Continuous isocratic development

The design procedure described above will, in theory, be applicable only to samples that are separated by isocratic development. Under gradient elution conditions the (k ) value of each solute is continually changing, together with the viscosity of the... 

In isocratic development, a solvent of constant composition is used to effect separation for example, 40% ethyl acetate m hexane. This may be extended to continuous development, in which the TLC plate is left in an isocratic system after the solvent front has reached the top of the TLC plate. This has the advantage that closely eluting bands may be resolved through the use of a nonpolar solvent over several hours. A major disadvantage of this technique is that unstable compounds may degenerate on the adsorbent during this lengthy time penod. 

Figure 1 shows a schematic drawing of a CAC apparatus. The apparatus consists of two concentric cylinders standing one inside the other, forming an annulus into which the stationary phase is packed. This annular bed is slowly rotating about its vertical axis. Under isocratic elution conditions the feed mixture to be separated is introduced continuously at the top of the bed at a space that remains fixed in space while the rest of the annulus is flooded with elution buffer. As time progresses, helical component bands develop from the feed point, with... 

The first step for further studies was to develop a better isolation procedure. Since one of the solvents used in the original solvent separation scheme was ethyl acetate, and the ethyl acetate did concentrate a significant amount of the active principals, a continuous liquid-liquid extraction of the ethanolic extract between ethyl acetate and water was employed as the initial step. After 24 h, all of the constituents of interest were extracted into the ethyl acetate layer. This layer was concentrated and subjected to HPLC on a partisil 10 column using an isocratic elution with 8% methanol in dichloromethane. Under these conditions, vanicoside A (1) eluted at approximately 14 min, vanicoside B (2) at approximately 20 min, and hydropiperoside (3) at approximately 40 min. In addition, there were numerous other peaks which contain phenolic moieties, as determined by stop-flow examination of the ultraviolet (UV) spectra of individual peaks. 

Since APCI and ESI interfaces operate at atmospheric pressure and do not depend upon vacuum pumps to remove solvent vapor, they are compatible with a wide range of HPLC flow rates. HPLC methods that have been developed using conventional detectors such as UV/VIS, IR, or fluorescence are usually transferable to LC/MS systems without adjustment. However, the solvent system should contain only volatile solvents, buffers, or ion-pair agents to reduce fouling of the mass spectrometer ion source. In the case of chlorophyll solvent systems, isocratic and gradient combinations of methanol, acetonitrile, water, acetone, and/or ethyl acetate have been used for APCI or ESI LC/MS. Unlike continuous-flow FAB/LSIMS, no sample matrix is necessary. 

If neither binary gradient nor three-solvent isocratics are successful, some systems will next try to perform a three-solvent gradient optimization. This development is very difficult to visualize. Assuming simultaneous optimization of %B, %C, and flow rate hinge points, it takes a long, computation-intensive time to carry out. It would be nearly impossible to carry out manually. The key is continually to use the rule of one change only one variable at a time and to carefully select limits for evaluation. 

Peptide mapping represents one of the fundamental tasks in protein analysis. The fact that most of the applications of CEC are restricted to isocratic elution has considerably limited the practical applicability of this technique in the past, particularly in the analysis of biopolymers. Two approaches have been developed to generate continuous gradients, namely, the merging of two endo-osmotic flows [78] or the application of two separate HPLC pumps. The latter presents practically no complication with regard to instrumentation as the same equipment can be used for HPLC,... 

The type of separation desired dictates the choice ofthe mobile phase. Isocratic separations can be carried out with a single solvent or a fixed proportion mlxtmre of two solvents. For gradient elution, however, the development solvent composition changes continuously. This is achieved by a gradient programmer usualty attached to the HPLC assembty. 

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