[Readers Insight] Why Do Ghost Peaks Appear?

Author: Chromatography Mound

Table of contents

Introduction Cause 1: Contamination of Mobile Phases Cause 2: Contamination from Instrument, Column, or Consumables Cause 3: Sample and Sample Solution Other Causes

Introduction

In HPLC analysis, ghost peaks are a vexing issue we occasionally encounter. Ghost peaks can appear at various positions across a chromatogram, and their response can sometimes be large. Although they generally do not interfere with the qualitative or quantitative analysis of target peaks, their presence is nevertheless unsettling.

There are many possible causes for ghost peaks, and even experienced practitioners may offer different explanations for the same experiment and chromatogram—hence the term “ghost peak.”

A chromatogram showing a peak caused by gradient fluctuation

Cause 1: Contamination of Mobile Phases

Determining the exact molecular identity of a ghost peak is often difficult, but contamination of the mobile phase is the most common source.

Even reagents labeled as Guaranteed Purity (>99.8%) or HPLC Grade can contain impurities such as plasticizers or antioxidants. The more additives present in the mobile phase—acids, bases, buffer salts, or ion-pairing reagents—the greater the chance of introducing such impurities.
Buffer components in the mobile phase can promote microbial growth during storage at ambient temperature; microbial metabolites may generate ghost peaks that interfere with analysis.
Because mobile phases are chemically complex, they may contain trace heavy metal ions that, during prolonged standing, can form complexes with solvents (water, methanol, acetonitrile) and produce coordination species with measurable UV absorption.

Cause 2: Contamination from Instrument, Column, or Consumables

Common contamination sources include the autosampler needle and the mixer. If a previously analyzed sample had a high concentration, excessive residues may remain on the needle.
When changing mobile phases, residual liquid in the pump head from the previous mobile phase can contaminate the new mobile phase.
Strongly retained components from a prior injection may not be fully eluted from the column and can be washed out on subsequent injections.
Low-polarity compounds may precipitate at the head of the column when the mobile phase contains a relatively high proportion of aqueous phase; these precipitates can be preferentially injected into the column on the next run.
Large, abrupt changes in gradient composition can also induce precipitation for certain compounds (for example, 5-methylcytosine).
Reusing autosampler vials can leave residues that contribute to ghost peaks.

An animation showing the installation of a Ghost-Buster column

Cause 3: Sample and Sample Solution

Typically, sample diluents are water, organic solvents, or buffer solutions. If the diluent composition is complex and the sample preparation lacks a cleanup step, impurities can be directly introduced.
Some sample components—both target and non-target substances—may degrade or transform over time under the influence of light, oxygen, or other factors, producing new species that give strong detector responses.
If the diluent is a pure organic solvent such as methanol or acetonitrile and it is stored for a long time, oligomeric species from septa or vial liners may dissolve into the solvent; these oligomers can exhibit significant absorption at low wavelengths.

Other Causes

Beyond the reasons listed above, ghost peaks may originate from:

Column bleed,
Aging or degradation of septa and tubing,
Insufficient or omitted post-run column flushing (which can allow microbial growth inside the column),
Accumulation of contaminants in the system dead volume, etc.

To prevent ghost peaks, routine cleaning of instruments is an effective way. When ghost peaks appear, it is recommended to check the above aspects one-by-one to locate the causes and perform corresponding countermeasures.

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