Reversed-phase columns flow rate

Figure 12.11 Coupled SEC-RPLC separation of compound Chemigum mbber stock (a) SEC ti ace (b) RPLC trace of fraction 1, dibutylphthalate (c) RPLC trace of fraction 2, elemental sulfur. Coupled SEC conditions MicroPak TSK 3000H (50 cm) X 2000H (50 cm) X 1000 H (80 cm) columns (8 mm i.d.) eluent, THE at a flow rate of 1 mL/min UV detection at 215 nm (1.0 a.u.f.s.) injection volume, 200 p-L. RPLC conditions MicroPak MCH (25 cm X 2.2 mm i.d.) column flow rate, 0.5 mL/min injection volume, lOpL gradient, acetonitrile-water (20 80 v/v) to 100% acetonitrile at 3% acetonitrile/min UV detection at 254 nm (0.05 a.u.f.s.). Reprinted from Journal of Chromatography, 149, E. L. Jolmson et al., Coupled column cliromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

Fig. 12. Separation of styrene oligomers by reversed-phase (left) and size-exclusion chromatography (right) (Reprinted with permission from [121]. Copyright 1996 American Chemical Society). Conditions (left) column, molded poly(styrene-co-divinylbenzene) monolith, 50 mm x 8 mm i.d., mobile phase, linear gradient from 60 to 30% water in tetrahydrofuran within 20 min, flow rate 1 ml/min, injection volume 20 pi UV detection, 254 nm (right) series of four 300 mm x 7.5 mm i.d. PL Gel columns (100 A, 500 A, 105 A, and Mixed C), mobile phase tetrahydrofuran, flow rate, 1 ml/min injection volume 100 pi, toluene added as a flow marker, UV detection, 254 nm temperature 25 °C,peak numbers correspond to the number of styrene units in the oligomers...

The influence of various structural and physicochemical parameters of the stationary and mobile phases on the tailing of a cationic dye in reversed-phase chromatography has been studied in detail. Measurements were performed in a C8 reversed-phase column (80 X 4.6 mm). The isocratic mobile phase was ACN-0.01 M aqueous HC1 (90 10, v/v). Analyses were carried out at 20°C and the flow rate was 1-5 ml/min. The concentration of the cationic dye, l,l -didodecyl-3,3,3, 3 -tetramethylindocarbocyanine perchlorate (Dil) in the model solutions varied between 0.9-309 pM. The dependence of the chromatographic profile of the dye on the injected concentration is illustrated in Fig. 3.112. Calculations and mathematical modelling indicated that the peak tailing of the dye can be... 

FIGURE 11.2 Separation of hydroxy-phenylacetic acids by displacement on a CIS reversed phase column (dimensions 4.6 X 250 mm). Carrier 0.1 M phosphate displacer 0.87 M aqueous -butanol. Flow rate 0.05 mL/ min temperature 25°C. (Reprinted with permission from Elsevier from Horvath, C. et al., J. Chromatogr., 218, 365, 1981. Copyright.)... 

The next major advance in LC-MS interfacing was developed by Blakely and Vestal (55, 56). To circumvent the solvent elimination problem, Blakely et al. (55) developed the thermospray interface that operates with aqueous-organic mobile phase at typical 4.6-mm i.d. column flow rates, 1-2 mL/min. The thermospray technique works well with aqueous buffers. This feature is an advantage when the versatility of the reversed-phase mode is considered. In fact, with aqueous buffers, ions are produced when the filament is off. A recent improvement in the thermospray technique is the development of an electrically heated vaporizer that permits precise control of the vaporization (56). This... 

Set the flow rate of the HPLC system to 1 ml/min across a 5-p.m x 250-mm x 4.6-mm C18 polymeric support reversed-phase column or equivalent at ambient temperature. Set the detector at 520 nm. Inject 50 ul sample into the HPLC system and start a gradient similar to that outlined in Table FI. 3.2. Analyze data as described (see Data Anaylsis in Critical Parameters and Troubleshooting). 

C18 reversed-phase column for HPLC separations of chlorophylls narrow-bore (2.1 -mm i.d.) column at flow rate of 50 to 300 p.l/min without splitting the flow, or analytical column (4.6-mm i.d.) at 1 ml/min with post-column solvent splitting of 5 1 (200 p,l/min entering the mass spectrometer) for APCI electrospray interfaces are available for use without solvent splitting over all flow rates from nl/min to 1 ml/min... 

Set up an HPLC system with a 5-pm x 250-mm x 4.6-mm C18 reversed-phase column and guard column. Set flow rate to 1.0 ml/min at constant room temperature (e.g.,... 

Figure 11.6.3 Gradient HPLC separation of isoflavone standards (see Basic Protocol 3). Peaks 1, daidzin 2, glycitin 3, genistin 4, malonyldaidzin 5, malonylglycitin 6, acetyldaidzin 7, acetylglycitin 8, malonylgenistin 9, daidzein 10, glycitein 11, acetylgenistin 12, genistein. Conditions Waters Nova-Pak C18 reversed-phase column (150 x 3.9 mm 4-pm i.d. 60 A pore size) mobile phase 1% acetic acid in water (solvent A) and acetonitrile (solvent B) flow rate 0.60 ml/min UV detector 260 nm column temperature 25°C. The dotted line represents the gradient of solvent B.

Analyses were carried out with an ODS guard column (30 X 4.6-mm ID) and a reverse-phase column of YMC-C8 (250 X 4.6-mm ID, particle size 5 /xm) maintained at 30°C the absorbance was monitored at 230 nm. All analyses were performed isocratically using acetonitrile-water as the solvent system at a flow rate of 1.2 ml/min. The pH of the solvents was maintained at 4.5 by adding 0.1 M HC1 to acetonitrile. 

On a reverse-phase column, separation occurs because each compound has different partition rates between the solvent and the packing material. Left alone, each compound would reach its own equilibrium concentration in the solvent and on the solid support. However, we upset conditions by pumping fresh solvent down the column. The result is that components with the highest affinity for the column packing stick the longest and wash out last. This differential washout or elution of compounds is the basis for the HPLC separation. The separated, or partially separated, discs of each component dissolved in solvent move down the column, slowly moving farther apart, and elute in turn from the column into the detector flow cell. These separated compounds appear in the detector as peaks that rise and fall when the detector signal is sent to a recorder or computer. This peak data can be used either to quantitate, with standard calibration, the amounts of each material present or to control the collection of purified material in a fraction collector. 

Motevalian et al. [62] developed a rapid, simple, and sensitive HPLC assay method for the simultaneous determination of omeprazole and its major metabolites in human plasma using a solid-phase extraction procedure. Eluent (50 /d) was injected on a /rBondapak Ci8 reversed-phase column (4.6 mm x 250 mm, 10 /un). The mobile phase consisted of 0.05 M phosphate buffer (pH 7.5) and acetonitrile (75 25) at a flow-rate of 0.8 ml/min. UV detection was at 302 nm. Mean recovery was greater than 96% and the analytical responses were linear over the omeprazole concentration range of 50-2000 ng/ml. The minimum detection limits were 10, 10, and 15 ng/ml for omeprazole, omeprazole sulfone, and hydroxyomeprazole, respectively. The method was used to determine the plasma concentration of the respective analytes in four healthy volunteers after an oral dose of 40 mg of omeprazole. 

Aqueous samples or aqueous extracts of nonaqueous samples analyzed by HPLC on a C-18 reverse phase column analyte detected by UV at 195 nm mobile phase, water, flow rate 2 mL/min pressure 38 atm. 

HPLC column C-18 reverse phase column, such as Zorbax ODS or equivalent mobile phase acetonitrile/water (70 30) flow rate 1 mL/min. 

FIGURE 4-4. Two approaches to the separation of polynuclear aromatics, (a) Reverse-phase separation of isomeric 4-ring polynuclear aromatics using a gradient of 70/30 (v/v) to 100/0 (v/v) acetonitrile/water as shown beneath the chromatogram. Column C,g detection at 254 nm. (b) Normal-phase separation of aromatic hydrocarbons. Column /uPorasil (silica, 10 /urn) 3.9 mm ID x 30 cm (2 columns) mobile phase hexane flow rate 8 mL/min. (Fig. 4-4b reproduced from reference 1 with permission.)... 

FIGURE 6-4. Column efficiency versus flow rate, (a) Column /xBondapak C 8 (10 Him) 3.9 mm x 30 cm. Mobile phase Acetonitrile water, 60 40. Sample Acenaph-thene, k = 3.5. (b) Comparison of reverse phase columns (10 jam). Conditions are the same as in a. (c) Comparison of normal phase columns silica (10 jam). Mobile phase hexane. Sample nitrobenzene. 

FIGURE 12-2. Example of reverse-phase separation of an APC tablet. Mobile phase MeOH. Flow rate 1 mL/min. Detector UV at 254 nm, 1.0 AUFS. Sample APC tablet in 100 mL MeOH, 10-p.L injection. Column Bondapak Ci8/Porasil B, 2 mm ID x 61 cm. (Note Actual separation will depend upon the quality of the mobile phase and column packing.)... 

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