Ballistic gradients

Romanyshyn L. Tiller P. Ultra-short columns and ballistic gradients considerations for ultra-fast chromatographic liquid chromatographic-tandem mass spectrometric analysis. Journal of Chromatography A, 2001,928,41-51. 

Primary screens were performed in duplicate on mixtures of 80 compounds (1 pM per compound, 5 pM protein), where samples in each mixture were orthogonally pooled so that no two compounds that are in one well are also together in another well. Primary hits were achieved when the same compound in two wells were observed. The GPC spin-column eluates were partially resolved using a reversed-phase C18 HPLC column (Waters Xterra 2.1 x 20.0 mm) with a 1-min ballistic gradient and total cycle time of 2 min. The HPLC eluates were analyzed with a Micromass eight-way MUX interfaced to an LCT Tof ESI-MS system. Achieved throughput was 100 000 compounds per day. 

Fig. 13.7 Post-column infusion study of a ballistic gradient. The matrix effects can be seen at the early part of the chromatogram, but the later part of the chromatogram where the analytes should elute did not show matrix effects. Adapted from [99], with permission from John Wiley and Sons.

Romanyshyn, L.A. Tiller, P.R. Ultra-short Columns and Ballistic Gradients Considerations for Ultra-fast Chromatographic Liquid Chromatographic-Tandem Mass Spectrometric Analysis, J. Chromatog. A 928,41-51 (2001). 

De Nardi, C. and Bonelli, F., Moving from fast to ballistic gradient in liquid chromatog-raphy/tandem mass spectrometry pharmaceutical bioanalysis Matrix effect and chromatographic evaluations, Rapid Commun. Mass Spectrom., 20(18), 2709, 2006. 

A variety of improvements with regard to HPLC-MS have occurred. The 2x 30 mm (4 p) supplanted the longer wider column and 2-3 min run times were achieved with ballistic gradients [9], Questions regarding matrix ion suppression and issues with coeluting endogenous and exogenous impurities resulted in issues with both discovery (non-GLP) and development (GLP) assays [10-12],... 

The use of short HPLC columns and ultrafast gradients has been explored in order to decrease HPLC-MS/MS analytical run times [27-30], Typical cycle times ranged from 85s to 2min and used 2x30mm, 4pm HPLC columns (Table 8.1). The lmin HPLC method is shown in Table 8.1. Mobile phases A and B are 0.01 M ammonium acetate in water/methanol (80 20) and methanol (100%), respectively. A ballistic gradient from 1% to 70% B was run over 0.01 min and held for 0.09 min. A linear gradient was then employed from 70% to 95% B for 0.6min and held for 0.1 min. The column was then reequilibrated to 1% B over 0.2 min at a constant flow rate of 0.8mL/min. 

FIGURE 8.3 Comparison of (a) the 2min linear gradient and (b) the lmin ballistic gradient. (From Dunn-Meynell, K.W. et al., Rapid Commun. Mass Spectrom., 19, 2905, 2005. With permission.)... 

A rapid separation means much more than an ultra-short (ballistic) gradient program. 

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