Silica column, microparticulate

Bulk vitamin Do may contain some of synthetic by-products shown in Scheme IV. Tartivita et al. (33) have reported an excellent chromatographic system which showed resolution of most of the photochemical isomers and reaction by-products. The chromatograms obtained on a 30-cm x 4-mm i.d. commercial microparticulate silica column using a 70 30 1 mixture of chloroform (free from ethanol and water), n-hexane, and tetrahydrofuran at a flow rate of 1 ml/min is shown in Figure 10. The detection was by a 254 nm UV detector. Using this system, vitamin D3 was quantitated in a resin sample containing 20 x 106 IU/g with a relative standard deviation of 1.37%. This procedure is essentially the basis for the USP XX (30) procedure for the analysis of bulk vitamin D3 which is reproduced below, in its entirety. 

Reverse Phase Chromatography. Microparticulate silica columns, and, more recently, more stable polymer matrices (e.g. vinyl alcohol copolymer gels (50) and polystyrene divinyl benzene resins) are modified with relatively nonpolar hydrocarbon chains (usually Cj g) to produce reverse phase (RP) columns. 

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. 

Analytical hplc these days is nearly always done with microparticulate column packings, which are small porous particles, usually spherical or irregular silica, with nominal diameters of 3,5 or 10 fxm. They combine the best features of the other two types, having high efficiency as well as a large surface area. In bulk, the appearance of a microparticulate silica resembles that of a fine talcum powder. With microparticulates, dry packing methods result in column beds that are unstable under pressure, so they are packed into columns using a slurry of the material in a suitable solvent and under considerable pressure. 

A number of specialised stationary phases have been developed for the separation of chiral compounds. They are known as chiral stationary phases (CSPs) and consist of chiral molecules, usually bonded to microparticulate silica. The mechanism by which such CSPs discriminate between enantiomers (their chiral recognition, or enantioselectivity) is a matter of some debate, but it is known that a number of competing interactions can be involved. Columns packed with CSPs have recently become available commercially. They are some three to five times more expensive than conventional hplc columns, and some types can be used only with a restricted range of mobile phases. Some examples of CSPs are given below ... 

Ionic solutes can be separated by ion-exchange chromatography using microparticulate resins or bonded ion-exchangers based on microparticulate silica. Such separations are often achieved more easily by ion-suppression or ion-pairing techniques, which use bonded phase columns in the reverse phase mode. 

Production of materials in which the daughter polymer and the template together form a final product seems to be the most promising application of template polymerization because the template synthesis of polymers requiring further separation of the product from the template is not acceptable for industry at the present stage. Possible method of production of commonly known polymers by template polymerization can be based on a template covalently bonded to a support and used as a stationary phase in columns. Preparation of such columns with isotactic poly(methyl methacrylate) covalently bonded to the microparticulate silica was suggested by Schomaker. The template process can be applied in order to produce a set of new materials having ladder-type structure, properties of which are not yet well known. A similar method can be applied to synthesis of copolymers with unconventional structure. 

Another example is the purification of a p-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 Jim particle size) Clg silica column, with a mobile phase of aqueous methanol having 0.1 M ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by liquid—liquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( l-2 mg/(g stationary phase-min) compared to 0.01—0.1 mg/(g-min) for proteins). 

The method involves chloroform extraction of acidified waste water samples and rotary evaporation without heat. After redissolving in chloroform the samples were analysed directly by high performance liquid chromatography on a microparticulate silica gel column. A number of solvent combinations are possible and 98 2 cyclohexane-acetic acid is preferred. The minimum detectable concentration is lppm (without sample concentration) and the coefficient of variation is 1-2%. The type of separation achieved with a microparticulate silica gel column is shown in Fig. 4.1. The first peak as determined by gas chromatographic-mass spectrometric analysis, consisted of a complex mixture of polychlorinated compounds, including octa-, hepta- and hexachlorodibenzo-/ -... 

High performance liquid chromatography on a small bore column packed with microparticulate silica based ion exchange material has been used [317] to determine down to 0.01 mg L 1 nitrate in water without interference from other ions associated with potable, pond, river and stream water. 

An early concern with the HPLC technique was the use of high pressures to achieve high flow rates of the mobile phase through a column packed with microparticulate silica. Recent improvements in column design and operating procedures, however, allow the purification of proteins at modest pressures (e.g., 500 psi) and flow rates (30-60 ml/h). Since it has been reported that C 3-alkyl chains are compatible with catalytic activity of adsorbed and eluted proteins, but larger alkyl substituents may cause denaturation (26), the use of reversed-phase columns of medium polarity, e.g., —C Hy-phenyl, when combined with a judicious choice of organic modifier and salt concentrations (e.g., isopropanol and phosphate) at pH... 

Achari and Theimer used methanol - dichloromethane (3 1), to which 1% of 29% ammonia was added, as mobile phase and a microparticulate silica gel column. Atropine, homatropine and scopolamine showed different capacity factors. 

Persson and Lagerstrom analyzed anti arrhythmic drugs in plasma by means of ion-pair partition chromatography. Quinidine and dihydroquinidine were separated on a microparticulate silica gel column loaded by an in-situ technique with the aqueous stationary phase ... 

Kates et al.8 used a solvent system consisting of 0.001 M tr1methyl amine hydrochloride and 0.001 M potassium hydroxide (pH 9) - methanol (1 4) and a microparticulate silica gel column to separate quinidine and dihydroquinidlne. Cinchonine was used as internal standard. 

Peat and Jenmson used a polar mobile phase in combination with a microparticulate silica gel column - as originally described by Jane - for the analysis of quinidine in plasma (Fig.5.14). Quinidine and quinine were not, however, separated cinchonidine was used as internal standard. A similar method was used by Pershing et al. . 

A microparticulate silica gel column and a mobile phase of dichloromethane - hexane - methanol - perchloric acid (65 35 5.5 0.1) was applied by Sved et al.28 to determine the quinidine and dihydroquinidine content in plasma. Perchloric acid was added to induce fluorescence, but it also had some influence on the retention times. 3-Hydroxyquinidine was separated from quinidine and dihydroquinidine however, 2 -quinidi none had the same retention time as quinidine. 51... 

A similar solvent system was also used by Rasmussen et al.51 for the analysis of morphine in organic poppy extracts (Fig. 7.13). The extracts could be analyzed without any purification prior to HPLC analysis. Gimet and Filloux performed analyses on alkaloids -including opium alkaloids - in pharmaceutical preparations, and used a microparticulate silica gel column and diethyl ether or diethyl ether saturated with water as mobile phase. In both cases 0.4% diethylamine was added to the mobile phase (Fig. 7.14). An increase... 

For the analysis of reserpine and hydrochlorothiazide, Butterfield et al. preferred a straight-phase separation. Because of the low concentration of the alkaloid compared to the thiazide drug, it was desirable to maximize the response of the former by eluting it from the column first. It was achieved by the straight-phase separation technique. On a microparticulate silica gel column, the drugs mentioned and a number of decomposition products could be analyzed (Table 8.13). 

Sitar et al. used straight-phase HPLC to analyse theophylline in biological fluids. A microparticulate silica gel column and a mobile phase consisting of chloroform - isopropanol -... 

Evenson and Warren developed an assay for theophylline using microparticulate silica gel columns and a mobile phase of water-saturated chloroform - heptane - acetic acid (300 200 0.4) to which 6% ethanol was added. Heptane was necessary to achieve a separation of theobromine... 

Jarvie et al. used a microparticulate silica gel column for the determination of colchicine in plasma. Iorio et al. isolated and identified some impurities of colchicine by means of HPLC, TLC and MS. Alkaloid separation was performed on silica gel with a gradient system of chloroform - methanol. 

Greving et al. analyzed some basic drugs and quaternary ammonium compounds by means of ion-pair chromatography in the straight-phase adsorption mode. Bromide or perchlorate were used as counter-ions in connection with microparticulate silica gel columns. Chloride and iodide were less suitable as counter-ions, because they caused, respectively, corrosion of the equipment or a too strong UV absorption background of the mobile phase. Methanol was used as mobile phase, containing 0.01-0.1 M of the counter-ion. 

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