Gradient generic

Speir, J. P. Perkins, G. Berg, C. Pullen, F. 2000. Fast, generic gradient high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry for the accurate mass analysis of mixtures. Rapid Commun. Mass Spectrom, 14,1937-1942. 

A generic gradient recommended for preparative separations is to start the elution at a concentration of B that corresponds to a value 10% earlier that the anticipated desorption then apply a change in concentration of B corresponding to 5% over 15 minutes. To exemplify this approach Table 5.2 shows a gradient, applied to an analytical column, used to purify a peptide that is anticipated to desorb at 20% B under overload conditions. 

The use of small particles also introduces another limitation in the use of microbore LC. The laige back-pressures that are produced can make operating conditions difficult. The use of fast-flow generic gradients [21, to improve resolution and reduce analysis time, has also emphasised this problem. 

Fig. 5.4. Example of fast generic gradient conditions for open-access LC/UV/MS. Column Supcico 3 uf ABZ+. 3.3 cm x 4.6 mm i.d. flow I ml/min split 50 I to deliver 20 pl/min to the electrospray source.

Fig. 8.14. Chromatograms from the retention probe pair for calibrated method B run on (a) the LC/MS system using the standard generic gradient, and (b) the autoprep system using calibrated method B.

The fifth major improvement was to use a fast generic gradient method for the analysis of the samples, which usually takes place in an overnight run of. sample vials from 24 compounds. The fast generic HPLC methods have been discussed in Chapter 2. The method applied here was a 3.5 min gradient from 0 to 95% acetonitrile using a Supelcosil-ABZ 30 x 4.6 mm column with a 1.5 ml/min flow rate. Together with the re-equilibration time the cycle time for each sample was 7.5 min. 

In conclusion, the application of a separation technique such as HPLC to the measurement of concentration in the determination of octanol-water partition coefficients greatly enhances the traditional methods. It is possible to automate the measurements and use much smaller amounts of compound without the need for very high purity. With the application of a fast generic gradient method, there is no need for method development and the analysis time can be reduced to 5-7 min per sample. 

Leroy, F., Presle, B., Verillon, F. and Verette, E. Fast generic-gradient reversed-phase high-performance liquid chromatography using short narrow-bore columns packed with small nonporous silica particles for the analysis of combinatorial libraries. ]. Chromatogr. Sci. 39 487-490, 2001. 

These two main fields of application for chromatography result of course in a dissimilar approach to selecting the chromatographic system. If time pressure minimizes the possible number of experiments the use of generic gradient systems (Section 4.3.1.3) is recommended. In such cases solubility problems are also important,... 

A fast solution for these separation problems is the use of generic gradients. Table 4.5 lists typical solvents and conditions for gradient runs for RP and NP systems. 

Generic Gradients for Fast Separations 130 4.3.2 Criteria for Choice of NP Systems 130... 

Analysis of the Saturated Solution and Residual Solid High performance liquid chromatography (HPLC) is the most commonly used analytical tool for the analysis of saturated solutions. Its advantage over the ultraviolet method is that it can detect impurities and any instability. A generic gradient method can be made readily available that is stability-indicating enough for multiple compound analyses without the need to make major adjustments in the column or mobile phase. 

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