Separator Hypersil Silica

Fig. 3.1. Separation of the unbonded silica test mixture using in-house packed (A) Hypersil silica, (B) Hypersil BDS silica, (C) Hypersil HyPURITY and (D) Kromasil silica capillaries (100 pm i.d., 33.5 cm total length and 25 cm effective). Conditions 8 2 v/v ACN-50 mM MES, pH 6.1, 20 kV, 20°C, 5 kV for 3 sec. injections, 254 nm. Peak identities 1 = biphenyl, 2 = benzamide, 3 = benzyl alcohol and 4 = thiourea. Adaptation of [20]. Reproduced with die permission of Chromatographia.

Benzyloxycarbonyl, N-(3,5-dinitrobenzyloxy carbonyl), 9-fluorenylmethoxycarbonyl, benzoyl, acetyl and N-(2,4-dinitrophenyl) derivatized amino acids and profens WAX (weak anion-exchange) type CSP tert. -butylcarbamoylquinine as chiral selector on Hypersil silica gel), 3 pm Acetonitrile-methanol (80 20)+400 mM acetic acid+4 mM triethylamine 335 mm x 100 pm i.d. 250 mm effective length, chiral separation... 

It should be pointed out that separation of more than one class of organic compounds can be achieved by SFC. For example. Fig. 1 shows the chromatogram of 35 PAHs, herbicides, and phenols from a contaminated water sample. Solid-phase extraction was used for sample preparation. Five Hypersil silica columns were coupled in series for separation of these contaminants. The percentage of methanol (as modifier) was varied from 2% (5 min) to 10% (29 min) at 0.5%/min. A pressure program was also applied. A diode-array detector was used in this work. 

Very high separation efficiencies (more than 100000 plates) can be achieved by coupling several packed columns in series (e.g.. Fig. 45 shows a separation of triazine pesticides with ten 2(X)x4.6 mm i.d. hypersil silica (5 pm) columns in series—note that the pressure drop over all these columns is only 12.4 MPa. This would not be possible in HPLC since, because of the higher viscosity of the solvents, the back-pressure over the eolumn would become too high (high-pressure shutdown). 

In order to determine the applicability of retention indices, based on the alkyl arylketone scale, as the basis of a reproducible method of reporting retentions, the separation of 10 barbiturates and a set of column test compounds were examined on an octadecylsilyl bonded silica (ODS-Hypersil) column with methanol-buffer of pH 8.5 as eluent [100]. The effects on the capacity factors and retention indices, on changing the eluent composition, pH, ionic strengthened temperature, showed that the retention indices of the barbiturates were much less susceptible to minor changes in the eluent than the capacity factors. 

Following extraction/cleanup, quinoxaline-2-carboxylic acid can be detected by electron capture, or mass spectrometric techniques, after gas chromatographic separation on capillary or conventional columns. A prerequisite of quin-oxaline-2-carboxylic acid analysis by gas chromatography is the derivatization of the molecule by means of esterification. Esterification has been accomplished with methanol (419, 420, 422), ethanol (421), or propanol (423) under sulfuric acid catalysis. Further purification of the alkyl ester derivative with solid-phase extraction on a silica gel column (422), thin-layer chromatography on silica gel plate (420), or liquid chromatography on Hypersil-ODS, 3 m, column (423), has been reported. 

In liquid chromatography, reversed-phase materials such as Cig and Cg are the most commonly used sorbents (429, 430, 434, 438, 446, 447, 453, 454). Examples of baseline separations with reversed-phase columns of several groups of anabolics including stilbenes, resorcyclic acid lactones, and other, frequently used anabolics have been reported (463-466). In addition to reversed-phase separations normal-phase separations of anabolics using either Hypersil (467) and Brownlee (456) silica or diol-modified silica have been reported. Although not all analytes were completely separated, the latter column could be efficiently used to differentiate between estrogenic and androgenic compounds within a mixture of 15 anabolics and their metabolites (468). 

Figure 25-12 Isocratic HPLC separation of a mixture of aromatic compounds at 1.0 mL/min on a 0.46 x 25 cm Hypersil ODS column (C,8 on 5-jxm silica) at ambient temperature ( 22 C) (1) benzyl alcohol (2) phenol (3) 3, 4 -dimethoxyacetophenone (4) benzoin (5) ethyl benzoate (6) toluene (7) 2,6-dimethoxytoluene (8) o-methoxybiphenyl. Eluent consisted ot aqueous buffer (designated A) and acetonitrile (designated B). The notation 90% B in the first chromatogram means 10 vol% A and 90 vol% B. The buffer contained 25 mM KH2P04 plus 0.1 g/L sodium azide adjusted to pH 3.5 with HCI.

The traditional Hypersil unbonded silica at pH 7.8 gave baseline separation within 14 minutes of the basic analyte mixture (Fig. 3.2). The poorer chromatographic performance of the procainamide analyte was attributed to its suspected metal chelating properties. The purer HyPURITY unbonded silica gave broader peak shapes with reduced analysis times than the traditional acidic silica, but complete separation of the analytes was not observed. The separation of the bases on Hypersil BDS was observed... 

Fig. 3.3. Separation of neutral, acidic and basic components in their ionised form with Hypersil unbonded BDS silica. Peak identities 1= Benzylamine, 11= Caffeine and 111= p-hydroxy benzoic acid. The arrow denotes the EOF. Conditions= 6 2 2 v/v/v ACN H2O 50 mM MES, pH 6.1, 20 kV, 20°C, 214 nm, 8 bar for 15 sec. inj. Adaptation of [20]. Reproduced with the permission of Chromatographia.

A purpose designed CEC stationary phase that gives excellent EOF character yet allows rapid simultaneous acidic, basic and neutral separations under isocratic conditions without tailing is yet to be discovered. Nevertheless, the Hypersil BDS unbonded silica used in this work was taken forward to explore such complex mixture separations as it combines features of both pure and traditional media. As a phase, it possesses a reasonable EOF (as it is based on traditional silica) with a lower number of activated silanol groups on the silica surface (due to the pre-treatment procedure used to remove surface metal contamination). Figure 3.3 shows the separation of benzylamine, caffeine and p-hydroxybenzoic acid in a single chromatographic analysis. 

The dynamic ion-exchange separations are performed successfully on a wide range of stationary phases, which include PS-DVB copolymers (e.g., Hamilton PRP columns), and chemically bonded silica materials (Cjg, Cg, and phenyl groups). Cjg supports are the most popular choice (e.g., hypersil, merck, and phase separation). Columns packed with 3 or 5 xm particles are used for analytical scale separation of lanthanides. 

Figure 22-22 Isocratic HPLC separation of a mixture of aromatic compounds at 1.0 mL/min on a 25 X 0.46-cm Hypersil ODS column (C18 on 5-p.m silica) at ambient temperature ( 22°C) (1) benzyl alcohol (2) phenol (3) 3, 4 -dimethoxyacetophenone ...

Fig. 1. (A) Separation of a mixture of dimethylphthalate, di-n-butylphthalate, and pyrene by reverse-phase HPLC using methanol water (85 15) as eluant comparison of HPLC profiles on ODS-silicas from different manufacturers. (B) Separation of dimethyl- and di-n-butylphthalate on short alkyl silicas (methanol water (85 15) as eluant). Trademarks Spherisorb Phase Separations Limited, U.K. Bondapak Waters Associates Inc., Massachusetts, U.S.A. Hypersil Shandon Southern Products Limited, U.K. Zorbax E. I. DuPont de Nemours, Wilmington, Delaware, U.S.A. LiChrosorb E. Merck, Darmstadt, West Germany Partisil Whatman Inc., New Jersey, U.S.A. These figures are used by kind permission of Hichrom, manufacturers of HPLC columns (% Bennett and Co., Brimpton Common, Reading, Berkshire, U.K.).

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