Ghost Peaks in HPLC (Liquid Chromatography) are a common issue in Analytical Laboratories.
Handling of Ghost Peaks in HPLC
The Ghost peaks in Liquid Chromatography (HPLC) are contaminant peaks that may appear even when no sample is injected. There are many causes for ghost peaks which are described below with troubleshooting.
- Dirty mobile phase
- Sample carryover
- Poor recovery / Peaks from an early isocratic run
Causes for Ghost Peaks in HPLC and Troubleshooting Guide
Please refer to the below causes for Ghost Peaks in HPLC and the respective Troubleshooting procedure.
Ghost Peaks in HPLC Causes – HPLC Columns
The primary cause of a ghost peak in HPLC is a dirty column or pre-column. Remove the pre-column and run a sample. If the ghost peaks are no longer present, replace the pre-column (or frit).
Column contamination may be another cause of ghost peaks. If the sample remains on the column, peaks will elute during the next run. Extend the runtime of the method and also flush with strong solvents to remove all compounds from the column.
Always contact the column’s manufacturer for best cleaning procedure recommendations. Contact Vendor for column care and troubleshooting.
Column contamination may be due to sample contaminants or unknown interferences from samples.
Improve sample cleanup prior to injection.
Ghost Peaks in HPLC Causes – HPLC Detectors
A defective detector may create ghost peaks in HPLC, by creating a noisy baseline on the chromatogram. To ruled-out, out a detector problem, turn the flow rate to zero and monitor the baseline. In case the baseline noise (fluctuation) is identified, it is due to the detector (bad lamp). Hence, replace the HPLC lamp.
Electronic noise (malfunction) may be the issue if the lamp does not solve the problem. In case of the problem is unresolved (non-lamp-associated detector-caused noise on the baseline), contact the relevant HPLC Vendor for “On-site Troubleshooting”.
Ghost Peaks in HPLC Causes – Carryover
The source of sample carryover caused for Ghost Peaks in HPLC is provided below in different ways. To test for sample carryover, inject the sample and then inject 3 blank samples (HPLC vials filled with Diluent / Mobile phase).
i) Wide peaks eluting at the beginning of the chromatogram
These peaks are still eluting from the last sample injection and the runtime is not long enough. In such cases, increase the runtime of the method by 15 minutes.
Another way of extracting the late eluting peaks from the column is to increase the solvent strength of the mobile phase. In the case of salt buffers, use water for longer periods of time to elute all compounds from the column.
ii) Sharp peaks at the same retention times (RT) of the sample
This is the reason why the sample is not eluted completely from the Liquid Chromatography system (LC/ HPLC). This can identify with blank injection, where the heights of the peaks decrease.
This might be due to the sample retained on the outside of the HPLC Injector needle. The inside of the needle is continuously flushed with solvent throughout the run because of the design of auto-samplers. Viscous samples may cling to the outside of the needle and tend to transfer analyte traces (contamination) to the next injection.
All the HPLC autosamplers have a function of needle wash that can program in most of the Chromatography Software (Empower/ Chromeleon/ ChemStation/ OpenLab). In the case of Agilent HPLC instruments, do not place a vial cap on the wash vial. However, each sample vial shall have a vial septum in place to avoid evaporation and sample carryover.
iii) Ghost peaks arise due to the impurities in the mobile phase (including the water)
Make sure that the water used for Mobile phase preparation is high-pure HPLC-grade water. If the observed peaks RT and the peak height are the same for each chromatogram, these impurities are observed due to the bad mobile phase. Hence, prepare a fresh mobile phase with fresh HPLC-grade water and HPLC-grade buffers. Ensure to filter/ degas the mobile phase properly. Thoroughly degas the mobile phase by using a Degasser Module, vacuum filtration, and or sonication.
Since the water and buffered water is the source for microbial growth, do not use the HPLC MP for not more than 48 hours. The validity period (use before date) of the Mobile phase can establish based on the study.
Another cause for the ghost peaks is the contaminant in the rotor seal of the 6-port valve of the autosampler. Replace the HPLC autosampler rotor seal.
In case of the HPLC instrument is not used for a long-time, there is a chance of contamination at the internal tubing and the flow cell. This is due to the salts deposition from the mobile phase. Hence, recommended to perform the below-mentioned HPLC washing procedure. if you suspect contaminants within the tubing. This procedure may help to eliminate Ghost peaks in HPLC.
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HPLC washing procedure
Proper implementation of the HPLC instrument washing procedure may arrest the Ghost Peaks in HPLC analysis.
1. Remove the column or pre-column and replace it with a zero dead volume fitting (union). Remove the solvent inlet frits (HPLC solvent filters/ suction filters) inside the mobile phase bottles.
A.Rinse the system with HPLC Water at a flow rate of 5 ml/min, and run each channel separately.
B. Then, rinse the system with diluted Nitric acid (1N) at 5 ml/min for all the HPLC channels.
C. After completion of the above process, rinse the HPLC channels with Water-Methanol-Water at a flow rate of 5 ml/min for all channels.
2. Finally reconnect the HPLC components and run the Liquid Chromatography system (LC) for further usage.
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