Where Do Interference Peaks in Liquid Chromatography Come From?

1. Reagents/Solvents

In HPLC, commonly used reagents include organic solvents, various salts, and water. Organic solvents are typically purchased in chromatography-grade form, so the chances of issues occurring are relatively low. However, organic solvents that have been dispensed for use and repeatedly exposed have a higher risk of contamination, thus increasing the likelihood of interference peaks.

Water, in particular, is one of the main sources of interference peaks, a problem that is often overlooked. For detection methods involving low wavelengths, it is recommended to use high-quality water to avoid many issues caused by water impurities. Additionally, various salts can also contribute to chromatographic interference. For example, as shown in Figure 2, compared to Figure 1, the addition of potassium dihydrogen phosphate in Figure 2 results in an interference peak at 16.5 minutes. Interference peaks caused by inorganic salts are generally addressed during the method development stage, and solutions are sought. Once the method is established, the type and grade of salts are also determined, so the impact of salts on the method becomes largely controllable.

A quick solution to interference peaks caused by water and inorganic salts is to install a ghost peak trapping column, which can effectively prevent such interferences. If ion-pairing reagents are used, a ghost peak trapping column that does not affect the ion-pairing reagent should be selected.

Figure 1: Spectrum without interference peaks

Figure 2: Spectrum with interference peaks introduced by potassium dihydrogen phosphate

Figure 3: Spectrum with interference peaks introduced by acetonitrile filtration

02. Equipment

The second source of interference peaks comes from the various equipment used during the preparation of the mobile phase and sample pretreatment.

First, the equipment used during mobile phase preparation, including beakers, glass rods, pH meters, filter cups, filter membranes, and mobile phase bottles. Beakers and glass rods are the first pieces of equipment to come into contact with the mobile phase, so it is recommended to use dedicated beakers and glass rods for mobile phase preparation. Filter cups, as long as they are thoroughly cleaned by laboratory personnel, generally do not introduce contamination. However, if filtering a mobile phase that contains ion-pair reagents, it is recommended to increase the number of cleaning cycles to avoid contamination of subsequent mobile phases. Therefore, basic cleaning procedures for filter cups should be established to ensure proper cleaning of the equipment used by laboratory personnel. Filter membranes are also a source of interference peaks.

As shown in Figure 3, after acetonitrile is filtered through an organic system filter membrane, significant interference peaks are introduced during gradient elution. Since chromatography-grade reagents have already been filtered, and most instruments are equipped with degassers, I recommend not filtering pure organic phases like methanol or acetonitrile again. The introduction of interference peaks from mobile phase bottles mainly occurs in summer due to high temperatures, which can cause bacteria to grow in pure water phases. If the mobile phase bottles are not properly cleaned afterward, interference peaks may be introduced. Therefore, a strict cleaning procedure for mobile phase bottles must be established.

Figure 4: Spectrum with interference peaks introduced by pipette tips

Figure 5: Spectrum with interference peaks introduced by disposable straws

Figure 6: Spectrum with interference peaks introduced by disposable syringes

Next, the equipment used during sample pretreatment. The equipment in contact with the sample during pretreatment mainly includes volumetric flasks, filter heads, syringes, and sample vials. Cleaning of volumetric flasks should follow the same procedures as mobile phase bottles and filtration devices, with proper cleaning protocols in place. Pipettes, filter heads, and syringes, which are mostly plastic, are chemically less inert compared to glassware, making them more likely to introduce interference peaks. As illustrated in Figures 4, 5, and 6, where the method detects at a wavelength of 210 nm and the solvent is a mixture of n-hexane and isopropanol (90:10), different interference peaks are introduced after the solvent comes into contact with a pipette tip, a disposable pipette, and a disposable syringe, respectively. If sample vials are used only once, they generally will not introduce interference peaks. However, if they are cleaned and reused multiple times, a cleaning procedure should be established, and the effectiveness of cleaning should be verified.

03. HPLC System

Compared to the previous two categories, solving interference peaks from the HPLC system is more challenging. The HPLC system is dynamic, so resolving these issues can be time-consuming and requires a high level of chromatographic expertise from the experimenter. These issues can mainly be attributed to three factors: strongly retained substances, ion-pair contamination, and bacterial growth in the system.

First, interference from strongly retained substances is characterized by broad peaks. This issue mostly occurs during isocratic elution, while during gradient elution, it typically appears in the high organic phase. This is because strongly retained substances are not eluted in the current injection and are carried over into subsequent injections, as shown by the chromatographic peak at 9 minutes in Figure 7. These interference peaks can be eliminated by extending the collection time or increasing the elution strength to prevent interference with subsequent samples.

Figure 7: Spectrum with interference peaks introduced by strongly retained substances

Figure 8: Spectrum after extending the collection time

The second aspect of system interference peaks is contamination of the HPLC system by difficult-to-elute reagents like ion-pairing agents or chelators. This contamination is typically due to incomplete washing of the system after using a mobile phase containing ion-pairing agents or chelators. These contaminants primarily interfere with methods using lower detection wavelengths, often manifesting as increased baseline noise or interference peaks, but the system tends to stabilize as the method progresses. The third aspect of system interference peaks is bacterial growth in the system. Many readers may find this surprising, as the system is typically flushed and stored with organic solvents after use. However, in many methods, mobile phase A is a water-based solution, and although the water phase is freshly prepared daily, if the tubing from the filter head to the proportioning valve remains in the water phase for an extended period, bacteria can grow. Over time, this can result in the gradual appearance of interference peaks during gradient elution. Although this issue is somewhat hidden, it is relatively easy to resolve by flushing the channel containing pure water with an organic solvent at the end of each day.

The greatest challenge chromatographers face in practice is how to identify and determine the source of interference peaks. If the root cause of the problem cannot be identified, it becomes difficult to develop targeted solutions.