What Do You Mean for Ghost Peaks?
When doing the gradient elution of liquid chromatography reversed-phase system, sometimes we will encounter the situation that the instrument does not feed the sample but also has the peak, affecting the quantitative results.
It can also make the analysis impossible because of its ghostly quality, called a ghost peak. Then we will understand the possible reasons for the appearance of ghost peaks and methods of elimination.
In the suspicion that the peak shape is abnormal is not a ghost peak, we can first carefully verify whether the operating conditions are consistent with the analytical method, whether the instrument is in normal working condition to see whether there is an operational error, to determine whether it is not the errors mentioned above, the instrument does not feed the sample, or into the mobile phase, or only run the method to see whether there is still an inexplicable peak in the baseline to determine whether it is a ghost peak. From the perspective of the frequency of ghost peaks, it can be considered from two major aspects: the mobile phase and liquid phase system.
Why Ghost Peaks are Various and Solutions
According to my experience and literature reports, the reasons are summarized as follows:
Reason 1: The mobile phase problem
The mobile phase, sample diluent, instrument or container impurities, when the gradient begins due to the organic phase ratio is not high, the elution capacity is not strong, impurities in the column head enrichment, with the mobile phase ratio changes, the elution capacity is enhanced, the enriched impurities are eluted, thus forming ghost peak.
Solution
As we know mobile phase impurities leading to ghost peaks is a common challenge in HPLC. The issue you described with impurities enriching at the column head and then eluting with a change in mobile phase ratio is a valid explanation. Luckily, there are several solutions you can try to address this problem:
1. Mobile Phase Purification:
- Filtration: Filter your mobile phases (especially organic solvents) through 0.45 µm filters to remove larger particles that could contribute to ghost peaks.
- Degasification: Ensure your mobile phases are degassed, as air bubbles can cause peaks and interfere with analysis.
- UV treatment: Consider using UV light filtration systems to degrade organic impurities in your mobile phases.
2. System Cleaning:
- Flush the system: Perform regular blank runs with the mobile phase to flush out any accumulated impurities from the pump, injector, and tubing.
- Clean system components: Regularly clean the injector needle, seal, and filters to remove potential contamination sites.
- Purge the column: If the ghost peaks persist, consider purging the column with a strong solvent (e.g., methanol) to remove accumulated impurities.
3. Optimize your analysis:
- Use high-purity solvents and sample diluents: Invest in HPLC-grade solvents and diluents to minimize impurity introduction.
- Adjust the gradient profile: Start with a higher organic phase ratio at the beginning of the gradient to avoid enrichment of impurities at the column head. Consider a more shallow gradient for better resolution and less peak distortion.
- Increase flow rate: Increasing the flow rate can shorten the retention times of analytes and impurities, potentially causing them to elute separately and reducing ghost peak interference.
4. Identify the source of the impurity:
- Run blank injections: Inject mobile phase only to see if the ghost peak appears, helping to pinpoint if it’s system-related or from the sample matrix.
- Try different sample preparation methods: Experiment with different sample preparation techniques to see if specific steps are introducing impurities.
- Use different detectors: If possible, try using different detectors (e.g., UV at different wavelengths, diode array, mass spectrometry) to gain more information about the ghost peak’s identity and potential source.
But the best solution will depend on the specific cause of your ghost peaks. By systematically trying these approaches and monitoring your results, you should be able to identify and eliminate the source of the problem and achieve cleaner chromatograms.
I hope this information helps! Don’t hesitate to ask if you have any further questions or need additional details on any of these solutions.
Reason 2: The mobile phase degassing is not complete.
The mobile phase degassing is not complete. The solubility of air in the organic phase and water is different. When the water and organic phase are mixed, bubbles are generated.
And incomplete degassing of the mobile phase can definitely lead to ghost peaks in HPLC. As you mentioned, the difference in air solubility between water and organic solvents plays a key role in this phenomenon. Let’s delve deeper into the issue and explore some solutions:
The Problem:
- Solubility Differences: Air is more soluble in organic solvents than in water. When aqueous and organic phases are mixed, the air trapped in the organic phase comes out of solution, forming bubbles.
- Bubble-Induced Peaks: These bubbles can interact with the stationary phase like chromatographic entities, causing peaks to appear on your chromatogram. They can also cause baseline fluctuations and instability, affecting peak integration and quantitation.
Solutions:
Thorough Degassing:
- Vacuum filtration: Filter your mobile phases (especially organic solvents) through 0.45 µm filters under vacuum. This helps remove both dissolved air and larger particles that can trap air bubbles.
- Helium sparging: Sparge your mobile phases with high-purity helium gas for 15-30 minutes. Helium displaces the dissolved air without significantly affecting the mobile phase composition.
- Online degassers: Invest in an online degassing system that continuously removes dissolved air from the mobile phase as it flows through the HPLC system. This is the most effective but also the most expensive option.
Optimize Mobile Phase Preparation:
- Pre-mix solvents: Mix the aqueous and organic phases before adding them to the reservoir bottle. This allows bubbles to form and escape before entering the HPLC system.
- Sonication: Briefly sonicate the pre-mixed mobile phase to facilitate bubble release and degassing.
- Use degassed water: Consider using commercially available degassed water to save time and effort.
Reduce Air Exposure:
- Minimize headspace: Use reservoirs with minimal headspace to reduce air contact with the mobile phase.
- Seal all connections: Ensure all tubing connections and bottle caps are tight to prevent air leaks.
- Purge the system: Flush the HPLC system with degassed mobile phase before starting your analysis.
Additional Tips:
- Monitor your baseline stability. If you see fluctuations, it could indicate the presence of bubbles.
- Consider using a different gradient profile with a higher organic phase ratio at the beginning. This can help prevent bubble formation during initial mixing.
- If the problem persists, consult your HPLC instrument manual or contact the manufacturer for specific troubleshooting advice.
By implementing these solutions and optimizing your degassing procedures, you can minimize the formation of bubbles and achieve cleaner, more reliable HPLC analyses.
Reason 3: Operation problem
A:) Reagent issues
1, Organic solvent
The methanol preparation process is relatively simple compared to acetonitrile, and the impurity content is also less. However, because the UV absorption range of methanol is wider than that of acetonitrile, so when using a UV detector, especially when the wavelength is lower, try to use acetonitrile as the organic phase, and the drift is too large with methanol.
The quality of acetonitrile used in gradient chromatography requires high quality. Generally, special acetonitrile with imported gradient chromatography can reduce the appearance of ghost peaks.
2, Water
Many units use homemade distilled water as the aqueous phase, but the results are not very good. The reason is that some low boiling point organic impurities can not be removed by distillation. As far as possible with HPLC-grade pure water, no conditions can also use in Wahaha or Watson’s water. However, if the chromatographic conditions are more stringent is also likely to be a problem.
3, Other reagents
Buffer salts, TFA, EDTA, and other reagents will also greatly impact the mobile phase. Try to use high-purity reagents to prepare the mobile phase.
4, mobile phase configuration after pre-sonication degassing
5, holding the mobile phase containers or measuring instruments is contaminated
This contamination may come from detergents, chromic acid wash, or other residual impurities after laboratory personnel use. Mobile phase container cleaning generally with clean water + organic solvents on the shabbing. Too many cleaning steps may cause secondary contamination. Sometimes the plastic cover fragments of the mobile phase container may be the source of impurities.
6, pH meter
Many pH meters use polycarbonate as a shell. However, polycarbonate is soluble in organic solvents, so try to use a glass pH meter to measure the pH of the mobile phase.
7, Filter membrane
Poor quality or the wrong type of filter membrane is often the reason for ghost peaks.
8, placed for too long
The mobile phase is exposed to air for too long, it may absorb organic impurities in the air, and sometimes the additives in the mobile phase, such as TFA, will oxidize over time and thus produce impurities.
B: Instrument problems
1, the mobile phase of the hplc tubing is contaminated.
Prolonged use of mobile phase with high water content (especially the addition of buffer salt), bacteria are easy to breed in the pipeline, bacterial metabolites or cell debris can cause ghost peaks.
2, transfer pump failure, resulting in system pressure instability, thus creating ghost peaks.
3, check valve blockage.
Causes system pressure instability, resulting in ghost peaks. The early Shimadzu liquid phase check valve seems unable to use pure acetonitrile as the mobile phase and now should have been improved.
4、The inline degasser is not completely degassed.
5, the ratio of the two mobile phases of the organic phase is too different.
For example, A is 100% water, and B is 100% acetonitrile. The gradient conditions allow for the minimization of the gap between the two mobile phases reducing the difference in the solubility of air in the two mobile phases, thereby reducing the generation of bubbles.
6、Contamination of solvent bottles and solvent inlet filter.
With the use of pure water phase or mobile phase with high water content for a long time (especially the addition of buffer salts), bacteria are easy to breed, and bacterial metabolites or cell debris stay, resulting in contamination. If the mobile phase is not replaced with a new one after a long time or if the mobile phase bottle is not cleaned but added directly to the replenishment method, it may also lead to ghost peaks.
In this case, developing a good mobile phase bottle cleaning and replacement plan to replace or clean is recommended.
In addition, solvent filter heads can also have contamination. Remove the solvent filter in the solvent bottle to see if the ghost peak can become smaller or disappear. If it becomes smaller or disappears, you can replace the hplc solvent inlet filter in the solvent bottle.
7, from the sample residue.
The sample can be residual in each plate of the instrument. We can check them one by one.
a. Confirm that the problem comes from the sample residue of the inlet.
Connect the inlet and outlet lines of the injector directly with two-way. If the ghost peak problem is solved, suspect the inlet needle, needle seat, and six-way valve rotor contamination problem and perform the relevant cleaning and replacement.
Tips: Before suspecting the residual problems of the injector, check whether the connection line is normal. Otherwise, it will also lead to ghost peaks.
b. Confirm that the problem comes from the sample residue of the hplc column.
The chromatographic column can be an enrichment for strongly retained contaminant components within the mobile phase or system flow path, flushed out as the organic phase ratio changes with the gradient program.
Replacing the column with a two-pass is a contaminated column if the ghost peak is eliminated. This can be solved by cleaning the column or replacing it with a new one.
On a side note, that is, uHPLCs has launched a product for ghost peaks, that is, the ghost peak trap column, in addition to the above steps, plus the ghost peak trap column is the icing on the cake!
| Area | Potential Cause | Solution |
|---|---|---|
| Reagent Issues | ||
| Organic solvent (methanol) | Wide UV absorption range; use acetonitrile for lower wavelengths. | – Consider using HPLC-grade acetonitrile for gradient chromatography, even if ghosting is minimal with methanol. |
| – If using methanol, consider running a blank injection at lower wavelengths to observe baseline drift and assess interference. | ||
| Organic solvent (acetonitrile) | Low quality for gradient chromatography. | – Purchase HPLC-grade acetonitrile specifically for gradient applications. |
| – Consider filtering the acetonitrile through a 0.45 µm filter before use. | ||
| Water | Low-boiling organic impurities. | – Use HPLC-grade pure water for optimal results. |
| – Avoid using homemade distilled water, as it may not remove all interfering impurities. | ||
| Other reagents | Low purity. | – Use high-purity buffer salts, TFA, EDTA, etc. from reputable suppliers. |
| – Consider filtering your prepared mobile phase through a 0.45 µm filter for additional purification. | ||
| Mobile phase configuration | Improper degassing after sonication. | – Ensure thorough degassing with a vacuum filtration system, helium sparging, or online degasser. |
| – Repeat degassing after sonication to remove any trapped air bubbles. | ||
| Container/instrument contamination | Detergents, chromic acid wash, residual impurities. | – Clean mobile phase containers with clean water and organic solvents, not detergents. |
| – Avoid over-cleaning containers, as excessive exposure to solvents can introduce new impurities. | ||
| – Check for plastic cover fragments in containers and remove if found. | ||
| pH meter | Polycarbonate shell soluble in organic solvents. | – Use a glass pH meter for mobile phase pH measurement. |
| Filter membrane | Poor quality or wrong type. | – Use high-quality, HPLC-grade filter membranes compatible with your mobile phase. |
| – Choose the appropriate pore size and membrane material based on your specific needs. | ||
| Mobile phase exposure | Absorbs air impurities, additives oxidize. | – Minimize air exposure of the mobile phase during preparation and storage. |
| – Prepare fresh mobile phase regularly to avoid accumulation of oxidized impurities. | ||
| Instrument Problems | ||
| HPLC tubing contamination | Bacteria growth, metabolites/cell debris. | – Flush tubing with organic solvents like methanol or acetonitrile. |
| – Consider replacing tubing if contamination persists or bacterial growth is suspected. | ||
| Transfer pump failure | System pressure instability. | – Troubleshoot and repair or replace the pump as needed. |
| – Consult the instrument manual for specific troubleshooting steps. | ||
| Check valve blockage | System pressure instability. | – Replace the check valve if necessary. |
| – Consult the instrument manual for valve replacement instructions. | ||
| Inline degasser inefficiency | Incomplete degassing. | – Optimize degassing settings (e.g., vacuum level, flow rate) for your mobile phase. |
| – Consider replacing the degasser membrane or internal components if necessary. | ||
| Mobile phase ratio imbalance | Large difference in air solubility between phases. | – Minimize the gap between A and B phases in your gradient program. |
| – Consider starting the gradient with a slightly higher organic phase ratio to reduce bubble formation. | ||
| Solvent bottle/filter contamination | Bacteria growth, residue buildup. | – Clean and replace solvent bottles regularly, especially with high water content mobile phases. |
| – Replace solvent inlet filters regularly to prevent build-up of contaminants. | ||
| Sample residue | Contamination in injector or column. | – Identify the location of contamination (injector vs. column) via specific tests. |
| – Clean or replace the contaminated component (injector needle, valve rotor, column) as needed. | ||
| Additional Resources | ||
| uHPLCs ghost peak trap column | Consider using this as an additional measure to capture and remove ghost peaks. | – Follow the manufacturer’s instructions for installation and use of the ghost peak trap column. |
Hope Those Solution Idea Can Help You In Your Lab System.
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