Gradient separations optimal

Particle class Protein Separation vs Concentration Separation Optimization criterion Purity Assoc/Dissoc in sucrose No Sedimentation coefficient 16.0 10-40% or 5-20% gradient 10-40 Sample form liquid/semi-solid Total sample volume (mL) 3.0 Sample concentration % w/w 1.0 Selected final location 45.0 Solvents No... 

Combining the appropriate equations for the retention volumes of the solutes 1 and 2 with adjacent bands and Equation 5.5, we obtain Equation 5.6 for resolution in gradient LC [4,6,28], which can be used in the optimization of gradient separations ... 

It is clearly beyond the scope of this chapter to consider further the selection of which variables to use in the simplex optimization. To summarize our own relatively limited experience, however (boxes in Table IV represent combinations examined to date), we recommend the following For a relatively simple separation, begin with a two-parameter simplex that includes either initial pressure (or density), using as many characteristics of the analytes and/or sample matrix to logically deduce which remaining variable to optimize. For a more complex separation, or one in which little is known about the sample, try a 4 or 5-variable simplex that includes the initial pressure and pressure gradient (or initial density and density gradient) as optimization variables. 

MUX inlet as possible. A flow rate of 12 ml/min on eight 2.1 x 50 mm Polaris Ci8 columns was optimal for general purposes in our study. This system could analyze more than 3000 compounds per day for a gradient separation with a cycle time of 3.5 min. 

All density gradient separations are carried out in aqueous cesium chloride solutions to which a nonionic surfactant has been added (polyoxyethylene-23-lauryl ether, Brij-35). The surfactant is necessary to disperse the fine coal particles in the aqueous medium. Without complete particle dispersion the separation will most likely not be optimal. 

Fig. 5 Typical chromatograms showing injection volume (V.n.) optimization for plasma samples spiked with 50 pg/mL of TACA and DEX-AC and extracted by selective SPE. The V.n. was (a) 0.1 pL (b) 0.5 pL (c) 2 pL (d) 5 pL. A 15 cm by 300 pm I.D. capillary column was used for separation the manufacturer-recommended V.n. was 0.1-0.2 pL. Selective SPE and two-segment gradient separation enabled a large Vin. on a 300 pm I.D. capillary column without deterioration of chromatographic performance and with greatly increased sensitivity (Reproduced with permission from American Chemical Society)...

Of course, the simultaneous optimization of different (primary) program parameters (initial and final composition, slope and shape of the gradient) and secondary parameters (nature and relative concentration of modifiers) may involve too many parameters, so that an excessive number of experiments will be required to locate the optimum. This problem may be solved by a separate optimization of the program (primary parameters) and the selectivity (secondary parameters) based on the concept of iso-eluotropic mixtures (see section 3.2.2). This will be demonstrated below (section 6.3.2.2). However, the transfer of... 

Gallant, S., Kundu, A., and Cramer, S. M. (1995). Optimization of step gradient separations —Consideration of nonlinear adsorption. Biotechnol. Bioeng. 47, 355-372. 

Cela, R. et al. PREOPT-W off-line optimization of binary gradient separation in HPLC by simulation IV phase 3. Comput. Chem. 1996, 20, 315-330. 

Optimization of the gradient separation could be expressed in the form of a mathematical algorithm. Usually from a couple of gradient runs of the same analyte mixture, this is sufficient to calculate empirical constants for the equation similar to equation (2-97). These algorithms are implemented in most of the optimization software, such as DryLab , ACDLabs , and ChromSword . 

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