Gradient time

The initial configuration proposed by Valko et al. for log P gradient determination was based on a gradient cycle time of about 15 min with a 150-mm column [40]. This procedure was modified by Mutton, who stated that resolution could be maintained when the gradient time and/or column length were reduced or the flow rate increased [59, 60]. 

In 2001, Valko et al. reduced the column length to only 50 mm and increased the flow rate to 2mLmin [42]. The gradient time was diminished to 2.5 min with a gradient cycle time of 5 min. Measurement of CHI and evaluation of log P were excellent with a 3-fold improved productivity. In these conditions, the system dwell volume (Vd) becomes essential and only dedicated chromatographic devices with Vjy lower than 0.8 mL can be used [42]. Special attention should be paid to the injected volume, which must remain lower than 3 pL to avoid any overloading or extra-column volume contributions. 

In this study, chromatographic experiments were 10 times faster with the monolithic column and results were equivalent to those obtained with the silica-based columns. This approach could be further optimized with faster gradient since flow rate should be increased by a factor 3 or 7 compared to conventional Cig supports [61, 62] and gradient time reduced by the same factor [63] to fully exploit the potential of monolithic supports. 

Fig. 5.3.6 j oint spatial-velocity images of xenon undergoing Poiseuille flow in a pipe (id = 4 mm, Vave = 27 mm s 1, D = 8 mm2 s 1) at 0.7 atm recorded with a protocol shown in Figure 5.3.4(A). Only particles at walls are selected by the edge enhancement filter . A modified imaging gradient time duration... 

Dolan, J.W., Snyder, L.R., Djordjevic, N.M., Hill, D.W., Waeghe, T.J. (1999). Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations. J. Chromatogr. A 857, 1-20. 

Where a, b, and c = van Deemter coefficients, dp = particle size of column, L = column length, Dm = diffusion coefficients of analytes, t = column dead time (depends on flow rate F), tg= gradient time (determines analysis time via tA = tg + t0), Ac = difference in concentrations of the organic modifier at the end and the beginning of the gradient (a continuous linear gradient is assumed), and B = slope of the linear relationship between the logarithm of the retention factor and the solvent composition. 

FIGURE 3.4 Calculated peak capacities dependent on flow rate and gradient time. Left conventional column using 5-flm particles. Middle same column dimension with sub-2-micron particles (1.8 flm). Right sub-2 -micron particles in column with same L/dp ratio as conventional column on left. Parameters for typical applications have been estimated. Note logarithmic scale of flow rate and time axis. 

By replacing conventional 3.5 or 5 jtm columns with sub-2-micron columns, gradient time can be reduced dramatically. The flow rate must be increased for optimal conditions as well but solvent consumption will be less than the amount used by the original method. To use the full power of these columns, an LC instrument must be thoroughly optimized toward lowest extra-column dispersion. The smaller the column (small ID and short length), the more sensitive the performance is to dispersion. With smaller internal diameter columns, the injection volumes and internal diameters of the capillaries should be reduced. 

Modem technologies provide many techniques for expanding the throughput of an analytical laboratory. The task that needs to be accomplished and the possible drawbacks should be carefully considered. Optimized LC equipment can utilize columns packed with much smaller stationary phase particles to achieve significant reductions in gradient time while still achieving the same or even better peak capacities than conventional methods. 

Fig. 20. Test of stability of weak cation exchange monolithic column (ISCO). Conditions column, 50 X4.6 mm i.d., mobile phase gradient of sodium chloride in 0.01 mol/1 sodium phosphate buffer (pH 7.6) from 0.1 to 0.5 mol/1 in 4.5 min and to 1 mol/1 in 6.5 min, overall gradient time 11 min, flow rate 10 ml/min. Peaks Ribonuclease (1), cytochrome c (2), lysozyme (3). The two separations shown in this figure were achieved 503 runs apart...

Fig. 21. Separation of cytochrome (peak 1), ribonuclease, (peak 2), carbonic anhydrase (peak 3), lysozyme (peak 4), and chymotrypsinogen (peak 5) by hydrophobic interaction chromatography on a molded poly(acrylamide-co-butylmethacrylate-co-N,AT,-methylenebisacry-lamide) monolithic column. (Reprinted with permission from [ 135]. Copyright 1998 Elsevier). Conditions column, 50 x8 mm i.d., 10% butyl methacrylate,mobile phase gradient from 1.5 to 0.1 mol/1 ammonium sulfate in 0.01 mol/l sodium phosphate buffer (pH 7) in 3 min, gradient time 3.3 min, flow rate 3 ml/min...

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