ambient temperature. Mobile phase constituents included diethylene dioxide/ethyl acetate/chloroform/hexane-pyridine... 

Reversed-phases are generally very stable, and do not dissolve in aqueous mobile phases, as do normal-phase silica-gel columns. After injection of many samples that contain highly retained, nonpolar materials, resolu-... 

SPE cleanup, online LC purification on Spherisorb S5W normal phase silica column... 

Normal phase Silica gel SS-05 1 % Isopropanol in hexane 380 chi, pheo, car Spinach 120... 

Normal phase silica column scouting is run the same way. Start gradients at 25% chloroform/hexane and run to 100% chloroform in 20min. For isocratic scouting, start at 80% chloroform/hexane and make dilutions with hexane. We will cover methods development in more detail in Chapter 11. 

Normal-phase silica packing requires only drying at a uniform temperature to be ready for packing. At 250°C, the fully hydrated silica is produced, while at 300°C water is lost between adjacent silica molecules forming the anhydride form normally packed in normal-phase columns. 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

Separation and purification of three turmerones, e.g. ar-turmerone, a- and P-turmerone, from turmeric oil extracted by supercritical carbon dioxide gave 71% purify by weight. Subsequently, purification using a normal-phase silica gel 60 column could separate and purify three major turmerones with 86% purify by weight of ar-turmerone and 81% purity by weight of a- and P-turmerone. These were identified by liquid-solid chromatography, NMR qualification and HPLC quantification, respectively (Li-Hsun Chang et al., 2006). 

Various carotenoids brown and red seaweeds Reverse Phase, C18 column Methanol and Water (9 1 v/v) isocratic Normal phase, Silica gel column Tetrahydrofuran and hexane (35 65 v/v) isocratic Maoka et al., 2002... 

Purified fucoxanthin Brown seaweed Normal phase, silica gel column Chloroform and Acetone (9 1) isocratic Yan et al., 1999... 

Fucoxanthin and its metabolites Egg yolk Normal phase, Silica gel column Acetone hexane gradient Normal phase, Silica gel column Hexane, Dichlormethane, 2-Propanol and Ethyl-diisopropylamine (90.9 7 3 0.1 v/v/v/v) isocratic Reverse phase, C18 column Methanol and Water (67 33 v/v) isocratic Bonded nitrile column Hexane, Isopropyl acetate, Acetone, Methanol (76 17 7 0.1 v/v/v/v) isocratic Strand et al., 1998... 

Chromatographic Mode Normal Phase (Silica, Florisil , Alumina, Diol, NH2, etc.) Reverse Phase (C18, CN, etc.) Ion Exchange (NH2, Anion Exchange, Cation Exchange, etc.)... 

The product was separated from the substrate on a normal phase silica gel column (Zorbax Sil) and eluted with methanol at 35°C as shown in Figure 9.621,II. The concentration of the reactants was determined by the fluorescence intensity at 535 nm, with an excitation wavelength at 340 nm. 

In recent years several normal-phase HPLC methods have been reported for the quantitative analysis of tocopherols and tocotrienols (Table 11.5). The best of these methods have been able to achieve baseline separation of all four tocopherols and all four tocotrienols, as shown in Figures 11.2 and 11.3. Kamal-Eldin et al. (2000) reported the optimal baseline separation of all eight common tocols using a Diol-bonded phase column and an isocratic mobile phase of hexane/methyl tert-butyl ether (MTBE), 96 4, v/v (Figure 11.2). Similar separations were reported by Moreau et al. (2007) using the same type of column and mobile phase. Schwartz et al. (2008) reported that, with a normal-phase silica column, plastochromanol-8 in rapeseed oil eluted between y-tocopherol and 5-tocopherol. 

Lewis acid-mediated ene reaction of di- —)- / ,25)-2-phenyl-1-cyclohexyl diazenedicarboxylate with cyclohexene using tin(IV) chloride in dichloromethane at —60 °C for 5 min afforded the azo-ene adduct in 80% yield after purification by flash chromatography (eq 2). The H NMR spectrum of the azo-ene adduct recorded at 380 K in deuterated toluene established the presence of only one diastereomer. Further analysis of the ene adduct by HPLC on a Whatman Partisil 5 normal phase silica column using hexane-ethyl acetate (9 1) as eluent confirmed the presence of only one diastereomer. 

The use of normal phase silica columns was also described but after acetylation of the flavonoids and then isocratic elution on silica gel. Polystyrene divinylben-zene as a stationary phase was also found to give satisfactory separation and good peak shapes without using... 

Since the functionality of most silica stationary phases is the same, the selection of the mobile phase is very important during the screening and method development of a normal-phase silica separation. As mentioned above, typical mobile phases involve a nonpolar solvent like heptane and a polar solvent like ethanol, but the combination of solvents is almost endless. A good screening (using TLC or analytical HPLC) should examine numerous solvent combinations utilizing different types of solvents, such as THF instead of alcohols, ethyl acetate or methylene chloride instead of heptane and should also examine the use of additives, e.g., acetic acid or triethylamine, to adjust the pH to either suppress or enhance interactions with the stationary phase. More information on the method development is provided in Section V. 

Normal phase silica Reversed phase silica... 

Most analytical separations are performed by reversed phase systems while most preparative separations are performed using an NP system. For example, 84% of the analytical separations at Schering AG, a mid-sized pharmaceutical company, are done in RP mode while 85 % of the preparative separations are performed on normal phase silica gels (Brandt and Kueppers, 2002). 

Normal phase Silica Heptane Ethyl acetate... 

This fact served as a test for the presence of the phytyl ester. Non-polar pigments, pheophytins and decarboxylated species, were purified over normal phase silica with increasing percentages of acetone in petroleum ether. 

Isolation of Metabolites from Poultry Bile for Spectral Identification. Pooled samples of bile from poultry receiving feed medicated at 25 ppm semduramicin sodium for 7 days were extracted with ethyl acetate followed by chloroform. Abundant metabolites were isolated by collection of fractions from repeated injections on a normal phase silica HPLC column. The recovered fractions were analyzed by Fast Atom Bombardment Mass Spectrometry and proton NMR spectroscopy. 

Silica gel may be chemically modified in a number of ways that alter both its chromatographic and physical properties. As shown in Fig. 4, the reactive silanol groups of silica gel may be blocked with a variety of silylchlorides to produce a nonpolar (reverse phase) or polar (bonded normal phase) chromatography support. The most commonly employed bonded phase silica gels are the reverse phase class although the use of bonded normal phase silica has increased with improved materials. 

Bonded normal phase silica is prepared in a manner analogous to the production of reverse phase supports, as shown in Fig. 5. Rather than straight-chain alkyl or phenyl silanes, short-chain, functionalized silanes are attached to the silica gel matrix. Amino-, diol-, and cyano-based bonded normal phases are commonly encountered. The main advantage of bonded normal phase supports over unmodified silica gel is the increased stability to polar solvents, including water, which allows the separation of very polar compounds that... 

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