The Influence of Stop-Flow on Band Broadening of Peptides in Micro-Liquid Chromatography
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Stop-flow techniques are occasionally needed in combinations of LC-NMR and LC-MS. During the interval when there is no flow on the column, axial diffusion of components not yet eluted can be expected to take place. In this paper the size of the band broadening which is caused by diffusion during stop-flow has been determined for two peptides on reversed-phase packed micro columns. Within a temperature range of 20–40 °C, stop-flow could be extended to 30 minutes for peptides having k values in the range of 0.7–5.1 with little increase in band width on 1.0 mm i.d. columns at isocratic conditions. Stop-flow for 6 h at 20 °C increased the peak width of bradykinin and leucine-enkephalin by 25% and 60%, respectively, depending on the secondary interactions of the peptides. The peak broadening increased with increasing temperature (from 20 to 40 °C), as expected, and the impact was significant at stop-flow times larger than 2 h. Stop-flow during gradient elution resulted in less increase in peak width than isocratic elution due to the peak compression obtained when re-entering the gradient. At 20 °C the effective diffusion coefficients of leucine-enkephalin and bradykinin were determined to 6.5 × 10−7cm 2/s and 5.5 × 10−7 cm2/s, respectively, on the packed micro column.
KeywordsColumn liquid chromatography LC-NMR Band broadening Stop-flow on micro column Bradykinin and leucine-enkephalin
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