Silica stationary phases

Stationary phase Silica gel, alumina, cellulose Silica gel (mainly), alumina, cellulose... 

The testing of impnrities in active pharmacentical ingredients has become an important initiative on the part of both federal and private organizations. Franolic and coworkers [113] describe the utilization of PLC (stationary phase — silica gel and mobile phase — dichloromethane-acetonitrile-acetone (4 1 1, v/v)) for the isolation and characterization of impurities in hydrochlorothiazide (diuretic drug). This drug is utilized individually or in combination with other dmgs for the treatment of hypertension. The unknown impurity band was scraped off the plate and extracted in acetonitrile. The solution was filtered and used for LC/MS and NMR analysis. The proposed procedure enabled the identification of a new, previonsly nnknown impurity. It was characterized as a 2 1 hydrochlorothiazide-formaldehyde adduct of the parent drug substance. 

In NPLC, which refers to the use of adsorption, i.e. liquid-solid chromatography (LSC), the surface of microparticulate silica (or other adsorbent) constitutes the most commonly used polar stationary phase normal bonded-phase chromatography (N-BPC) is typified by nitrile- or amino-bonded stationary phases. Silica columns with a broad range of properties are commercially available (with standard particle sizes of 3, 5 and 10 im, and pore sizes of about 6-15nm). A typical HPLC column is packed with a stationary phase of a pore size of 10 nm and contains a surface area of between 100 and 150m2 mL-1 of mobile phase volume. 

Native PEGs represent a rather difficult class of compounds for either RP-HPLC or NP-HPLC due to their high polarity [3]. On polar stationary phases (silica, diol, CN, aminopropyl) they are very strongly retained and strong solvents like methanol or ethanol are needed to elute them. However, such an elution leads to very poor resolution of... 

Above we have considered the examples of separation according to functionality on silica gel as the stationary phase. Silica gel has been prefered for certain functional groups and because of the existence of a well-founded correlation theory for silica gel64. At present, however, most separations are performed on chemically modified phases. All the basic regularities of the chromatography of macromolecules close to the critical conditions are also valid for these stationary phases. 

Actually, solid-phase extraction is used not only as a rough preliminary fractionation procedure. Prieto et al. described the complete fractionation of the total lipids from wheat into eight neutral lipid, two glycolipid, and four phospholipid classes in addition to PC and LPC, TV-acyl PE and A-acyl LPE were detected (37). However, two separate stationary phases (silica and aminopropyl) as well as seven different mobile phases were needed. Moreover, 14% crosscontamination of PC and LPC was observed, and the recovery of the phospholipids was limited to about 85%. Hence, SPE is a rapid and efficient technique for preliminary fractionation, but loses its advantages if more complex separations are tried. 

Stationary phase silica gel G, 250 /im thick, impregnated with 0.1 M potassium hydroxide in methanol, and dried. 

TLC the thin layer chromatogram of the tested compound may not differ from the chromatogram of the reference compound stationary phase, silica gel GF254R, mobile phase chloroform-methanol - ammonia 25% (70 40 10 vol. parts), saturated chamber, detection 254 nm. 

Fig. 17. Gradient HPLC chromatogram of the 16-component star polymer mixture, stationary phase silica gel, mobile phase i-octane/THF,linear, 20-100% THF (from [92] with permission)...

Table 6 Column Chromatographic Conditions Used to Purify Cryptands (Stationary phase silica gel)...

Classical separations by open column chromatography with different stationary phases (silica gel, reversed-phase C-18 or C-8, polyamide, cellulose) and elution with appropriate solvent mixtures are also useful for flavonoid fractionation and purification. Different column systems can be used. The classical open column chromatography uses relatively large particle sizes (0.2-6 mm), with limited resolution, and solvent filtration through the column proceeds by the pressure of the solvent column placed on top of the stationary phase. In other cases, smaller... 

Conditions Solvent benzene n-butanol pyridine water, 1 5 3 3 (upper layer) stationary phase silica Gel, 0.25 mm thick development ascending multiple development (t ce), one-half distance (7.0 cm) first run, full length (14.0 cm) second run. 

Stationary phases (silica, reversed phases, Amberlite, and Sephadex) or by solid-phase extraction techniques using different adsorbents, which in the last decade has been found very convenient for the isolation of flavonoids from complex matrices. 

Stationary Phase Silica Gel 60 TLC plate Mobil phase Chloroform/Methanol/Water = 65 25 4 UV light is used for general visualization. Rf value of initial NGPE = 0.37... 

Stationary Phase Silica Gel 60 F g TLC plate Mobil phase 65/25/4 (or 80/20/2) Chloroform/ Methanol/Water... 

The optimization strategy will be exemplified by a rather difficult isomer separation. The product to be separated was a relatively strong base and first experiments using neat solvents on different types of stationary phases (silica gel, amino-, cyano-, and diol-modffied silica gel) revealed that it would be very difficult to separate the two isomers on a larger scale. Therefore, synthetic chemists were contacted to modify one of the secondary base functions with an easily removable BOC (tert-butoxycarbonyl) group. 

Figure 4.9 Experimental adsorption isotherms of pure cis- and trans-androsterone. Stationary phase silica treated with a phosphate buffer mobile phase acetonitrile-dichloromethane solutions. Symbols, experimental data soUd lines, best fit of experimental data to the Langmuir model. 1 and , cis-androsterone 2 and o, frans-androsterone. Reproduced with permission from f.-X. Huang and G. Guiochon, J. Colloid and Interf. ScL, 128 (1989) 577 (Fig. 2).

Figure 11.23 Qualitative prediction of the elution order reversal of cis- and frans-androsterone. (Left) Anal5dical and preparative sample sizes. High concentration band profiles calculated with the LeVan-Vermeulen isotherm model. Inset experimental data [23], Stationary phase silica modified with a pH 6.8 phosphate buffer. Mobile phase (9 1) acetonitrile-dichloromethane, 0.98 mL/min. Samples, cis-androsterone 0.026 and 5.2 mg, trans-androsterone 0.15 and 1.8 mg. (Right) Band profiles calculated with the competitive Langmuir model. Reproduced with permission from S. Golshan-Shirazi, J.-X. Huang and G. Guiochon, Anal. Chem., 63, (1991) 1147 (Fig. 8). ( )1991, American Chemical Society.

In general, the eluting strength of commonly used solvents for normal phase chromatography is stationary phase = silica gel neutral alumina increasing order proceeds as follows petroleum ethers < hexanes < cyclohexane < toluene < diethyl ether < dichloromethane < chloroform < ethyl acetate < acetone < ethanol < methanol < acetic acid. 

The principle of adsorption chromatography (normal-phase chromatography) is known from classical column and thin-layer chromatography. A relatively polar material with a high specific surface area is used as the stationary phase, silica being the most popular, but alumina and magnesium oxide are also often used. The mobile phase is relatively nonpolar (heptane to tetrahydrofuran). The different extents to which the various types of molecules in the mixture are adsorbed on the stationary phase provide the separation effect. A nonpolar solvent such as hexane elutes more slowly than a medium-polar solvent such as ether. 

Figure 2.28 Separation of phenothiazine derivatives on a short column (Prolabo). Conditions column, 5cm x 4mm i.d. stationary phase, silica, 6.2 im mobile phase, 0.8 ml min diisopropyl ether-methanol (1 1) containing 2.6% water and 0.2% triethylamine pressure, 15 bar UV detector, 254 nm number of theoretical plates, 850 (final peak). Peaks 1 =3-chlorophenothiazine 2 = chlorophenethazine 3 chloropromazine 4 = promazine 5 = 5,5-dioxy-chlorpromazine 6 = oxy-chlorpromazine 7 = 2-chloro-10-(3-methylaminopro-pyD phenothiazine 8= N-oxychlorpromazine.

Figure21.2 Preparative separation ofthe reaction mixture. Conditions sample, 1 ml of solution containing 250 mg guard column, 5 cm x 4.5 mm i.d., dry-packed with silica (40gm) column, 25cm x 21.5mm i.d. stationary phase, silica, 7 xm ...

To the silica are bound linear chains bearing aminopropyl, cyanopropyl, benzyl groups or dipolar ligands (zwitterions) which confer an intermediate polarity to the stationary phase (Figure 3.10). An improvement is noted in the separation of very small polar molecules which require mobile phases rich in water (cf. paragraph 3.8). For example, sugars, peptides and other hydrophilic compounds become separable under these conditions (see, e.g. Figure 3.14). For these modified stationary phases, silica gel acts as a support. 

Fig. 3. Elution of polystyrene as a function of the degree of polymerization P and the eluent composition stationary phase silica gel mobile phase chloroform-carbon tetrachloride. (From [8] with permission)...

Fig. 6. Separation of hydroxy-terminated poly(butylene glycol terephthalate) as a function of the mobile phase composition stationary phase silica gel mobile phase THF-heptane peaks 1.2 solvent, peaks 3-7 nonfunctional homologues (n = 0-4), peaks 8-12 monofunctional homologues (n = 0-4), peaks 13-17 bifunctional homologues (n = 04). (From [2] with permission)...

Fig. 24. Chromatograms of PS-PMMA blands in different chromatographic modes, and critical diagram log molar mass vs retention time for PMMA upper right comer) stationary phase silica gd Si-100 mobile phase methylethylketone-cyclohexane. (From [53] with permission)...

Fig. 22.1 Separation of bile acids using micro-HPLC. (Reproduced with permission from D. Ishii, S. Murata and T. Takeuchi, J. Chromatogr., 282, 569 (1983).) Conditions sample, 11 nl of solution containing 20 ng each of the acids guard column, 5cm x 0.2 mm i.d. fused silica column, 20cm x 0.26 mm fused silica stationary phase, silica ODS CS-01, 5 im mobile phase, 60 mM phosphate buffer containing NAD and acetonitrile, gradient with increasing acetonitrile content, 2.1 pi min - detector, derivatization with immobilized enzyme, then fluorescence, 365/470 nm. For abbreviations, see text.

Table 6.5 Examples of TLC systems for separating phospholipids (stationary phase = silica gel G) (data from Spanner, 1973)...

Although new materials are rapidly emerging for chromatographic stationary phases, silica is still the principal medium used as the material for liquid chromatography (LC) packing [1-3]. 

---