Cleaning and Passivation of Liquid Chromatography

If the chromatographic pump inlet filters, inlet/outlet valves, and tubing (including the injector and detection cell) of the liquid chromatography system become contaminated, they should undergo cleaning and passivation. Cleaning should be performed first, followed by passivation.

The purpose of cleaning is to remove dirt from the stainless steel tubing and the system; cleaning must be completed before passivation. The purpose of passivation is to form a smooth and uniform oxide layer on the inner surface of the stainless steel tubing.

Contaminants inside the HPLC system may come from operators touching it, exposure to the laboratory environment, residues on instrument parts, or residues from the mobile phase and samples. Although the system can clean itself with the mobile phase, certain released impurities can adsorb to the liquid phase system, thereby reducing overall performance.

So, how do you maintain a high-performance liquid chromatography system? Let's take a look.

1）Acid Cleaning of Liquid Chromatography

Typically, a 30% phosphoric acid aqueous solution is used for cleaning. The specific procedure is as follows:

Remove the chromatography column and use a two-way connector instead.
Fill the pump head with ultrapure water.
Rinse with a 30% phosphoric acid aqueous solution at a flow rate of 1 mL/min for 1 hour (for UPLC, reduce the flow rate and increase the rinsing time).
Switch to ultrapure water and rinse until the water at the outlet has a neutral pH.
Transition to pure methanol to wet the pump head and tubing, then set aside for future use.

2）Passivation of Liquid Chromatography

1. Purpose of Passivation

Prevent Corrosion: The passivation layer can prevent metal from coming into contact with corrosive media, thereby reducing the rate of metal corrosion.
Increase Durability: The passivation layer can enhance the durability and wear resistance of the metal, extending its service life.
Improve Peak Shape: Interactions between metal ions and analytes can cause chromatographic peaks to disappear or broaden. This is particularly noticeable for substances containing phosphate or carboxyl groups. Passivation mainly addresses the tailing issues of substances, especially polar anionic analytes such as those with phosphate functional groups, organic acids, glycosides, and biomolecules (including peptides, proteins, nucleotides, etc.).

2. Specific Passivation Procedure

Remove the chromatography column and use a union to connect the system, filling the pump head with pure water.
Passivate with a 6M nitric acid aqueous solution at a flow rate of 1 mL/min for 1 hour (for UPLC, reduce the flow rate and extend the passivation time).
Replace with pure water and rinse until the outlet water shows a neutral pH.
Use pure methanol to flush the system, soaking the pump head and tubing, and then prepare for future use.

3. If Passivation Does Not Resolve the Issue, Address the Remaining Metal Ions

There are two main approaches: competing with metal ions or eliminating metal ions.

Strategies to Compete with Metal Ions:
- Add EDTA, citric acid, or methylene diphosphonic acid to the mobile phase.
- Saturate the tubing with a matrix sample.
- Saturate the tubing with a high concentration of the analyte.
- Acidify the sample.
- Note: In mass spectrometry systems, EDTA may cause ion suppression, leading to a reduction in analyte signal and potential precipitation at the tip of the spray needle.
Strategy to Eliminate Metal Ions:
- The simplest approach is to replace the metal tubing with biocompatible instrument components and column hardware, such as using diamond-coated column hardware like Welch DLC columns.

Overall, using biocompatible tubing is the best choice to prevent protein substances from adsorbing onto instrument components and chromatographic columns. If hardware replacement is not feasible, a viable approach is to use PEEK tubing to replace existing metal flow paths, but care must be taken regarding the system's pressure tolerance.