Flow Rate Gradients

Some kinds of chromatography require relatively little optimization. In gel permeation chromatography, for example, once the pore size of the support and number of columns is selected, it is only rarely necessary to examine in depth factors such as solvent composition, temperature, and flow rate. Optimization of affinity chromatography is similarly straightforward. In RPLC or IEC, however, retention is a complex and sensitive function of mobile phase composition column type, efficiency, and length flow rate gradient rate and temperature. 

Several methods are applied to reduce the separation time. The best way is the selection of a suitable column and an eluent using isocratic elution. However, much skill and knowledge are required to make such a system. A flow rate gradient, step-wise elution, or eluent composition gradients are commonly applied to reduce separation times. 

A. Key Gradient Parameters (Flow Rate, Gradient Time [t ], Peak Capacity [P])... 

Gradient parameters (flow rate, gradient time, peak capacity, and dwell volume)... 

Flow Rate Gradient Program Detection Reagents ... 

Very detailed separations have been obtained by numerous authors (61-66) based upon the method originally developed by Christie (67). This method is based mainly on iso-octane (similar to hexane), 2-P, water containing 500 /jlM serine adjusted to pH 7.5 with ethylamine, and trace amounts of tetrahydrofuran (THF) as a mobile-phase modifier. Lutzke and Braughler modified slightly the mobile-phase system proposed by Christie by including a flow rate gradient to maintain low column backpressure (62). According to the authors, this positively affected detector response to PLs. Markello et al. used the procedure described by Christie, albeit without the addition of serine or ethylamine (65). Melton proposed the use of two solvent mixtures only, but they included exactly the same solvents as proposed by Christie (66). However, PI and PA were not resolved. 

Robustness Systematically change chromatographic conditions (examples column temperature, flow rate, gradient composition, pH of mobile phase, detector wavelength). Check influence of parameters on separation and/or peak areas. 

Optimize Chromatography (column, solvents, flow rate, gradient) using a solution of the analyte in mobile phase... 

Column uBondapak NH (300x4 mm ID), mobile phase acetonitrile - water (92 8), flow rate gradient from 2 ml/min to 6 ml/min in 18 min, detection UV 200 nm. Peaks 1, allantoin 2, symphytine N-oxide 3, echimidine N-oxide 4, acetyl-lycopsamine N-oxide 5, lycopsamine N-oxide. (Reproduced with permission from ref. 23, by courtesy of Hippokrates Verlag)... 

Combinatorial compounds are highly diverse, although the choice of solid phase is usually limited. The separation of different kinds of the compounds can nevertheless be accomplished by choosing the right mobile phase. The solvent type, flow rate, gradient slope, and chemical modifiers can influence the separation efficiency, product recovery, product purity, purification speed, and the purification cost. 

Change of the flow direotion -Flow-rate gradient -Time-based flow injeotion... 

Retention in LC depends on retention mechanism and conditions used such as stationary phase type, mobile-phase composition including its pH, flow rate, gradient applied if any, etc. 

Tested different stationary phases, mobile phases, compositions, and flow rates gradient elution... 

Column dimension, particle size, flow rate, gradient system, injection solvent, and carryover are important factors to be considered for successful development of a bioanalytical assay ... 

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