Mobile chromatographic test optimization

Another important issue in the optimization of a chromatographic test is the selection of the most appropriate chromatographic conditions to analyze the test compounds. More specifically, depending on the mobile phase composition, different kinds of information on the chromatographic performance of stationary phases can be gleaned. In a first attempt, columns were tested with two mobile phases composed of pH 7.0 phosphate buffer (one with acetonitrile and the other with methanol) and one composed of a pH 3.0 phosphate buffer. 

Mobile phase optimization using HPLC can easily be carried out using fully automated instruments, but this approach is extremely time-consuming. Conversely, modern thin-layer chromatographic techniques allow several mobile phases to be tested in parallel within a very short period of time. Furthermore, different methods are available to visualize the sample components, and the thin-layer chromatogram immediately provides information about the presence of products in the sample that remain at the point of application. 

There are also other trial and error approaches, the simplest of which is the so-called spot test. The sample is applied as several spots on a TLC plate. Then specified volumes of different solvents are applied to the centers of the spotted samples. The resulting circular chromatograms can give preliminary information about solvent strength and selectivity required for separation of the sample. With modern instruments for sample application this test can be automated. However, actual optimization of the mobile phase must still be performed in a suitable chromatographic chamber. 

Using Acquity UPLC HSS T3 column (50 x 2.1 mm i.d., 1.8 pm particle size) reversed-phase chromatography, water, 10 mM ammonium formate, 0.1% ammonium formate, 10 mM ammonium acetate, and 10 mM formic acid were tested for the aqneons bnffer component of the mobile phase, while methanol and acetonitrile were examined as the organic constituent Best peak shape, resolntion, and signal-to-noise ratio was achieved using a shallow gradient of 10 mM ammoninm formate and acetonitrile with an initial ratio of 95 5. Optimal chromatographic separations occurred at a flow rate of 0.3 mL/min, and a column temperature of dO C. The mass spectrometer was operated via positive ion mode ESI, and the vitamins were... 

A chemometric approach where the /ty-values of forty-seven flavonoids in seven TLC systems were studied using principal component and cluster analyses, has made it possible to choose the minimum number of chromatographic systems needed to perform the best separation (20). Another method (the PRISMA model) based on Snyder s solvent selectivity triangle has been described to aid mobile phase optimization (21). This model is reported to give good separation of flavonol glycosides from Betula spp. (1). When tested in our laboratory no improvements were obtained in comparison with established systems (22) such as the solvent ethyl acetate-formic acid-acetic acid-water (100 11 11 27) on silica support, which can be used for separation of a wide range of flavonoids. 

Being an enantiomer does not provide specific qualities other than optical to conventional organic compounds. Therefore, their analysis does not impose any particular requirements with respect to equipment. Any HPLC apparatus, provided with the appropriate chiral column, can be used for enantiomer determination. Only the availability of some accessories, such as multiple port valves to permit the programmed test of several columns and mobile phases, can be of help to speed up the search and optimization of analytical chromatographic conditions. 

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