HPLC, gradient

For ESI, the HPLC gradient started with acetonitrile-10 mM ammonium acetate (3 17) for the first 3 min and was changed to 9 1 acetonitrile-10 mM ammonium acetate in 31 min (held for 4 min). The HPLC column was a Zorbax RX-C8, 2.1-mmi.d.x 150 mm, 5-p.m particle size, with a flow rate of 0.15 mLmin and a 20-pL injection. 

FID (1) Flame ionisation detector GE-HPLC Gradient-elution high-performance... 

FIGURE 1.2 2D liquid protein expression map of the HCT-116 human colon adenocarcinomacell line. The x-axis is in p/units from 4.0 to 7.0 in 0.2 increments. They-axis is percent B of the RP-HPLC gradient. The gray scale of the bands represents the relative intensity of each band by UV detection at 214 nm. From Yan et al. (2003) with permission of the American Chemical Society. (See color plate.)... 

Preparative reversed-phase HPLC purification was carried out using Gilson liquid handlers and HPLC equipment controlled by Unipoint Version 3.2 software. Initial and final HPLC gradient... 

Hsieh et al. (2003) and Zeng et al. (2003a), used direction injection to quantitate multiple drug discovery compounds simultaneously with good robustness. In one application, the flow rate of the HPLC gradient was programmed. The flow rate was first increased (4 to 8 mL/min) for fast protein clean-up, then decreased (1.2 mL/min) for better sensitivity. The column maintained similar efficiency after several hundred injections. 

FIGURE 21 Two HPLC gradient chromatograms (tryptic maps of lysozyme) illustrating the dramatic effect of flow rate (F), gradient time (t ), and void volume (Vg) on analysis time. Figure reprinted with permission from Reference 22. 

The case stndy of the SP development of an HPLC assay method for an OTC prodnct derived from natural materials illustrates the importance of optimizing both the extraction and filtration conditions to fully recover all the labile active components in the formulation. The HPLC gradient assay method, which separates all active ingredients from other... 

The results are qualitatively evaluated by making overlay plots. An example of an HPLC gradient separation for a typical assay and impurities analysis is shown in Figure 13a. The selectivity is evaluated on five different lots of the selected stationary phase. As can be observed, apart from two minor selectivity issues (indicated by the arrows in Figure 13a), the reproducibility on different lots of stationary phases is acceptable. 

The HPLC gradient system consisted of two pumps for solvent delivery, an injector, a 120 x 4.6 mm Polyspher AA-NA column (Merck, 75°C), a pump for reagenf delivery, a mixing tee with reaction coil, and a fluorescence detector lined to an integrator (Waters). 

Figure 4B. Chromatogram of RP-HPLC gradient elution separation of OPA—Ac—Cys derivatives of standard protein amino acid enantiomers.

HPLC Gradient System with Column Switching for Blue-Fluorescing Pterins... 

Table 6.1.3 Gradient program used to control the HPLC gradient system with column switching for blue-fluorescing pterins ... 

Average recoveries for model compounds from five repetitive 500-L experiments are shown in Tables I and II. Table I lists data for the low-to-medium polarity organics (broadly classified by retention through more than half of the reverse-phase HPLC gradient) of low aqueous solubility, whereas Table II summarizes data for the organic acids and hydrophilic neutrals of higher polarity. Although these are not... 

Table F1.3.1 HPLC Gradient for Anthocyanin Separation on Silica Cis Columns...

Table F1.3.3 HPLC Gradient for Anthocyanidin Separation on C-is Silica Columns...

For phenolics in fruit by-products such as apple seed, peel, cortex, and pomace, an HPLC method was also utilized. Apple waste is considered a potential source of specialty chemicals (58,62), and its quantitative polyphenol profile may be useful in apple cultivars for classification and identification. Chlorogenic acid and coumaroylquinic acids and phloridzin are known to be major phenolics in apple juice (53). However, in contrast to apple polyphenolics, HPLC with a 70% aqueous acetone extract of apple seeds showed that phloridzin alone accounts for ca. 75% of the total apple seed polyphenolics (62). Besides phloridzin, 13 other phenolics were identified by gradient HPLC/PDA on LiChrospher 100 RP-18 from apple seed (62). The HPLC technique was also able to provide polyphenol profiles in the peel and cortex of the apple to be used to characterize apple cultivars by multivariate statistical techniques (63). Phenolic compounds in the epidermis zone, parenchyma zone, core zone, and seeds of French cider apple varieties are also determined by HPLC (56). Three successive solvent extractions (hexane, methanol, aqueous acetone), binary HPLC gradient using (a) aqueous acetic acid, 2.5%, v/v, and (b) acetonitrile fol-... 

For removal of the Fmoc protecting group, the peptide T4-(4a14,4COCH2ONHFmoc) (13 mg, 1.56 pmol) was dissolved in 10% piperidine/DMF (200 pL) and stirred at rt for 30 min. After precipitation with cold Et20, the product was isolated by centrifugation (5 min, 4000 rpm), lyophilized from MeCN/H20 and purified by HPLC (gradient 5—95% MeCN) yield 10.3mg (90%). 

Which type of instrument should be used Is the method for one specific model from a specific vendor or should it be used by all models from all vendors This is especially important for HPLC gradient methods, because different instruments may have different delay volumes, ranging from 0.5 up to 8 ml. This can have a tremendous impact on the separation and elution order of the compounds. 

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