Hydrophilic interaction liquid chromatography (HILIC) has become popular for separating polar components in combination with mass spectrometry because of the increased relevance of metabolomics.
Acetonitrile (ACN) is probably the most common organic solvent for HPLC in general, owing to the lower viscosity relative to other organic solvents like methanol. While ACN is ideal for preventing damage to your column and systems due to increased backpressure from changes in eluent viscosity ꟷ especially during gradient separations ꟷ this solvent also comes with some known problems, like particle contamination and polymerization.
If you are struggling with using ACN in your HILIC separations, rest assured there are a few ways to troubleshoot and quickly resolve the issues.
Acetonitrile, the perfect organic solvent for HPLC?
Acetonitrile, also known as methyl cyanide, has essential chemical properties favorable for HPLC.
- ACN has a stronger elution strength than methanol, is miscible with water, and produces low backpressure in a mix with water.
- ACN has minimal UV absorbance at low wavelengths (<250nm) and is compatible with solutions containing aromatic compounds like tyrosine and benzoic acid.
- ACN is an industrial byproduct from the manufacture of acrylonitrile, which makes the compound widely available and affordable.
Common issues you can encounter when using ACN in HPLC
Although ACN sounds like a perfect solvent for HPLC, some flaws exist.
For starters, UV transparency depends on the purity/quality of your ACN. Depending on the specific requirements, there is a spectrum of different grades of ACN for your HPLC and LC-MS analysis. Unfortunately, these different classifications are not harmonized between suppliers, which results in significant quality differences leading to performance reduction or even increased wear of your instrument.
As mentioned earlier, ACN is an industry byproduct and requires purification and filtration to remove significant particle contaminations from the source. For this reason, filtration quality is key to preventing small particles that cause check valve leakages in the HPLC pump or higher wear on pistons and seals, respectively.
Another often-discussed phenomenon is the so-called polymerization. Publications indicate that ACN can polymerize under extreme conditions or in the presence of strong acids not found in your HPLC system.
Notably, the effect of sticky check valves on the ACN channel is a commonly known issue described by John Dolan in 2008.
How to mitigate ACN-related issues with your HPLC analysis
Following best practices for HPLC systems is a good starting point for unwanted downtime:
1. Keep your HPLC instrument (and components) clean
Just as water can biologically contaminate over time, ACN contaminations may accumulate with aging. For this reason, plan regular sanitization cycles with a suitable and clean wash solvent, such as Isopropanol (IPA) or mixture with water, every 4-6 weeks.
Best practice includes flushing the entire fluidic path from the solvent reservoir, through the pump to the autosampler. Setting up an automated flush procedure can also allow more frequent cleaning cycles between sequences.
Additionally, daylight exposure of the HPLC solvent bottles might accelerate the growth of biological contamination. In such cases, utilize a suitable light cover for the bottles, like wrapping with aluminum foil, or switch to amber bottles, if applicable.
2. Use high-quality solvents to help prevent contaminating your HPLC system
Using LC-MS grade solvents with high filtration quality (<0.2 µm) helps prevent the introduction of particles. Yes, high-quality solvents are more expensive than normal ones, but consider the productivity losses and costs associated with excess downtime of your LC system.
Be aware that mobile phase filters inside your HPLC reservoirs DO NOT protect against sub-micron particle contaminants. These filters only have typical pore sizes between 10-20 µm and are intended to protect your HPLC from larger debris that could severely damage the system.
- Small sub-micron particles can lead to leaking check valves, blocking internal filters, and high wear of pistons and high-pressure seals.
Standard additives, like formic acid (FA) or trifluoroacetic acid (TFA), should be treated like your solvents. A large old reservoir bottle opened several dozen times is probably not a suitable match for high-quality solvents.
- Ideally, you should purchase these additives in suitable container sizes, like ampules.
3. Consider the cleanliness of your glassware
Get in the habit of exchanging your eluent bottles when empty to prevent the accumulation of contaminants in the bottle. And, before using, rinse the bottles with a clean mobile phase to wash out any residues.
Where do you leave the bottle cap with the attached solvent line while exchanging the bottle? Directly transfer the bottle cap assembly from the empty bottle to a fresh one.
You should also regularly inspect the content of your mobile phase bottles against light while gently agitating the bottle to discover dust and dirt floating in the mobile phase.
4. Do not store your pump under ACN for a longer period (overnight)
Not planning to use your HPLC for a couple of days? No problem. You can put your system into standby mode at a lower flow rate (50µL/min) or completely shut down the instrument.
In the latter case of shutting down, flush the pump with a suitable solvent like IPA before turning off your pump. If the pump is immersed in ACN, the check valve ball can stick to the seat preventing liquid from aspirating. Although clogged valves can operate again, this fix requires de-installation of the check valves from the pump module.
If your instrument needs to be restarted quickly, a standby condition at low flow is recommended.
5. Avoid mixing ammonium hydroxide additives with pure ACN
A common HILIC mobile phase under basic elution conditions contains Ammonium hydroxide (NH4OH)/ACN. This specific mobile phase may deteriorate the standard high-pressure seals in your HPLC pump. Adding a low percentage (around 5%) of water into the ACN phase is a good way to mitigate deterioration. Since HILIC applications typically start with a significant amount of water, you can premix water into the ACN mobile phase.
If eluent adaptation is not possible, consider changing the piston seals. For instance, you can exchange standard UHMW-PE (ultra-high molecular weight polyethylene) seals with graphitized PTFE (polytetrafluoroethylene), which are not impacted by the NH4OH/ACN mobile phase.
Note that PTFE seals are applicable up to 700 bar and can be utilized without compromise since HILIC applications normally operate at much lower pressures.
Important: Due to the DLC (diamond-like carbon) coating of Vanquish Horizon UHPLC System pistons, you must not use graphitized Teflon seals!
6. Troubleshoot low backpressure-related instrument issues
Pump check valves in LC systems are often passive components requiring sufficient pressure differences to ensure the ceramic ball closes the flow path without leakage.
While many HILIC applications only produce around 100 bar (1,500 psi) of backpressure, state-of-the-art (U)HPLC systems have much higher pressure limit capabilities. Consequently, these systems and key components, like check valves, will operate below the ideal working pressure.
The low operating pressure might not be high enough to ensure complete tightness of the check valve, or not able to perform a pressure-driven seal-cleaning. A simple fix is to introduce a small inner diameter capillary between your pump module and autosampler. This switch will increase the backpressure for the pump and achieve a better operating condition while the pressure experienced by the HILIC column is unaffected.
Want more resources for troubleshooting your HILIC methods?
Find more background information on HILIC applications in our HILIC Separations Technical Guide and HILIC Learning Center.
You can learn ways to monitor your solvent quality in our Technical Note.
A resource MUST for your lab: View the Column Troubleshooting Guide for HILIC
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