No peak
T3. No peaks
Confirm the cause by checking t0 (peak of non-retained compound) first.
| Analysis result of t0 | Cause | |
|---|---|---|
| t0 is not detected (no peak). | Detector may be defective. | |
| Retention time of t0 has shifted. | Pump may be defective. | |
| Retention time of t0 is the same as usual. | Column may be defective. | |
● Solution for Each Column Type
| Column Type | Cause | Solution | |
|---|---|---|---|
| Reversed phase column | Sample is still in the column due to its high hydrophobicity. | Increase elution strength of mobile phase until the sample elutes. For example:
|
|
| Metal coordination or basic compounds may be adsorbed to the column. | Basic compounds may interact with residual silanols in the packing material. Use 3C18-EB or 5C18-MS-II, which have fewer residual silanols, or add 0.1-1% acid (e.g., trifluoroacetic acid, acetic acid) to the mobile phase. | ||
| Metal coordination compounds may interact with a small amount of metal in the packing material. Add 5 mmol/l di-sodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA ・ 2Na) to the mobile phase. | |||
| Normal phase column | Sample is strongly hydrophilic and is still in the column. | Increase elution strength of mobile phase until the sample elutes. Replace with strongly eluting solvent (e.g., ethanol) or increase its concentration. | |
| Metal coordination or basic compounds may be adsorbed to the column. | Add 0.1-1% acid (e.g., trifluoroacetic acid, acetic acid) to the mobile phase. | ||
| Columns for saccharide analysis (Sugar-D, NH2-MS) or hydrophilic column (HILIC) | (5NH2-MS) Sample is adsorbed to amino groups. | Use Sugar-D, which prevents sample adsorption and has high recovery rates. | |
| Sample is strongly hydrophilic and is still in the column. | Increase concentration of water in mobile phase until the sample elutes. Sugar-D and HILIC are compatible with 100% aqueous mobile phase. 5NH2-MS is compatible with up to 50% water (e.g., acetonitrile : water = 50 : 50). | ||
| Gel filtration column (Diol series) | Sample has ionic effect on silanol group. | To increase ionic strength of mobile phase, add approx. 0.3 mol/l of salt (e.g., sodium chloride). | |
| Adjust mobile phase pH to 5.5 or less to prevent ionic interaction. | |||
| Sample may have adsorbed to stationary phase due to hydrophobic interaction. | Add 10-50% organic solvent (e.g.,acetonitrile) to mobile phase. | ||
| Hydrophobic chromatography column (HIC) | Hydrophobicity of sample is too strong | Add 5% organic solvent (e.g., methanol or acetonitrile). | |
| Sample may have ammonium sulfate precipitation before injection. | Decrease concentration of ammonium sulfate to 0.5 mol/l or less, until no precipitation is observed. | ||
● Defective Pump
| Cause | Solution | |
|---|---|---|
| Bubbles generated in the pump | Degas. Please refer to T7. | |
| Solvent leaking | Tighten connectors and/or replace tubing. | |
● Defective Detector
| Cause | Solution | |
|---|---|---|
| Detector is not connected correctly. | Follow the detector user manual to connect correctly. | |
| Defective signal from the detector | Contact detector manufacturer. | |
| UV absorption range is not suitable for your sample. | Analyze at suitable UV absorption wavelength for the sample. If the sample has little or no UV absorption, use refractive index (RI) detector or evaporative light scattering detector (ELSD), or label the sample. | |
T4. Unstable base line
| Cause | Solution | |
|---|---|---|
| Sample may be adsorb to the column, and elute on the next analysis. | Wash with strongly eluting solvents. Please refer to Q13. | |
| The bonded phases of PE-MS, πNAP, PYE, NPE, PBr, and Cholester have UV absorption, and slight shedding may cause baseline noise. | Wash with strongly eluting solvents. Please refer to Q13. A small amount of bonded phase shedding does not significantly affect column performance. |
|
| Sudden changes in the pump pressure may create bubbles. | Degas. Please refer to T7. | |
| When using refractive index (RI) detector | ||
| Large temperature variation | Use a thermostatic bath to keep a constant temperature. Be aware of air conditioners or other wind sources blowing on the RI detector or tubing. Cover equipment and tubing for insulation. | |
| Residual gas in mobile phase | Degas mobile phase (by ultrasonic waves or aspirator). | |
| Needle-like peaks from an ultraviolet-visible detector | ||
| Bubbles in the column or detector | Increase pressure to remove bubbles by blocking outlet of the detector. Caution: Too much pressure may break the detector cell. If the problem persists, disconnect the column and flow a viscous solvent (e.g., 2-propanol) for 15 min. |
|
| Column temperature may be above or near the boiling point of the mobile phase, creating bubbles. | Analyze at suitable temperature. Generally, 20-50°C is suitable temperature
for columns. To get the best results, analyze at least 20°C lower than the boiling point of the mobile phase (for methanol [boiling point: 64.7°C], analyze at 45°C or less). |
|
| When using ion-pair reagents or buffers | ||
| Inadequate equilibration of the column | Use longer equilibration time. When using ion-pair reagents or buffers, longer equilibration time is required compared to mobile phases without them. | |
| Salt may precipitate in the mobile phase (the mobile phase reservoir may become cloudy). | (a) Decrease concentration of buffer. (e.g., 100 mmol/l → 20 mmol/l) (b) Replace with a different buffer solution. (e.g., phosphoric acid buffer → acetic acid buffer) (c) Reduce concentration of organic solvent. (e.g., acetonitrile : water = 70 : 30 → 50 : 50) (d) Replace with a different organic solvent. (e.g., acetonitrile → methanol) |
|
T5. Unstable retention time
● Cause by Equipment
| Equipment | Cause | Solution | |
|---|---|---|---|
| Pump | Bubbles in the check valve of pump. | Degas. Please refer to T7. Normal phase solvents have lower boiling point, so bubbles are easily created. Furthermore, its low viscosity prevents bubbles from eluting. | |
| Solvent leaking | Tighten the leaking part. If the problem is not resolved, replace it. | ||
| Thermostatic Bath (for adjusting temperature) |
Column temperature may vary by season or the time of day (without the use of thermostatic bath or column oven). | Use thermostatic bath or column oven to keep a consistent column temperature. Set thermostatic bath or column oven to 5°C above room temperature when doing room temperature analyses to prevent temperature fluctuation. | |
● Cause by Columns
| Column Type | Cause | Solution | |
|---|---|---|---|
| Reversed Phase Columns | Inadequate column equilibration when using ion-paring reagents. | Make equilibration time longer. When using ion-pairing reagents, longer equilibration time is often required. | |
| If 100% water is used as mobile phase on a C18 column, phase collapse may occur. |
Use C18-PAQ, which is compatible with 100% aqueous mobile phases. To fix phase collapse, wash the column with high-organic solvents (e.g., methanol : water = 70 : 30). | ||
| Normal Phase Columns | A small amount of water in the organic solvent may affect retention time. | Remove water from mobile phase. If the sample solvent contains water, change the sample solvent or decrease the injection volume. If water is trapped in the column, wash with ethanol to recover. | |
| Columns for Saccharide Analysis (Sugar-D, NH2-MS) or Hydrophilic Chromatography column (HILIC) | A small amount of stationary phase has detached. | Sugar-D and HILIC can be recovered by washing with 100% water for 15 minutes. 5NH2-MS may be recovered by washing with 50% water (e.g., acetonitrile : water = 50 : 50) for 15 minutes. | |
