Liquid does not pass through TopTips before loading plant samples
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The slit on TopTips is too narrow
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Increase the centrifugal force to make liquid pass through. Or, switch to a new TopTip.
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Liquid does not pass through TopTips after loading plant samples
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Plant debris blocks the TopTip
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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.
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Drift of GC retention time
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Deuterium labeled compounds are analyzed
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It is normal for deuterium labeled compounds to have slightly shorter retention times.
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Carrier gas leaks significantly
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Use a leak detector to find out where the leak is. Often, a worn Merlin Microseal septum is the source of the leak.
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Change of carrier gas
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If the drift of GC retention time occurs after the change of carrier gas cylinders, check if correct gas cylinders are used.
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Low yield of both endogenous IAA/IBA and IAA/IBA internal standard
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Water in the methanol eluate reduces the methylation efficiency
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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.
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Wrong NH2 resin
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Make sure that correct NH2 resin is used to extract IAA/IBA. Some brands of NH2 resin do not bind IAA/IBA sufficiently.
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The pH of solutions loaded onto PMME TopTips is too high
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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.
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Low yield of endogenous IAA/IBA, but normal yield of IAA/IBA internal standard
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Insufficient tissue homogenization and/or equilibration
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Make sure that plant tissues are well homogenized. Allow longer time period for equilibration, e.g., overnight in the dark at 4 °C.
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Low endogenous IAA/IBA content in plant tissues
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Collect more plant material for extraction. Usually, more plant material is required for IBA analysis as compared with IAA analyses.
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Broad/tailed peaks
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The GC liner is dirty
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Change the liner, and cut ~30 cm from GC column from the injector end.
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The GC column is dirty
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Turn off the MS, and change the GC column.
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Overlapping peaks
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The GC may need maintenance
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See above.
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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
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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.
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Reduced MS sensitivity
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The tune file is out of date
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Auto-tune the MS system.
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The EI source is dirty |
Turn off the MS, and remove the parts of EI source. Reassemble the EI source after cleaning the parts. |