Mobile-phase gradients, chromatographic

Figure 7.13 Separation of a test eixture using automated multiple development with a universal mobile phase gradient from acetonitrile through dlchloromethane to carbon disulfide on a silica gel HPTLC plate. The chromatogram was scanned at different wavelengths to enhance the chromatographic information.

Fig. 9. Reversed-phase separations of cytochrome c digests obtained with trypsin-modified beads (left) and trypsin-modified monolithic reactor (right) in a tandem with a chromatographic column (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Conditions digestion (left curve) trypsin-modified beads reactor, 50 mm x 8 mm i.d., 0.2 mg of cytochrome c, digestion buffer, flow rate 0.2 ml/min, 25 °C, residence time, 15 min (right curve) trypsin immobilized onto molded monolith other conditions the same as with trypsin-modified beads. Reversed-phase chromatography column, Nova-Pak C18,150 mm x 3.9 mm i.d., mobile phase gradient 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid in 15 min, flow rate, 1 ml/min, injection volume 20 pi, UV detection at 254 nm...

Precolumn Lichrosorb RP2 10 pm (40x2.1 mm ID), column Ultrasphere ODS 5 pm (250x4.6 mm ID), mobile phase gradient with solvent A 0.01 M sodium acetate and 0.005 M tetrabutylammonium hydrogen sulfate in water (pH 4.9), solvent B same salt concentrations in 50% methanol (pH 4.8). Gradient 0-7.5 min 0 B, 7.5-15 min 0-T5% B, 15-25 min 15-30% B, 25-33 min 30-32% B, 33-38 min 32-45% B and 38-41 min 45-0% 6. Detection UV 280 nm. Peaks 1, xanthine 2, uric acid 3, 3-methyluric acid 4, 7-methyl xanthine 5, 3-methyl xanthine 6, 1-methylxanthine 7, theobromine 8, 3,7-dimethyl uric acid 9, 7-methyluric acid 10, 1-methyluric acid 11, 1,3-dimethyluric acid 12, 1,7-dimethyl xanthine 13, theophylline 14, e-hydroxyethyltheophylline (internal standard) 15, 1,7-dimethyluric acid 16, 1,3,7-trimethyluric acid 17, caffeine, (reproduced with permission from ref. 192, by the courtesy of Journal Chromatographic Science)... 

The optimization of reversed phase separation by gradient runs is sometimes unavoidable even in process scale. Mobile phase gradients can reduce the development time dramatically as the solvent strength is varied during the chromatographic... 

FIGURE 10.7 Chromatographic comparison mobile-phase gradients (dotted line) using a linear (a) vs. a step (b) gradient. (Reprinted from Waybright, T.J. et al., J. Liquid Chromatogr. Related Technol., 29, 2475, 2006. With permission.)... 

Running a sample of the natural product extract on analytical gradient HPLC (Fig. 1) using a mobile phase gradient of wide ranging polarity should serve to separate the mixture and elute all the components. On the basis of retention time, It should be possible to select general chromatographic conditions (either HPLC or low-pressure columns) for further preparative purification. 

In HPLC the mobile phase is also easily manipulated indeed, the use of mobile phase gradient systems provides HPLC with one of its greatest advantages over other chromatographic methods. In GLC no equivalent technique exists, while in TLC and paper chromatography... 

Fig. 4.6. Anion-exchange chromatography of carbohydrates as their borate complexes. Chromatographic conditions column, Aminex A-28 10 /im (50x0.3 cm I.D.) mobile phase, gradient of 40 ml of 0.124 M borate, pH 7.0 to 40 ml of 0.58 M borate, pH 10.0 flow rate, 10 ml/h temperature, 55°C detection, orcinol reagent reacted at 100°C for 20 min and measured by UV at 420 nm. Reproduced from Morrison et al. (1976), with...

Although infrequently used, good chromatographic separations of the HETEs can be obtained using normal phase chromatography. A separation on a /iPorasil silica gel column with a linear mobile phase gradient from 100% hexane-acetic acid (100 0.8) to 100% cMoro-form-acetic acid (100 0.8) has been reported to resolve 12-HETE,... 

HETE, 9-HETE and 5-HETE unfortunately, 15-HETE coeluted with 12-HETE and 8-HETE with 9-HETE (Boeynaems et al., 1980). A second chromatographic separation describes the resolution of 5-HETE, 8-HETE, 9-HETE, 11-HETE and 12-HETE produced by human neutrophils in the presence of arachidonic acid (Goetzl and Sun, 1979) the separation used a normal phase column with a linear mobile phase gradient from 100% hexane-acetic acid (125 1) to chloroform-methanol-acetic acid (125 5 1). 

FIGURE 19.9 (a) Chromatographic separation of 50 ng/mL EPI-hNE4 on a Zorbax SB-C18 with a gradient mobile phase, b) Chromatographic separation of IS (alkylated EPI-hNE4) in the same conditions. [Reprinted from Becher et al. (2006) with permission of the American Chemical Society.]... 

Various fiingi from the genus Phoma were extracted with ethyl acetate and characterized by their chromatographic profiles using a C,g column (photodiode array detector from A = 200-400 nm, monitor at A = 230nm) with a 60-min 100/00/100 water/methanol mobile phase gradient. Marine versus terrestrial strains were differentiated. Two compounds positively identified by thermospray MS were mellein and hydroxymellein [415]. 

Sander and Field (16) used a liquid chromatograph (solvent programmer in conjunction with two pumps) to generate a mobile-phase gradient. The eluent was introduced into the developer trough and distributed across the layer. 

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