Gradient elution shapes

The application of trifluoroacetic acid (TFA) for ion pairing purposes in LC separation led to a retention shift of all constituents contained in the formulation. The AS and AES that eluted first, but were delayed compared with RP-Ci8 gradient elution, could be observed with a tremendous improvement in peak shape (RT = 9.0—12.5 min) in the total ion mass trace after ESI(+) ionisation (Fig. 2.5.11(c)). AE and amine oxides were not observed during the recording time of 30 min. 

In addition, the use of fast gradients elution mode has become the bioanalytical mainstream as a possible way to improve peak parameters (shape and symmetry) and to minimize method development time, especially for the multi-analytes methods. 

FIGURE 10.21 Comparison between experimental and simulated peaks in gradient elution (Langmuir adsorption isotherm). Injected concentration 2.7g/L injected volume 0.163 xL. Continuous line experimental profile dotted line simulation. The shape of the gradient program is also represented. (Reprinted from Marchetti, N. et ah, J. Chromatogr. A, 1079, 162, 2005. With permission from Elsevier.)... 

Since other secondary interactive effects, e.g. electrostatic effects, can also contribute with a particular type of RPC sorbent to the retention of peptides as the mobile-phase composition is changed, the shape of these In k versus ip plots then over the range 0.0 < ip < 1.0 takes on the characteristic U-shaped dependencies.112,211 As a consequence of these dependencies of In k on ip, for mixtures of peptides elution usually requires the use of gradient elution conditions, even when closely related or homologous peptides are being separated. 

A gradient elution should be described by the initial and the final mobile-phase compositions, by the gradient shape, and by the gradient steepness. A step gradient is often convenient when strongly retained solutes are not the solutes of interest. 

Fig. 1.28. Effects of the gradient steepness (gradient time) (three top chromatograms), of the initial concentration of the stronger eluent, methanol or acetonitrile in water (three middle chromatograms) and of the gradient shape (two bottom chromatograms, (A) convex, (B) concave) on the reversed-phase gradient-elution separation of ten fluorescent derivatives of homologous n-alkylamines (methyl- to n-decyl-) on a LiChrosorb RP-18, 10 pm. column (3(X) x 4.0 mm i.d.). Other operation conditions and compounds as in Fig. 1.25.

Gradient elution in RPC, NPC or lEC systems can be optimised using principally the same strategies as in isocratic chromatography, which are briefly described in Section 1.4.7. Simultaneous optimisation of gradient time (steepness), initial concentration and — if necessary — gradient shape can use Eqs. (1.32)-(1.37) for predictive calculations of the retention and of the resolution of the individual pairs of sample compounds from the isocratic data acquired in a few mobile phases of different composition or in a few initial gradient-elution runs. 

---