Mobile phase screening

A concentrated hexane extract (11.5 g/4 ml) of B. carterii resin and B. serrata resin, respectively, was applied to a column hlled with 80-g silica gel (Merck LiChroprep Si 60 No. 9390) conditioned by hexane. The fractionation was achieved by a gradient of 200-ml hexane followed by 200-ml hexane-dichloromethane (1 + 1 v/v), 200-ml dichloromethane and 200-ml dichloromethane-acetone (1+1 v/v) as eluents to give four subfractions of 200 ml each. These fractions were collected, concentrated, and applied to a TLC plate for a screening with the mobile phase dichlormethane-diisopropylether (9+1 v/v). 

In the procedure with mobile phase 1 and 2, the visualization is accomplished by screening under uv light (254 and 366 nm) and in addition by spraying with Dragendorff s reagent, modified by Munier and Deboeuf, followed by 30 percent hydrogen peroxide. 

The series of regioisomeric amines 48-50, methamphetamine (29) and phentermine (31), can be identified in forensic screening analyses by RP-HPLC-UVD (254/280 nm dual accessory) using a Cis stationary phase and a mobile phase buffered at pH 3.0. The capacity factors and retention times increase in the order 48 < 49 < 29 < 31 < 50. Other methods for identifying these compounds failed for example, the base peak in MS is m/z = 58 for all five compounds, corresponding to a loss of a benzyl group from the molecular peak also their IR and UVV spectra are too similar to be useful for this... 

Initial screening conditions are suggested in Table 6.1. Multiple pH values are included because mobile-phase pH can significantly affect retention. Major selectivity shifts such as transpositions in elution order are fairly common changes in resolution are much more so.2,14-16 Changes in retention due to pH variation relate to protein hydration. Proteins are minimally charged at their isoelectric points (pis). This means that they carry the minimum of electrostricted hydration water. Both protein surface hydrophobicity and HIC retention should therefore reach their maximum at a protein s pi.6 As pH is either increased or... 

Cyclic amines (including local anesthetic drugs) and amides were among the first classes of chiral compounds investigated in the early stages of the application of macrocyclic antibiotics as chiral selectors therefore, they were screened on vancomycin [7], teicoplanin [30], and ristocetin A [33] CSPs, under RPmode systems. Cyclic imides (including barbiturates, piperidine-2,6-diones, and mephenytoin) have been separated on a vancomycin CSP [157], under NP and RP mobile phase conditions. 

A screening approach was described in subcritical fluid chromatography for basic compounds by Stringham [52]. The analyzing conditions of the screening step are the following a flowrate of 2 mL/min, 180 bars of backpressure, and room temperature. The column for screening was Chiralpak AD-H, i.e. 5-p.m material was used. The mobile phase contained CO2, 20% of ethanol, and 0.1 % ethanesulfonic acid (ESA)... 

Geiser et al. [50,51] illustrated the screening of different chiral stationary phases and the separation of highly polar amine hydrochlorides using EEL methanol/C02 mixtures and the columns, Chiralpak-AD-H, Chiralpak-AS. This method is advantageous because no acid or base additive was required to achieve base line separation of the racemates and conversion to free base form for enantiomer separation was not required. Preparative-scale separations of the amine-hydrochloride were accomplished using similar mobile phase conditions [51], Furthermore, this is believed to be the first chiral separation of highly polar solutes without the addition of acid or base additive to effect the separation. 

In summary, the use of RPLC is ideal for pharmaceutical analyses because of the broad range of commercially available stationary phases because the most common RPLC mobile phases (buffers with acetonitrile or methanol) have low UV cut-off wavelengths, which facilitate high sensitivity detection for quantitation of low-level impurities and because selectivity can readily be controlled via mobile phase optimization. Additionally, the samples generated for selectivity screening (as detailed above) are typically aqueous based. In subsequent phases of pharmaceutical development, aqueous-based sample solvents are ideal for sample preparation and are, under limited constraints, compatible with MS detection required to identify impurities and degradation products. 

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