Segmented gradient

For complex samples, segmented gradients with varying steepness may provide the best resolution across the separation. However, multisegment gradients often have problems with precision and transferability. 

Figure 25-13 Gradient elution of the same mixture of aromatic compounds in Figure 25-12 with the same column, flow rate, and solvents. The upper trace is the segmented gradient profile, so named because it is divided into several different segments.

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)...

The loaded pre-columns were connected between the injector and the analytical column. Linear two segment gradients were used 0% to 50% (v/v) methanol/water in 20 minutes, followed by 50% to 100% (v/v) methanol/water in 50 minutes. 

Non-linear gradients with curved profile (concave or convex, rarely used, mostly substituted by segmented gradients)... 

Segmented gradients, including several subsequent steps, usually linear with different slopes... 

The simulation and optimization modules are used for the simulation of various experiments and for the optimization of isocratic and gradient HPLC methods. Parameters that can be optimized are concentration of organic solvent in the mobile phase, linear and multi-segment gradient profile, pH value, temperature, type of reversed-phase column, and organic solvent. 

Fast optimizing of the multi-segment gradient profile in reversed-phase high performance liquid chromatography using the Monte Carlo method, LC-GC Int. Mag. 8, N 10 (1995), 581-587. 

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