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Table 4 Common problems and troubleshooting guide.

From: Protocol: analytical methods for visualizing the indolic precursor network leading to auxin biosynthesis

Problem Possible reasons Solutions
Liquid does not pass through TopTips before loading plant samples The slit on TopTips is too narrow Increase the centrifugal force to make liquid pass through, or switch to a new TopTip
Liquid does not pass through TopTips after loading plant samples Plant debris blocks the TopTip slit Always try to avoid transferring plant debris into TopTips
Increase the centrifugal force to make liquid pass through
Use a dissecting probe to remove visible plant debris
Drift of LC retention time Injection volume is too large Evaporate eluate or extract to a smaller volume with vacuum concentrator
Reduce the sample volume injected onto the LC
Change of LC mobile phase Check that correct, freshly prepared mobile phase solvents are being used
Purge the LC lines after changing solvents
LC solvents are blocked or leaking Replace the precolumn filter
Check column connections and reinstall if needed
Check LC lines for leaking solvent
Low yield of labeled compounds, but normal yield of endogenous compounds and/or internal standards Slow turnover Increase the labeling time period
Insufficient intake of labeled precursors Increase the concentration of labeled precursors in the labeling solution
Make sure plants are bathed in sufficient volume of labeling solution
Broad/tailed LC peaks Injection volume is too large Evaporate eluate or extract to a lower volume with vacuum concentrator
Reduce the sample volume injected onto the LC
The precolumn filter is dirty Replace the filter
The column is dirty Disconnect the LC line from the MS source and wash the column with solvent according to manufacturer’s instructions
Analyte m/z values do not match calculated values The MS needs to be calibrated Perform mass calibration with calibration standard mixture according to instrument manual
Reduced MS sensitivity The MS needs to be calibrated See above
The tune file needs to be modified Adjust source parameters for optimal ion intensity using standard compounds