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How to clean an ACQUITY UPLC using Acid / base cleaning protocol after severe PEG contamination or other contamination? - WKB16167

Article number: 16167


If an ACQUITY UPLC is contaminated with PEG due to bad solvents, then the background stays visible. Even if the switch is made to good solvents, the PEG background stays visible on sensitive mass spectrometers like Vion, Xevo G2-XS, and Synapt G2-Si. Also if spectral peak fronting is observed on intact monoclonal antibody analysis on e.g. BioAccord (RDa) or Vion this protocol can be used.  This protocol can of course also be used for other contaminants. This procedure describes how to clean the ACQUITY according to the acid / base cleaning protocol.




  1. Remove the UPLC column and replace for a union. Place ALL solvent lines into each solvent bottle for each flushing step. Use a Critical Clean glass bottle kit (Ordering_Brochure). Sinker stones that are already in use for a while should be removed as well. 
  2. Purge line A and B for five minutes per line with solvent in the exact order as stated below. Purge FTN 60s for needle wash, 40 cycles for sample syringe. Create an analysis or sample list and run 20 injections of 10 uL of the same solvent as is running on the machine. Runtime one min, flow rate 0.8 mL/min on A1:B1 50:50. Direct the LC flow to waste and not to the mass spectrometer. In case of an optical detector like UV or PDA the flow can go through the detector. The water flushes are only needed to remove the previous solvent from the system. No injections are needed with that. 
    1. Water flush to clear customer's mobile phases from the system.
    2. Magic mix; water - acetonitrile - methanol - IPA 1:1:1:1 with 2-5% FA
    3. Water flush to clean acid from the system.
    4. 10% NH4OH in water (this removes PEG and other polymers).
    5. 10% NH4OH on line A1 and A2 and 100% IPA on line B1 and B2 (or for an H-class 10% NH4OH on A and B and 100% IPA on C and D). This is a very strong combination of solvents to remove PEG.
    6. Water flush to remove the base from the system
    7. 100% IPA
    8. Water flush as final rinse
  3. If methanol or IPA are not being used as eluants, then replace old sinker stones with fresh SS stones or the titanium stones 289002172).


The 10% ammonia solution really needs to be the final concentration. So if a stock of 28-30% ammonia is available it is only a 3 times dilution. Due to the very intense smell use a fume hood to prepare and additionally take good care when placing the bottle on the machine. 

Do not use this protocol on M-class. The PeekSil and silica tubings cannot handle a pH this high.

At step 8, monitor the pH of the water rinse to ensure it is back to lab pH prior to hooking column and MS back in flow path.

Remake all aqueous mobile phase weekly, triple rinsing the bottle with Magic Mix (25/25/25/25 water/isopropyl-alcohol/methanol/acetonitrile) or 100% organic like methanol or acetonitrile then rinsing with the solvent to be used before refilling. Never top off a mobile phase solvent, never put mobile phase bottles in a dish washer.

eluent, isopropanol

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