HPLC-DAD post column DPPH assay
Posted: Sat Jul 20, 2013 6:39 pm
Dear colleagues,
recently I have been trying to set up an HPLC post column DPPH assay. Though i have made the required set up and came up with DPPH concentration (5 *10-5 M) in a methanol-buffer mixture (40 mM citrate, pH 6.0) 75:25 v/v there is the following predicament.
When injecting the sample (1:1 water/methanol) I observe after some minutes (rather after the dead volume of the column) a high positive peak which then becomes negative and end with a positive (N). My mobile phase is acetonitrile:water (0.1% acetic acid) and i use a gradient as recommended starting from 10% acetonitrile (0.5 ml/min flow rate). The size of the peaks that i describe are depended on the injected volume and when i use as sample solvent 1:1 acetonitrile/water in order to be closer to the mobile phase, then I get only the first positive and the negative peak. The spectrum of the positive peaks is that of the radical. I would not mind for these peaks but 2 of the peaks from a test extract elute at the same time so that i cannot have them clearly reacting with the radical. The compounds are polar but introduction of an isocratic run of ~ 2 min does not change their retention time, whereas starting the gradient from 5% acetonitrile causes a significant increase and then decrease in the base line within a ~5 min duration creating a new problem. I thought of using methanol which may cause a shift of the retention time in comparison to acetonitrile but still one of the peaks elute when the detector gives the negative signal. I use a 14 m coil of blue teflon 0.25 mm i.d. and the radical solution is introdued via a T piece with an HPLC pump (0.15 ml/min flow). The radical solution is transferred via a vacuum degasser line. Any recommendations?
recently I have been trying to set up an HPLC post column DPPH assay. Though i have made the required set up and came up with DPPH concentration (5 *10-5 M) in a methanol-buffer mixture (40 mM citrate, pH 6.0) 75:25 v/v there is the following predicament.
When injecting the sample (1:1 water/methanol) I observe after some minutes (rather after the dead volume of the column) a high positive peak which then becomes negative and end with a positive (N). My mobile phase is acetonitrile:water (0.1% acetic acid) and i use a gradient as recommended starting from 10% acetonitrile (0.5 ml/min flow rate). The size of the peaks that i describe are depended on the injected volume and when i use as sample solvent 1:1 acetonitrile/water in order to be closer to the mobile phase, then I get only the first positive and the negative peak. The spectrum of the positive peaks is that of the radical. I would not mind for these peaks but 2 of the peaks from a test extract elute at the same time so that i cannot have them clearly reacting with the radical. The compounds are polar but introduction of an isocratic run of ~ 2 min does not change their retention time, whereas starting the gradient from 5% acetonitrile causes a significant increase and then decrease in the base line within a ~5 min duration creating a new problem. I thought of using methanol which may cause a shift of the retention time in comparison to acetonitrile but still one of the peaks elute when the detector gives the negative signal. I use a 14 m coil of blue teflon 0.25 mm i.d. and the radical solution is introdued via a T piece with an HPLC pump (0.15 ml/min flow). The radical solution is transferred via a vacuum degasser line. Any recommendations?