Step gradients

Step 4) Precolumn clean-up not shown in Figure 5.4. After the heart-cut analytes have been transferred to the analytical column, a step-gradient programme is used to flush the precolumn of the more strongly retained compounds. An additional pump configuration makes precolumn clean-up possible while the analysis is running. 

The methanol gradient from 50 to 60% releases quite a lot of interfering components. Omitting the step gradient does not provide enough selectivity and so the best conditions were obtained with a pH gradient. The experimental conditions are shown in Table 13.1. 

Figure 13.7 Selectivity effected by employing different step gradients in the coupled-column RPLC analysis of a surface water containing 0.40 p-g 1 bentazone, by using direct sample injection (2.00 ml). Clean-up volumes, (a), (c) and (d) 4.65 ml of M-1, and (b) 3.75 ml of M-1 transfer volumes, (a), (c) and (d), 0.50 ml of M-1, and (b), 0.40 ml of M-1. The displayed cliromatograms start after clean-up on the first column. Reprinted from Journal of Chromatography, A 644, E. A. Hogendoom et al, Coupled-column reversed-phase liquid chromatography-UV analyser for the determination of polar pesticides in water , pp. 307-314, copyright 1993, with permission from Elsevier Science.

With dicamba, a more polar chlorobenzoic acid herbicide, a gradient step is needed to elute all of the compounds in one chromatographic run. Depending on the buffer and selectivity of the detector, the baseline can be severely disturbed. If this happens, a step-gradient elution is recommended (52), and in this way the method can detect all of the compounds at very low levels. 

The submitters mixed active anhydrous silica gel with water (12% v>/w) and stored it in a sealed container for at least 24 hours prior to use. A ratio of 60-80 g. of silica gel per gram of crude product was used for column chromatographic separations, and a column was chosen that would give a 10 1 height diameter ratio of adsorbent. Columns were wet-packed with distilled petroleum ether (b.p. 60-68c), and after the crude product had been applied a step-gradient was run rapidly through 2% vjv ether in petroleum ether, 5% ether, and 10% ether. The column was then eluted with 20% vjv ether in petroleum ether until the bromohydrin acetate was obtained. 

Figure 6, High pressure liquid chromatogram of creatine kinase isoenzymes. First peak, MM second peak, BB. Conditions 50 cm X 4.8 mm (i.d.) column with yydac porous layer bead anion exchange mobile phase, step gradient Solvent A, 10 mmol/liter Tris buffer, pH 8.3 solvent B, 10 mmol/liter Tris buffer, pH 7.0,0.5 mol KCl flow rate, 2 ml/min detection, collected fractions assayed (45).

FIGURE 13.7 Total ion chromatogram reproducibility for three 2DLC (S AX-Step Gradient/ RP)/MS analyses of an E. coli cytosolic fraction. 

Millea, K.M., Kass, I.J., Cohen, S.A., Krull, I.S., Gebler, J.C., Berger, S.J. (2005). Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension. J. Chromatogr. A 1079, 287-298. 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

An ESRI system can be built with small modifications of commercial spectrometers by, for example, gradient coils fixed on the poles of the spectrometer magnet, regulated direct current (DC) power supplies, and required computer connections [40,53,55]. Gradients can be applied in the three spatial dimensions, and a spectral dimension can be added by the method of stepped gradients. The spectral dimension is important when the spatial variation of ESR line shapes (as a function of sample depth) is of interest this situation will be described below, in the ESRI studies of heterophasic polymers. In most systems, the software for image reconstruction in ESRI experiments must be developed in-house. 

Basic procedure LDL is isolated from fresh EDTA(Na2) plasma by sequential ultracentrifugation in KBr step gradient at 120,000 rpm (microultracentrifuge HITACHI CS 120) and a temperature of 10°C for 2 X 2 h. The LDL was covered with nitrogen in a screw-cap tube and stored in the refrigerator at 4°C until further use. 

Their method included a Waters 2795 Alliance HT (high throughput) HPLC system with an integrated autosampler. The stationary phase was a Supelco C18 column (250 x 4.6 mm, 5 fim). The mobile phase consisted of solvent A (water containing 2mM ammonium acetate and 0.1% formic acid) and solvent B (methanol containing 2mM ammonium acetate and 0.1% formic acid). The mobile phase was delivered at a flow rate of 0.6 mL/min in a step gradient mode 50% solvent B from 0 to 0.4 min and 100% solvent B from 0.4 to 0.8 min. 

Epirubicin — Anthracyclines have been used in cancer chemotherapy for more than 30 years and epirubicin (EPI) is one of the most widely used agents.55 Li et al. developed a high-throughput method for the analysis of epirubicin in human plasma using UPLC-MS/MS.56 A Waters Acquity UPLC system was coupled with a Micromass Quattro Premier MS. The stationary phase was a Waters Acquity BEH C18 column (50 x 2.1 mm, 1.7 jum). The column was maintained at 30°C. Solvent A was 0.1% formic acid in water and solvent B was acetonitrile. The mobile phase was delivered at a flow of 0.20 mL/min in a step gradient mode at 85% solvent A at 0 min, 70% solvent A at 1.00 min, and 85% solvent A from 2.50 to 4.00 min. Epidaunorubicin (EPR) was the IS. 

THE TWO- AND THREE-STEP GRADIENT PROGRAMMES USED FOR THE ANALYSIS OF EXTRACTS FROM VACCINIUMMYRTILLUS L. AND VACCINIUM V1TIS-IDAEA L. MIXTURE OF TOLUENE, HEXANE, ETHYL ACETATE, AND METHANOL WERE USED AS MOBILE PHASES... 

Three-step gradient elution for quercetin (Stepwise gradient elution hR = 52) ... 

RP-HPLC-vis measurements were performed in an ODS column (250 X 2 mm i.d. particle size 5 jum). A 10 X 2 mm column filled with lead(IV) oxide was employed for the post-column oxidation of the analytes. The method of post-column oxidation allowed the simultaneous detection of each dye and metabolite in the visible range. Dyes were eluted with a two-phase step gradient programme mobile phase 1 (0.05 M aqueous ammonium... 

---