Increased column pressure
T6. Increased column pressure
Please refer to Technical Information, Troubleshooting for Increased Pressure (PDF 568 KB)
T7. Unstable pump pressure
| Cause | Solution |
|---|---|
| Bubbles in the check valve of a pump. | Degas the check valve (open drain valve, and let mobile phase through) according to the pump's instructions. If the problem persists, wash check valve (e.g., ultrasonic cleaning in water). |
Notes:
- If the bubbles occur often in normal phase chromatography, connect pre-column to increase pressure, and let bubbles elute.
- Degas mobile phase by ultrasonic waves or an aspirator.
T8. Poor resolution on C18 columns
| Solution | Features |
|---|---|
| Use a longer column | A longer column results in sharper peaks. The pressure will increase, and the retention time will be longer. |
| Change mobile phase condition (e.g., pH, type/concentration of organic solvent) |
Knowledge of sample chemistry is required. Complicated conditions may lack reproducibility and are not recommended. |
| Use packing material with non-hydrophobic interactions. | Separate sample by molecular shape selectivity, π-π- electron interactions, or others. Please refer to specialty columns web pages. |
T9. No retention on reversed phase columns
| Solution | Features |
|---|---|
| Use ion-pairing reagents | Ion-pairing reagents enable separation by forming ion pairs with the sample to increase hydrophobicity. Therefore, it is not suitable for non-dissociating samples. |
| Use hydrophilic columns (HILIC). Alternatively, use a reversed phase column with better retention for polar compounds (such as PBr). | Hydrophilic analytes are retained longer compared to C18. |
T10. Excessive retention on reversed phase columns
| Solution | Features |
|---|---|
| Use gradient elution. | Gradient elution shortens analysis time by changing the organic solvent concentration during analysis. Disadvantages: need for a suitable instrument, increasing baseline, and the need for equilibration time between runs. |
| Use UHPLC Columns. | Please see our UHPLC page for details. |
| Change mobile phase conditions. | Problem may be solved by changing pH and/or type/concentration of organic solvent. |
| Use column with low hydrophobicity. | CN-MS is recommended. Please see our catalog or web site for details. |
T11. Different separation performance compared to the past>
| Symptom | Cause | Solution |
|---|---|---|
| Decreased theoretical plates | Natural deterioration of the packing material | No method to recover column |
| Decrease of retention time or separation | Impurities may be adsorbed to the packing material. | The column can be recovered by washing. |
| Stationary phase shedding | No method to recover column |
T12. Different separation performance with a new column
| Cause | Solution | ||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Analytical conditions not suitable for sample | Adjust pH of mobile phase to sample pKa ±2 or greater. | ||||||||||||||||||||||||||||||||||||||
| Use mobile phase with high repeatability. | |||||||||||||||||||||||||||||||||||||||
| Column deteriorated | If the column deteriorates, retention time may decrease, and peak shape may change. Replace the column. | ||||||||||||||||||||||||||||||||||||||
| Lot-to-lot variation |
Contact us with the column name, serial numbers, and present separation
status.
(b) Develop a robust analytical method that is less influenced by lot-to-lot variation. |
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| Column is not the cause (e.g., mobile phase, flow rate, temperature) | Use our FAQ and troubleshooting guide to find the cause. |
T13. Colored elute from columns (colorless sample)
| Cause | Solution |
|---|---|
| Small amount of stationary phase shedding, impurities or previous samples. | Wash column with strongly eluting solvent (e.g., methanol), or use cleaning solution kit for reversed phase HPLC columns (product no. 08966-30). |
A small amount of stationary phase shedding does not affect retention time.
T14. Air inside the column (dry column)
Flow solvent with low viscosity (e.g., methanol) through column at half of normal flow rate for 1 hour. Store it tightly plugged in a cool, dark place.
