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. |