Table 1.

Common problems and suggested solutions

Problems	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	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 GC retention time	Deuterium labeled compounds are analyzed	It is normal for deuterium labeled compounds to have slightly shorter retention times.
	Carrier gas leaks significantly	Use a leak detector to find out where the leak is. Often, a worn Merlin Microseal septum is the source of the leak.
	Change of carrier gas	If the drift of GC retention time occurs after the change of carrier gas cylinders, check if correct gas cylinders are used.
Low yield of both endogenous IAA/IBA and IAA/IBA internal standard	Water in the methanol eluate reduces the methylation efficiency	If it takes a long time to evaporate the solvents to dryness, the samples are likely to contain residual water. Re-methylate samples by adding ethereal diazomethane and 10% methanol, and run samples again after drying and re-suspension.
	Wrong NH2 resin	Make sure that correct NH2 resin is used to extract IAA/IBA. Some brands of NH2 resin do not bind IAA/IBA sufficiently.
	The pH of solutions loaded onto PMME TopTips is too high	The pH has to be between 3 and 3.5. Cut a pH strip into narrower strips, and dip a strip in the solution to check the pH. Make sure that correct concentration of PA is made (pH ≤ 1.8), and avoid adding too much SA which increases the pH.
Low yield of endogenous IAA/IBA, but normal yield of IAA/IBA internal standard	Insufficient tissue homogenization and/or equilibration	Make sure that plant tissues are well homogenized. Allow longer time period for equilibration, e.g., overnight in the dark at 4 °C.
	Low endogenous IAA/IBA content in plant tissues	Collect more plant material for extraction. Usually, more plant material is required for IBA analysis as compared with IAA analyses.
Broad/tailed peaks	The GC liner is dirty	Change the liner, and cut ~30 cm from GC column from the injector end.
	The GC column is dirty	Turn off the MS, and change the GC column.
Overlapping peaks	The GC may need maintenance	See above.
	The plant sample may contain other metabolites that elute at similar GC retention time and produce ions with m/z values the same as the analytes	Run samples again using a different GC temperature gradient program. The temperature gradient commonly used is 20 °C/minute, and a slower or faster gradient can be used. Note that the retention time will be different when a different gradient is used. Run a standard to obtain the new retention time.
Reduced MS sensitivity	The tune file is out of date	Auto-tune the MS system.
	The EI source is dirty	Turn off the MS, and remove the parts of EI source. Reassemble the EI source after cleaning the parts.