Cleaning of HPLC System:
Here is a practical list of cleaning solvents commonly used for cleaning HPLC
systems, including what they remove and how/when to use them.
🧴 Common Cleaning Solvents for HPLC System Maintenance
Purpose / What It
Solvent or Mixture When to Use
Removes
Removes salts, buffers, After using buffer-based
HPLC-grade water
aqueous contaminants mobile phases
Removes organic
General system flush,
100% Methanol residues, moderately
column cleaning
polar compounds
Removes nonpolar
Routine flushing or after
100% Acetonitrile (ACN) organics, hydrophobic
long runs
compounds
Removes strongly
retained hydrophobic Deep cleaning,
Isopropanol (IPA)
materials, dissolves especially LC-MS
oils/grease
Universal flush, good General cleaning
Water:ACN (50:50 or 60:40)
miscibility between runs
After aqueous or
Water:Methanol (50:50) Gentle cleaning solvent
organic phases
Water:IPA:ACN:Methanol Removes organic, ionic, Deep cleaning; monthly
(25:25:25:25) (aka Waters Magic and hydrophobic or when contamination
Mix / AAA Wash) residues is suspected
Removes basic or ionic For LC-MS systems;
0.1–2% Formic Acid in
contaminants, improves removes ionizable
Water:ACN
MS baseline residues
For strong Only for stainless steel
0.1 N NaOH or H3PO4 (CAUTION) contamination or systems and only if
microbial cleaning approved
Purpose / What It
Solvent or Mixture When to Use
Removes
(system only, NOT
column!)
Removes salts,
Weekly/biweekly; only if
Hot Water (50–60°C) microbial
system supports it
contamination
🔶 Notes and Best Practices
General Guidelines
• Always use HPLC- or MS-grade solvents
• Start cleaning with weaker solvents (e.g., water), then move to stronger
solvents (e.g., methanol, IPA)
• Use gradual transitions between immiscible solvents (e.g., water → methanol
→ IPA)
Cautions
• Never switch directly between water and 100% IPA or ACN without a miscible
intermediate like methanol
• Do not use strong acids or bases in silica-based columns unless specifically
recommended
• Avoid chlorinated solvents (e.g., DCM, chloroform); they are too aggressive
and not HPLC-compatible
🧪 Recommended Cleaning Sequence (General)
1. Flush with water (especially if using buffers)
2. Flush with 50:50 water: organic solvent (e.g., MeOH or ACN)
3. Flush with 100% organic solvent (e.g., MeOH, then IPA if needed)
4. (Optional) Flush with Magic Mix for deep cleaning
5. Return to mobile phase or storage solvent
Waters Magic Mix for Cleaning HPLC System:
The cleaning solution known as Waters Magic Mix (also known as AAA Wash) is an
informal but widely adopted practice for cleaning and maintaining HPLC and UPLC
systems, especially in LC-MS workflows. However, there is no formal regulatory
guideline (such as from FDA, USP, or ICH) that directly mandates or describes this
specific mixture.
Key Points:
• Waters Magic Mix is primarily recommended in instrument vendor
documentation, such as from Waters Corporation, and is commonly shared in
technical notes, application notes, and user forums.
• It is especially useful for removing sample residues, salts, and other
contaminants that can affect sensitivity or cause carryover in LC-MS systems.
• The mixture is composed of:
o 25% HPLC- or MS-grade water
o 25% acetonitrile
o 25% methanol
o 25% isopropanol
o With 0.1–2% formic acid optionally added to enhance the cleaning of
acidic or ionic residues.
Guidance Source
• This cleaning mixture is not part of a regulatory guideline, but it is often
recommended by LC-MS instrument manufacturers, particularly:
o Waters Corporation
o Possibly other vendors (like Thermo, Agilent) in their service or support
documentation
• You may find this mixture referenced in:
o Waters’ Service Notes
o Technical manuals
o Community best practices
o Journal articles or presentations involving LC-MS system maintenance
Conclusion
Waters Magic Mix is not part of a formal regulatory guideline, but is a vendor-
recommended and widely accepted best practice for maintaining LC-MS systems. If
you're in a regulated environment (e.g., cGMP lab), it's important to document and
justify its use in your Standard Operating Procedures (SOPs), possibly referencing
manufacturer recommendations or internal validation.
Hot Water for Cleaning HPLC System:
Flushing an HPLC system with hot water is indeed a widely accepted maintenance
practice, particularly for reversed-phase systems, but like the Waters Magic Mix, this
procedure is not governed by a specific regulatory guideline (e.g., FDA, USP,
ICH). Instead, it is based on instrument manufacturer recommendations and
general chromatographic best practices.
What Supports the Practice?
1. Instrument Manufacturer Recommendations:
o Vendors like Waters, Agilent, Thermo Fisher, and Shimadzu often
recommend hot water flushing in their user manuals or technical notes,
especially to:
▪ Remove buffer salts
▪ Eliminate microbial growth in aqueous lines
▪ Clear out residues from aqueous mobile phases
2. Chromatography Best Practices:
o Flushing with hot water (typically 50–60°C, not boiling) helps:
▪ Restore baseline stability
▪ Remove water-soluble contaminants
▪ Prevent column clogging and prolong lifespan
o These are described in textbooks (e.g., “Practical HPLC Method
Development”) and peer-reviewed articles on LC maintenance.
3. Good Laboratory Practice (GLP):
o While GLP (from OECD, FDA, or EPA) doesn't dictate specific
maintenance procedures, it emphasizes:
"Regular maintenance of instruments must be documented and justified to
ensure system suitability."
o Hot water flushing can be considered part of such maintenance, if
documented in an SOP.
What Do Guidelines Like USP or ICH Say?
• USP <1058> Analytical Instrument Qualification:
o Talks about instrument qualification and maintenance, but does not
prescribe hot water flushing directly.
• ICH Q2(R1) (Validation of Analytical Procedures) and ICH Q9 (Quality Risk
Management):
o Do not cover routine maintenance like flushing, but emphasize system
suitability and risk-based approaches, which can support the inclusion
of hot water flushing in SOPs as a preventive measure.
Conclusion
Hot water flushing is supported by:
• Manufacturer guidance (Waters, Agilent, etc.)
• Chromatographic best practices
• GLP/ISO principles requiring documented maintenance
It is not mandated or described in formal guidelines (e.g., FDA, USP chapters,
ICH).
Recommendation
If it is in a regulated environment:
• Include hot water flushing in your SOPs
• Reference manufacturer manuals or internal validation data
• Document maintenance in logbooks
