Common Abnormal Peak Patterns in HPLC and Relationships with HPLC Column
In the process of HPLC analysis, some abnormal peak shapes often appear due to various reasons, which seriously affect the analysis of the results and bring great trouble to the experimenters. HAWACH introduces some common peak shapes and possible causes here, and we will see whether there is a relationship with the HPLC column.
HPLC Abnormal Peaks and Possible Causes
| Abnormal Peak Type | Possible Causes |
|---|---|
| 1. No Peak | – Sample not injected or decomposed, not dissolved, or concentration too low – LC system failure (e.g., pump, detector) – Inappropriate mobile phase (e.g., too polar in reversed-phase LC) – Inappropriate detector (e.g., UV detector used for non-UV absorbing compounds) |
| 2. Flat Head Peak | – Sample concentration or injection volume is too high (main cause) – Detector settings are incorrect |
| 3. Negative Peak | – The mobile phase has high background absorbance – Air bubbles entered during injection – Sample absorbance is lower than the mobile phase – Sample solvent mismatched with mobile phase |
| 4. Shoulder Peak | – Injection volume or sample concentration is too high – Guard column or column head clogged – Guard column or main column contaminated or degraded – Column head collapsed |
| 5. Split Peak | – Solvent effect: large difference in polarity between the sample solvent and the mobile phase – Guard column or column head clogged – Column is contaminated or invalid – Column head collapsed |
| 6. Ghost Peak | – Residuals in the injection valve; insufficient cleaning between injections – Unknowns in sample; inadequate pretreatment – Mobile phase contamination; needs replacement or filtering – Small air bubbles in the flow path; purge required |
| 7. Protruding Peak | – High sample concentration or injection volume – The sample solvent is more polar than the mobile phase – Column contaminated or degraded – Inappropriate mobile phase; needs optimization (pH/type/ratio) |
| 8. Tailing Peak | – High sample concentration or injection volume – Unsuitable mobile phase (pH/type/ratio) – Silanol interaction (can add triethylamine or buffer) – Clogged frit; replace or add an online filter – Column head collapse or reduced column efficiency |
1. No peak
Possible reasons:
a. The sample was not successfully injected, or the sample was not dissolved in the solvent, or the sample was decomposed or deteriorated, or the sample concentration was too low.
b. The problem of the liquid chromatography instrument itself: pump or detector failure.
c. The mobile phase selection is not appropriate. For example, for reversed-phase silica gel columns, if the mobile phase is too polar, some small polar substances cannot be washed off, and the substances remain in the column, so no peaks can be emitted.
d. The detector is not suitable. For example, some products have no ultraviolet absorption or very weak absorption, but a UV detector is used for detection.
2. Flat Head Peak
Possible reasons:
a. The sample concentration is too large, or the injection volume is too large, which is also the main reason.
b. The detector setting of the liquid chromatography is incorrect.
3. Negative peak
Possible reasons:
a. The mobile phase absorption background value is too high.
b. Air enters during the sampling process.
c. The absorption of the sample components is lower than that of the mobile phase.
d. The solvent of the prepared sample is inconsistent with the mobile phase.
4. Shoulder peak
Possible reasons:
a. The injection volume is too large, and the sample concentration is too high.
b. The guard column or the column head is blocked.
c. The protection column or the chromatographic column is contaminated or invalid.
d. Collapse of the column head.
5. Split peak
Possible reasons:
a. Solvent effect: the polarity of the solvent used to dissolve the sample and the initial mobile phase is too different.
b. The guard column or the column head is blocked.
c. The protection column or the chromatographic column is contaminated or invalid.
d. Collapse of the column head.
6. Ghost Peak
Possible reasons:
Ghost peaks appear in a certain spectrum, and may not appear again after doing the same conditions. Sometimes there are no peaks.
a. For the residual peak of the injection valve, take sufficient time to balance and clean the system after each injection.
b. There are unknowns in the sample; improve the pretreatment of the sample.
c. If the mobile phase is contaminated, replace it with a new mobile phase (or change the brand); prepare as much as possible for current use, and filter the overnight mobile phase when it is used again.
d. There are small bubbles in the flow path; open the Purge valve to increase the flow rate to eliminate.
7. Protruding Peak
Possible reasons:
a. The injection volume or sample concentration is high.
b. The solvent that dissolves the sample is more polar than the mobile phase.
c. The protection column or the chromatographic column is contaminated or invalid.
d. If the mobile phase is not suitable, replace it with a new one (adjust the pH or change the type, ratio, etc.).
8. Tailing peak
Possible reasons:
a. The injection volume or sample concentration is high.
b. If the mobile phase is not suitable, replace it with a new one (adjust the pH or change the type, ratio, etc.).
c. Silanol effect: add triethylamine, increase the concentration of buffer or salt with alkali-induced passivation column to reduce the pH of the mobile phase, and passivate the sample.
d. Failure of the sintered stainless steel in the column: replace the sintered stainless steel, add an online filter, and filter the sample.
e. The column head collapses, or the column efficiency decreases: replace the chromatographic column.
