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Gradient Slope/Time

The larger the analyte molecular weight, the more it will be affected by small changes in the gradient slope, or the % change of mobile phase per minute. Small molecules are only slightly affected, mid-sized molecules, such as peptides, are affected to a greater degree, and large peptides/proteins are dramatically affected.

The degree by which analytes are affected is governed by their s value – defined as the gradient when the log of solute retention time is plotted against the change in mobile phase composition, Φ.

In Figure 1, the s values for three different analytes are plotted, a small molecule, benzene (MW 78), a mid-sized molecule, leucine enkephalin (MW 555.6), and a large molecule, lysozyme (MW 14,300). It is for these reasons that a full gradient, 0 - 100% MeCN, is never run for peptides and only very shallow gradients are required for protein analysis.

‘s’ value plots for small, medium and large molecules

Figure 1: ‘s’ value plots for small, medium and large molecules.


Peak capacity, the number of peaks that can be theoretically separated during a chromatographic run, is inversely proportional to the gradient slope/duration. Therefore, the shallower the slope, or longer the gradient duration, the greater the resolving power
(Figure 2).

However, this is not a linear relationship as demonstrated by peak capacity vs. gradient time in Figure 3. After 100 minutes, the benefit, in terms of peak capacity, becomes much less as the gradient time continues to increase, and is negligible after 150 minutes. This, like a large number of parameters in analytical chemistry, is a balancing act.

It may appear that setting a default gradient time of 150 minutes would be the ideal scenario, however, analytical productivity is markedly reduced at this time, and so it is recommended that the gradient time and/or slope is empirically optimized for each separation.

More information pertaining to gradient separations can be found in the Gradient HPLC module in the HPLC Theory channel.

Peak Capacity vs. Gradient Time

Figure 2: Peak capacity vs. gradient time – chromatograms. Bovine serum albumin digest. Flow rate 0.4 mL/min. Gradient 0-50%B in 6.25 min (8%/min), 12.5 min (4%/min), 25 min (2%/min), and 50 min (1%/min). C18 150 x 2.1 mm, 1.7 µm, mobile phase A: 0.10% TFA in water/acetonitrile 98/2 v/v, mobile phase B: 0.08% TFA in acetonitrile, temperature 60 °C, DAD 214 nm.

 
Peak Capacity vs. Gradient Time – plot

Figure 3: Plot peak capacity vs. gradient time (left) and production rate vs. gradient time (right).


It should also be noted, that differences in selectivity may be encountered when varying gradient slopes or times. This is exhibited as peak inversions or co-elutions and great care should be exercised.

In the trastuzumab peptide map it can be seen that whilst reducing the slope/extending the gradient time is of benefit in certain instances, as for T32, it can be detrimental in others, as with T5 (Figure 4).

Trastuzumab peptide maps at differing gradient slope/times

Figure 4: Trastuzumab peptide maps at different gradient slope/times

 
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