What is Ghost Peaks?

Ghost peaks can occur in several different situations. In the following, we will discuss the various causes of these ghost peaks. 

The Problem of Ghost Peak

One of the brain-racking challenges in LC analysis is the presence of ghost peaks (see Fig.). Ghost peaks are of unknown origin in a chromatogram, are easily misidentified when they are close to peaks of interest, and can result in quantitative errors when they overlap peaks of interest. Uncertainty in data quality and reliability is of course the result. 

To make matters worse, considerable time can easily be expended in determining the source of an apparent impurity, even if there is none associated with the compound of interest. 

With the increase in UV detector sensitivity that has come about in recent years, detection has become possible at very low concentrations, bringing the problem of ghost peaks into the spotlight. 

Sources of Ghost Peaks

The sources of ghost peaks are typically divided into three categories: those due to impurities other than the target substance, internal contamination of the LC instrument, and impurities in the mobile phase, as shown below.

1. Impurities in instrument
2. Impurities in sample
3. Impurities in mobile phase

ALSO READ: Abnormal Peak Shapes

Ghost Peaks Due to Instrument

• The most common reason for ghost peaks originating in the instrument is carryover attributable to the autosampler (or manual injector). When the needle is dipped into the vial to aspirate the sample, substances in the sample, which become the source of carryover, are adsorbed to the inside and outside surfaces of the needle. 
• Those substances which are not eliminated even after needle rinsing are carried over to the next analysis, and appear as ghost peaks. Since substances that exhibit particularly strong adsorption will appear even after several blank analyses (analysis in which only the mobile phase is injected), it may be difficult to identify the autosampler as the source of the ghost peaks
• On the other hand, ghost peaks may also be attributed to instrument contamination.

Ghost Peaks Due to Sample

• When there is an impurity in the sample having the same absorption wavelength as the compound of interest, it results in a ghost peak. Since a ghost peak that originates in the sample does not appear in a blank injection, determining the source is relatively easy.
• The remedy is to devise a sample preparation procedure to eliminate the impurity, such as an extraction or use of a disposable filter, etc.
• Further, when the appearance of a ghost peak is due to the degradation of the sample, that degradation can be inhibited by using an autosampler with a vial cooling feature.
• On the other hand, the source of the ghost peak may not be in the sample itself, but rather, from dissolved oxygen in the sample solvent. This is especially noticeable when using a UV detector. 

Ghost Peaks Due to Mobile Phase

• Ghost peaks can also be generated from the mobile phase in various ways, including the following.

1. Generation of organic substances in the mobile phase due to prolonged use, or dissolution of organic substances from the air in the mobile phase.
2. Mobile phase vial contamination due to topping off the existing mobile phase with new mobile phase over a long period of time instead of preparing a fresh bottle each day or for each set of samples.
3. Use of contaminated organic solvent and/or water to prepare mobile phase
4. Use of contaminated mobile phase reagent

• Due to the high consumption of the mobile phase and the relatively long periods of time it is used before being replaced, pretreatment measures such as filtering that are typically used for samples are not practical for eliminating contaminants in the mobile phase.
• Mobile phase contaminants adhere to the inside of the instrument and tubing, small amounts are inevitably transported to the column along with the mobile phase during the solvent delivery process and are likely to be detected as ghost peaks.

• In reversed-phase analysis using gradient elution, such substances that cause ghost peaks are retained in the column at the start of analysis, and are then eluted as the organic solvent ratio increases, making separation very difficult when they exhibit behavior similar to that of the compound of interest.
• Thus, it is difficult to specify the cause when ghost peaks are of mobile phase origin, and furthermore, it is difficult to address the problem by making changes to the method.

How do I get rid of ghost peaks?

Possible Cause	Solution
Contamination	Flush column to remove contamination; use HPLC-grade solvent
Elution of analytes retained from previous injection	Flush column with strong solvent at end of run; end gradient at higher solvent concentration
Ion-pair chromatography - upset equilibrium	Prepare sample in mobile phase; reduce injection volume
Oxidation of trifluoroacetic acid in peptide mapping	Prepare trifluoroacetic acid solutions fresh daily; use antioxidant
Reversed-phase chromatography - contaminated water	Check suitability of water by running different amounts through column and measure peak height of interferences as function of enrichment time; clean water by running it through old reversed-phase column; use HPLC-grade water
Reversed-phase chromatography - contaminated water	Check suitability of water by running different amounts through column and measure peak height of interferences as function of enrichment time; clean water by running it through old reversed-phase column; use HPLC-grade water
Unknown interferences in sample	Use sample cleanup or pre-fractionation before injection

ALSO READ: Peak Purity and Peak Identification