Stepwise gradients

W. Markowski and K. L. Czapiriska, Computer simulation of the separation in one- and two-dimensional tliin-layer chromatography hy isocratic and stepwise gradient development ,/ Liq. Chromatogr. 18 1405-1427 (1995). 

G. Matysik and E. Soczewiriski, Stepwise gradient development in thin-layer cltro-matography. II. Two-dimensional gradients for complex mixtures , ]. Chromatogr. 369 19-25 (1986). 

FIGURE 6.16 Rp values of aromatic amines obtained on silica gel plate 1, 3 — isocratic development with 5 and 50% solutions of methyl ethyl ketone in cyclohexane, respectively, 2 — two-stepwise gradient development with both solvents open squares, N, N-dimethyla-niline, open triangles, iV-ethylaniline, open circles, aniline, diamonds, 2-phenylenediamine, filled squares, 3-phenylenediamine, filled triangles, 4-phenylenediamine, filled circles, 3-aminopyridine. (From Soczewinski, E. and Czapinska, K., J. Chromatogr. 168, 230-233, 1979. With permission.)... 

FIGURE 6.17 Densitograms of Azulan extract scanned at 410 nm divided into fractions a to i (a) isocratic development, ethyl acetate in chloroform (1 5), (b) stepwise gradient development, 10 to 40% v/v of ethyl acetate in chloroform. (From Matysik. G., Soczewinski, E., and Polak, B., Chromatographia, 39, 497-504, 1994. With permission.)... 

Systems with different selectivity were nsed for the separation of 10-deacetyl-baccatin III (10 DAB 111) from yew extracts [69]. A silica column with stepwise gradient elution with aqneous methanolic mobile phases can be nsed for separation of the taxoid fraction from nonpolar materials, partial separation of the taxoid fraction into a polar one (containing 10-DAB 111), and for a medinm polarity taxoid fraction (containing paclitaxel and cephalomannine). Most polar material (tannins... 

Figure 8.43 Separation of enantiomers using complexation chromatography. A, Separation of alkyloxiranes on a 42 m x 0.2S mm I.O. open tubular column coated with 0.06 M Mn(II) bis-3-(pentafluoro-propionyl)-IR-camphorate in OV-ioi at 40 C. B, Separation of D,L-amino acids by reversed-phase liquid chromatography using a mobile phase containing 0.005 M L-histidine methyl ester and 0.0025 M copper sulfate in an ammonium acetate buffer at pH 5.5. A stepwise gradient using increasing amounts of acetonitrile was used for this separation.

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

It is possible to combine stepwise gradient and continuous gradient, i.e., the starting elution buffer has a higher ionic strength than the binding buffer, and after a short time the elution of the protein(s) of interest starts by application of a flat continuous gradient. 

The preparation of cell nuclei from rat liver is an example of the application of a pre-formed stepwise gradient The tissue homogenate is placed on top of a cushion of concentrated sucrose solution, and only the dense nuclei cross this cushion during centrifugation. 

System (5) has been reported for the determination of C21-hydroxy corticosteroids in human urine, and uses prednisone as the internal standard [153]. The column (25 cm x 4.6 mm) was packed with TSK gel ODS-120 T (5 pm). The mobile phase consisted of a stepwise gradient elution at 1 mL/min with methanol-IM ammonium acetate-acetonitrile... 

If ionic interactions are important for complex formation, a change in pH or ionic strength weakens the interaction by altering the extent of ionization of ligand and macromolecule. In practice, either a decrease in pH or a gradual increase in ionic strength (continual or stepwise gradient) is used. 

Horizontal and descending TLC are other forms of chromatography which require special equipment. The advantages of these systems are not clear. They are usually more expensive than ascending equipment, but often have other features for stepwise gradient elution or continuous solvent programming. 

The insecticides were extracted and 0.5 ml of extract equivalent to lOg of sample was placed on the column and eluted with the above stepwise gradient. The results are represented by Figure 6. 

Mori S (1996) Characterization of styrene-acrylonitrile copolymers by size exclusion chromatography/stepwise gradient elution-liquid precipitation chromatography. Int J Polym Anal Charact 2 185-92. 

Nagy et al. [11] reported the use of RPLC-MS on a Cig column using a stepwise gradient of partly miscible solvents, methanol/water and methanol/n-hexane, for the analysis of TAG in blood and/or plant oil. 

Either stepwise gradient or continuous gradient elution is possible. 

No principal difference distinguishes the analytical and preparative uses of focusing FFF. Both types of fractionations can be carried out under conditions of continuous operation [10], which represents the high-performance experimental arrangement for preparative FFF. The fractionation channel, equipped with several outlet capillaries at various positions (and occasionally with several inlets to preform a stepwise gradient in the direction of the focusing), allows one to fractionate the sample which is introduced continuously into the channel and to collect the focused layers eluting by the individual outlets. A schematic representation of such a fractionation channel is shown in Fig. Id. 

The use of a stepwise gradient of the mobile phase is well described by theoretical models [1,3,4]. The migration of zones is shown in Fig. 2a. In the ideal situation, the spots of solutes overtaken by the consecutive fronts of increased modifier concentrations accelerate their migration so that the strongly retained solutes also start to migrate. Depending on the polarities of the solutes, they migrate all the time in the first concentration zones or are overtaken by the consecutive... 

---